Charles University in Prague

Faculty of Science
Department of Cell Biology

Crosstalk of integrin and mTOR signalling

Diploma thesis

Lucie Teglová

2010

Supervised by RNDr. Daniel Rösel, Ph.D.

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 Hereby, I declare that this thesis is my own work based on consultation with my
supervisor and literature.

Prague, May 2010

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 Acknowledgements
In the first place, I would like to express my gratitude to my supervisor, RNDr. Daniel
Rösel, Ph.D. for showing me the way and helping me with this project whenever I needed it.
I also thank to RNDr. Jan Brábek, Ph.D. for his valuable comments and never-ending
enthusiasm.

Especially, I address my thanks to all members of Laboratory of Cellular and Molecular

biology for stimulative atmosphere and to Mgr. Zuzana Gerndtová for her assistance with
cell cytometry.

Finally, I thank my fantastic family for their support throughout my life.

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 Abstract
Crosstalk of integrin and mTOR signalling is an essential process that monitors cellular
interaction with extracellular matrix and transmits these inputs to cell growth signalling.
Although adhesion status of the cell monitored by integrin signalling is clearly important
for regulation of cellular growth, a little is known about the crosstalk of integrin and mTOR
signalling. In this study, we employed two different approaches to describe and elucidate
character of this crosstalk. p130Cas is an adaptor protein phosphorylated by Src kinase and
focal adhesion kinase upon integrin ligand binding and implicated in cell adhesion, motility
and survival in both Src-transformed and untransformed cells. Recently, p130Cas was also
described in cellular pathology, mainly by its ability to stimulate cell invasion and
metastasis. In this study, we described that p130Cas affects mTOR signalling
in Src-transformed cells. Substrate domain of p130Cas was found to be indispensable for this
effect and is also responsible for serum-induced activation of mTOR signalling. In addition,
we prepared cell lines overexpressing various Rheb protein versions and characterized them
in context of mTOR signalling, integrin signalling and cell cycle progression. Interestingly,
a cell line overexpressing constitutively active Rheb harbouring mutations S16H and Q64L
stimulated Paxillin phosphorylation (Y118) independently of focal adhesion kinase. This
result suggests that integrin signalling might be affected by activation of mTOR complex 1.

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 Table of contents
ACKNOWLEDGEMENTS ...................................................................................................... III

ABSTRACT ............................................................................................................................... IV

ABBREVIATIONS ................................................................................................................. VII

1 THEORETICAL OVERVIEW ........................................................................................... 2

1.1 MTOR PATHWAY ................................................................................................................ 2

1.1.1 MTOR COMPLEX 1............................................................................................................. 2
1.1.2 UPSTREAM REGULATION OF MTORC1.............................................................................. 3
1.1.2.1 Growth factors signalling ............................................................................................... 3
1.1.2.2 Cellular energy status ..................................................................................................... 5
1.1.2.3 Nutrients ......................................................................................................................... 6
1.1.2.4 Oxygen availability ........................................................................................................ 6
1.1.3 MTOR COMPLEX 2............................................................................................................. 7
1.2 INTEGRIN SIGNALLING ....................................................................................................... 8
1.2.1 INTEGRINS – MODULATORS OF CELL ADHESION ............................................................... 8
1.2.2 P130CAS/BCAR1 .............................................................................................................. 9
1.3 CROSSTALK OF INTEGRIN AND MTOR PATHWAY .......................................................... 11
1.3.1 MTOR/INTEGRIN CROSSTALK ......................................................................................... 11
1.3.2 MTOR/INTEGRIN CROSSTALK IN CANCER ....................................................................... 12

2 MATERIALS AND METHODS ....................................................................................... 15

2.1 MATERIALS ....................................................................................................................... 15

2.1.1 ORGANISMS USED............................................................................................................ 15
2.1.2 CELL LINES USED............................................................................................................. 15
2.2 PLASMIDS CONSTRUCTED AND USED ............................................................................... 16
2.2.1 LIST OF PRIMARY AND SECONDARY ANTIBODIES USED .................................................. 17
2.2.2 SOLUTIONS FOR CELL CULTURES HANDLING .................................................................. 18
2.2.3 SOLUTIONS FOR PROTEIN ANALYSIS ............................................................................... 18
2.2.4 SOLUTIONS FOR DNA MANIPULATION AND BACTERIAL CULTURES HANDLING ............. 19
2.3 METHODS .......................................................................................................................... 19
2.3.1 CELL CULTURE TECHNIQUES ........................................................................................... 19
2.3.1.1 Cell culture maintaining ............................................................................................... 19
2.3.1.2 Cell cultures preservation ............................................................................................. 20
2.3.1.3 Transfection procedure and stable cell lines establishment ......................................... 20
2.3.1.4 Cell lysis ....................................................................................................................... 21
2.3.2 DNA MANIPULATION TECHNIQUES ................................................................................. 21
2.3.2.1 Plasmid DNA purification............................................................................................ 21
2.3.2.2 DNA extraction from agarose gels ............................................................................... 22
2.3.2.3 Plasmid restriction ........................................................................................................ 23
2.3.2.4 Electrophoresis in agarose gel ...................................................................................... 23
2.3.2.5 DNA ligation ................................................................................................................ 24
2.3.2.6 Transformation of E.coli by electroporation ................................................................ 24
2.3.3 PROTEIN MANIPULATION TECHNIQUES ........................................................................... 24
2.3.3.1 Protein concentration determination ............................................................................ 24
2.3.3.2 Protein electrophoresis ................................................................................................. 25

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2.3.3.3 Western blot ................................................................................................................. 26
2.3.3.4 Immunodetection ......................................................................................................... 26
2.3.3.5 Cytometry..................................................................................................................... 27
2.3.3.6 Microscopy................................................................................................................... 27
2.3.3.7 Statistical analyses ....................................................................................................... 28
2.3.3.8 Densitometry ................................................................................................................ 28

3 RESULTS ............................................................................................................................ 29

3.1 THE ROLE OF P130CAS IN MTOR SIGNALLING .............................................................. 29

3.1.1 THE ROLE OF P130CAS IN MTORC1 SIGNALLING ........................................................... 30
3.1.2 THE ROLE OF P130CAS IN MTORC2 SIGNALLING ........................................................... 33
3.2 RHEB PROTEIN AND ITS EFFECTS ON MTOR SIGNALLING, INTEGRIN SIGNALLING AND
CELL CYCLE PROGRESSION ....................................................................................................... 35
3.2.1 CHARACTERIZATION OF RHEB VARIANTS IN CONTEXT OF MTORC1 SIGNALLING ......... 42
3.2.2 MTORC2 IS NOT INFLUENCED BY RHEB VERSIONS OVEREXPRESSION ........................... 43
3.2.3 EXPRESSION OF RHEB VARIANTS HAS NO EFFECT ON CELLULAR MORPHOLOGY ........... 45
3.2.4 RHEB AFFECTS INTEGRIN SIGNALLING ............................................................................ 46
3.2.5 ROLE OF P130CAS IN CELL CYCLE PROGRESSION........................................................... 48

4 DISCUSSION ...................................................................................................................... 51

5 SUMMARY ......................................................................................................................... 56

6 REFERENCES ................................................................................................................... 57

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Abbreviations
4E-BP1 4E-binding protein 1

AMPK AMP-activated protein kinase

APS ammonium persulfate

BCAR1 breast cancer resistance 1

bp base pair

BSA bovine serum albumin

CA constitutively active version

DABCO 1,4-diazabicyclo (2.2.2.) octane

DMEM Dulbecco‟s modified eagle medium

DMSO dimethylsulfoxide

DN dominant negative version

DTT dithiothreitol

ECM extracellular matrix

EDTA ethylenediaminetetraacetic acid

EGFP enhanced green fluorescent protein

EGTA ethyleneglycoltetraacetic acid

eIF4E eukaryotic translation initiatio factor 4E

ERK extracellular-signal-regulated kinase

FACS fluorescence-activated cell sorting

FAK focal adhesion kinase

FBS fetal bovine serum

FKBP12 FK506-binding protein 12 kDa

FSC forward scatter

GAP GTPase-activating protein

GEF guanine nucleotide exchange factor

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IRS insulin receptor substrate 1

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LB Luria Bertani broth

LKB1 liver kinase B1

MEF mouse embryonic fibroblasts

mSIN1 mammalian stress-activated protein kinase interacting protein

mTOR mammalian target of rapamycin

mTORC1 mTOR complex 1

mTORC2 mTOR complex 2

p130Cas Crk-associated substrate of 130 kDa

PBS phosphate buffer saline

PDK phosphoinositide-dependent kinase

PI3K phosphatidylinositol-3-OH kinase

PIP3 phosphatidylinositol 3,4,5 triphosphate

PIPES piperazine-N,N′-bis(2-ethanesulfonic acid)

PKC protein kinase C

RSK 90-kDa ribosomal S6 kinase

RT room temperature

S6K p70 S6 kinase

SD substrate domain of p130Cas

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SH2 Src homology 2

SH3 Src homology 3

TBS Tris buffer saline

TEMED N, N, N‟, N‟-tetramethylethylendiammin

TSC tuberous sclerosis complex

TTBS Tris buffer saline+Tween 20

WT wild type version

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1 Theoretical overview

1.1 mTOR pathway

The serine/threonine protein kinase mTOR (mammalian target of rapamycin) plays a
central role within a signalling cascade that regulates multiple fundamental cellular processes
including cell growth, cell migration and survival. In mTOR pathway, both extracellular
stimuli and intracellular signals are integrated to regulate e.g. mRNA translation, autophagy
and cell cycle progression. Moreover, deregulations of mTOR pathway occur in number of
diseases, e.g. cancer and diabetes attracting even more attention to the mechanisms of mTOR
regulation and its elucidation. Macrolide rapamycin and its analogs have been widely used to
anticancer and transplantional therapy with promising results. The mTOR kinase belongs to
PI3-K related kinase family and resides in two functionally and structurally distinct
complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).

The protein kinase mTOR is a 289-kDa serine-threonine kinase that remains conserved
from yeasts to mammals. The TOR genes (tor1 and tor2) were originally identified in yeast
genetic screen for genes mutated upon rapamycin treatment (Heitman et al., 1991). Further
studies revealed TOR genes also in genomes of higher eukaryotes and proposed mechanisms
of rapamycin binding to TOR protein. Upon entering the cell, rapamycin interacts with
FK506-binding protein 12 kDa (FKBP12) and specifically binds to TOR, thus interfering
with its function (Chiu et al., 1994; Sabatini et al., 1994).

1.1.1 mTOR complex 1

The mTORC1 is sensitive to bacterial macrolide rapamycin and consists of mTOR kinase
(also known as FRAP1) and two associated proteins: raptor and GβL (also known as LST8).
Raptor (regulatory associated protein of mTOR) determines the specifity of mTORC1, in
part by interaction with its substrates by a 5-amino acid motif termed TOS (TOR signalling
motif) which is found at the N-termini of S6K1 and 4E-BP1 (Schalm and Blenis, 2002). The
role of LST8 is still unknown. Although RNAi experiments in cell cultures showed that
mTORC1 is affected by LST8 suppression (Jacinto et al., 2004), no change in mTORC1
signalling was observed in lst-/- mouse primary fibroblasts (Guertin et al., 2006).

The phosphotransferase activity of mTORC1 regulates its major substrates: p70 ribosomal
S6 kinases – p70S6Ks (including S6K1 and S6K2) and 4E-BP proteins. The mTORC1
positively controls proteosynthesis by phosphorylation of S6K1, a member of AGC kinase
family. Phosphorylated S6K1 stimulates cap-dependent mRNA translation initiation, mRNA

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 biogenesis and also ribosome biogenesis. S6K1 was proposed to phosphorylate translation
initiation factor eIF4B and protein PDCD4 and both of them converge on eIF4A to regulate
the helicase function of eIF4F during translation initiation. Another S6K1 substrates, SKAR
and CBP80 (cap-binding protein 80) have been shown to associate with newly synthesized
mRNA, but their exact function remains to be investigated. The activation of mTORC1 also
leads to phosphorylation of translation initiation factor 4E-BP1 that prevents eIF4E from
binding to eIF4F. Released eIF4E can interact with eIF4G and eIF4A at 5„end of mRNA.
Subsequently, a small ribosomal subunit, ternary complex (comprising eIF2, Met-tRNA and
GTP) and eIF3 assemble at 5‟end and promote ribosome scanning and initiation of
translation. The 40S ribosomal protein S6, another S6K1 substrate, has been thought to
mediate translation of mRNA transcripts containing 5„ terminal oligopyrimidine tract (5„
TOP motif), but data from s6-/- mouse embryonic fibroblasts (MEF) do not support this
notion (Ruvinsky et al., 2005). In addition to regulation of protein biogenesis, mTORC1 is
also involved in regulation of lipid synthesis. It has been demonstrated that mTORC1
regulates the activity of at least two transcription factors, SREBP1 and PPARγ that control
expression of genes involved in lipid and cholesterol homeostasis as reviewed in (Laplante
and Sabatini, 2009). Least but not last, mTORC1 is able to phosphorylate and thus
negatively regulate insulin receptor substrate 1 (IRS1) (Harrington et al., 2004).

Besides its main function as activator of diverse anabolic processes, mTORC1 also
inhibits catabolic processes, such as mRNA degradation or autophagy. In response to limited
nutrients, cells undergo sequestration of organelles and their degradation in lysosomes, by a
process called autophagy. Autophagy provides biological material in conditions of reduced
nutrients availability. When activated, mTORC1 was reported to inhibit autophagy by
phosphorylation of autophagy-related gene 13 (ATG13) and unc-51-line kinase 1 (ULK1).

1.1.2 Upstream regulation of mTORC1

As a key regulator of cell growth, mTOR pathway is under control of sophisticated

mechanisms integrating signals from growth factors and monitoring nutrient availability,
energy status of the cell and oxygen sufficiency (Fig. 1.1).

1.1.2.1 Growth factors signalling

In response to growth factors, receptor tyrosine kinases and G-protein coupled receptors
become activated in the first line. Phosphatidylinositol-3-OH kinase (PI3K)/Akt and
Ras/ERK pathways belong to the most important signalling cascades that become stimulated

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after growth factor binding to the receptor. Upon Akt/PKB stimulation by PI3K and
phosphorylation by phosphoinositide-dependent kinases (PDKs), a complex of tumor
suppressors TSC1/2 undergoes phosphorylation by Akt/PKB on several residues, including
S939 and T1462. Tumor suppressor complex TSC1/2 which includes proteins tuberous
sclerosis complex 1 - hamartin (TSC1) and tuberous sclerosis complex 2 - tuberin (TSC2) is
an intracellular sensor that integrates inputs from extracellular environment (growth factors,
amino acids, oxygen availability) and monitors intracellular energy status. Mutations in both
genes, TSC1 and TSC2 were described to cause a tuberous sclerosis disease. In a cell, both
proteins form a heterodimeric complex that acts as negative upstream regulator of mTORC1.
TSC2 protein includes a GTPase-activating protein (GAP) domain that promotes GTP
hydrolysis in small G-protein Rheb. As a result, Rheb protein resides in a GDP-bound
inactive state, which prevents Rheb-mediated activation of mTORC1. Phosphorylation by
Akt/PKB impairs TSC2 GAP activity towards Rheb, thus allowing mTORC1 activation by
Rheb. Similarly, extracellular-signal-regulated kinase (ERK) and 90-kDa ribosomal S6
kinase (RSK) were shown to negatively regulate TSC2 by phosphorylation (S664 and
S1798), enabling mTORC1 activation (Ballif et al., 2005; Ma et al., 2005). Although the
Ras/ERK pathway has an established role in transcription regulation, its contribution to
regulation of translation requires further investigation.

Recently, a novel mTORC1 binding protein PRAS 40 (40 kDa Pro-rich Akt substrate,
Akt1S1) has been showed to link Akt/PKB to mTORC1 signalling. PRAS40 contains a
TOR–signalling motif and functions as mTORC1 inhibitor that competes with its substrates
4E-BP1 or S6K. Activated Akt/PKB phosphorylates PRAS40 evoking its sequestration by
14-3-3 proteins. Finally, mTORC1 is released and can phosphorylate PRAS40 creating a
positive feedback mechanism.

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 Figure 1.1: A brief graphical overview of mTOR pathway and its upstream regulation (Yang and Guan,
2007). Upon insulin binding to its receptor, IRS1 (insulin receptor substrate 1) becomes activated and
stimulates PI3K phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol
3,4,5 triphosphate (PIP3). This leads to recruitment of Akt/PKB to the plasma membrane and its activation.
Subsequently, Akt/PKB inhibits TSC1/2 GTPase activity towards Rheb by phosphorylation of TSC2.
However, negative feedback loop represses PI3K/Akt signalling after prolonged mTORC1 activation. S6K
phosphorylates IRS1 (Harrington et al., 2004) reducing its signalling potential to PI3K and promoting
insulin-resistance with implications in both diabetes type II and cancer.

1.1.2.2 Cellular energy status

During intensive proliferation, translation may consume up to 40% of the energy

generated by the cell. Therefore, it‟s essential to carefully monitor cellular energy status and
suppress translation when necessary. AMP-activated protein kinase (AMPK) is believed to
be the master regulator of cellular energy status. AMPK monitors intracellular AMP level or
AMP/ATP ratio (Kahn et al., 2005). Under condition of reduced cellular energy levels,
AMPK becomes phosphorylated in the activation loop by liver kinase B1 (LKB1) and thus
activated. Active AMPK suppresses ATP-consuming processes of the cell, e.g. fatty acids

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 and cholesterol synthesis, inhibiting mTORC1 signalling as well. TSC1/2 undergoes
phosphorylation by AMPK on S1345 that enhances its GTPase activity towards Rheb and
thereby reduces mTORC1 signalling. Recently, AMPK was shown to control mTORC1
independently on TSC1/2. Gwinn and co-authors described that AMPK directly
phosphorylates raptor on S792 and S722 which induces binding of 14-3-3 proteins (Gwinn et
al., 2008). Taken together, AMPK dispose of variety of mechanisms to suppress mTORC1
signalling in order to prevent depletion of cellular energy resources.

1.1.2.3 Nutrients

In the last decade was repeatedly shown that mTORC1 is inhibited by amino acids
withdrawal and that readdition of amino acids can trigger its signalling (Hara et al., 1998;
Wang et al., 1998). However, the mechanism by which amino acids signal to mTORC1
remains to be elucidated. Earlier, TSC1/2 complex was considered to integrate information
on amino acids availability and transduce it to mTORC1 (Gao et al., 2002). But recently,
amino acid signalling was shown to be independent on TSC1/2 (Smith et al., 2005) and
various studies implicated a class 3 PI3K, hVPs34 in the amino acids response (Nobukuni et
al., 2005). In addition, Rag proteins were described to interact with mTOR in amino
acid-dependent manner (Kim et al., 2008; Sancak et al., 2008). Sancak and co-authors (2008)
further proposed that Rag proteins stimulate amino acid-induced localization of mTOR
within the endomembrane system where it might be available for its activator Rheb.

1.1.2.4 Oxygen availability

Cellular responses to hypoxia (deprivation of oxygen supply) are controlled and mediated
by three main pathways: hypoxia-inducible factor (HIF) pathway, mTORC1 pathway and
unfolded protein response (UPR) pathway. In 2004, Amrsham and co-authors described
mTORC1 inhibition after brief exposures to modest hypoxia (Arsham et al., 2003). Later
was described that mTORC1 inhibition was mediated by AMPK-dependent activation of
TSC1/2 (Liu et al., 2006). In general, hypoxia inhibits mTORC1 signalling and mRNA
translation initiation by many independent mechanisms.

Numerous studies observed that decreased oxygen availability can stimulate transcription
of REDD1 gene (Brugarolas et al., 2004; Sofer et al., 2005). REDD1 protein can indirectly
stimulate TSC1/2 activity by preventing its binding to 14-3-3 proteins. Thus, Rheb protein
remains GDP-bound and mTORC1 remains inactive. In addition, Bcl2/Adenovirus E1B
19 kDa protein-interacting protein 3 (BNIP3) was shown to associate with Rheb protein and

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 thus inhibits mTORC1 signalling (Li et al., 2007). Similarly, promyelocytic leukemia tumor
suppressor (PML) was shown to inactivate mTORC1 by its sequestration in nuclear bodies
(Bernardi et al., 2006).

1.1.3 mTOR complex 2

mTOR complex 2 was identified in 2004 (Jacinto et al., 2004; Sarbassov et al., 2004) and
although this rapamycin-insensitive complex has been very intensively studied, its function
and regulation is in comparison to mTORC1 still poorly understood. Both mTORC1 and
mTORC2 comprise shared components, mTOR kinase and LST8 (GβL). In addition, rictor
(rapamycin insensitive companion of mTOR), mSIN1 (mammalian stress-activated protein
kinase interacting protein) and Protor-1 (protein observed with rictor-1) are found in
mTORC2. Except for Protor-1, all components are essential, as inactivation of any of them
in mice causes embryonic lethality (Guertin et al., 2006; Yang et al., 2006a). There is some
evidence, that mentioned components stabilize each other in the complex but the mechanism
is poorly understood.

The mTORC2 was originally described as rapamycin-resistant complex, because

rapamycin-bound FKBP12 cannot directly interact with mTORC2 (Jacinto et al., 2004;
Sarbassov et al., 2004). However, in certain cell lines prolonged rapamycin treatment was
found to affect also mTORC2, probably by inhibiting its assembly (Sarbassov et al., 2006).

Our understanding of mTORC2 upstream regulation is still elusive. Various research

groups reported that mTORC2 can phosphorylate its substrate Akt/PKB upon growth factor
stimulation (Frias et al., 2006), implicating that PI3K pathway may regulate mTORC2, but
the direct mechanism remains to be determined. Recently, TSC1/2 tumor suppressor
complex was proposed to stimulate phosphotransferase activity of mTORC2 towards its
substrate Akt/PKB by direct binding (Huang et al., 2008). Unlike mTORC1 that is
negatively regulated by TSC1/2 complex in Rheb-dependent manner, mTORC2 activation
by TSC1/2 seems to be independent of both Rheb protein and GTPase activity of tuberin.
Recently, Kamimura and co-authors proposed a novel insight in PIP3-independent mTORC2
activation in Dictyostelium discoideum. They suggest that Ras GTPases and heterotrimeric
G-proteins act as upstream regulators of TORC2 (Kamimura et al., 2008). Further evidence
is required to elucidate whether this regulation is conserved in mammals.

In 2005, Akt/PKB was identified as mTORC2 substrate (Sarbassov et al., 2005), thus
employing mTORC2 as an important regulator of cell growth, proliferation, metabolism and
survival. Full activation of Akt/PKB requires its phosphorylation at threonine 308 by PDK1

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 and at serine 473 by mTORC2. Interestingly, some studies suggested that T308
phosphorylation does not depend on prior S473 phosphorylation, which is in contrast to
hierarchical phosphorylation of another AGC kinase, p70S6K (Biondi et al., 2001; Yang and
Guan, 2007). Inhibition of Akt following mTORC2 depletion reduces Akt/PKB activity to
certain but not all substrates suggesting that Akt/PKB phosphorylation at S473 by mTORC2
determine Akt substrates specifity (Guertin et al., 2006; Jacinto et al., 2006)

As mentioned above, mTORC2 was found to phosphorylate kinases of AGC family,

similarly to mTORC1. Shortly after this fact was observed, new AGC kinases were
identified as mTORC2 substrates. In 2008, mTORC2 was shown to phosphorylate serum and
glucocorticoid-induced kinase 1 (SGK1) that controls cellular processes such as ion transport
and growth (Garcia-Martinez and Alessi, 2008). Different studies focused on protein
kinase C (PKC) phosphorylation at the hydrophobic motif, that is mTORC2 dependent, but
probably not mediated by mTORC2 (Ikenoue et al., 2008). No PKC phosphorylation by
mTORC2 has been observed in vitro yet.

When discovered in 2004, mTORC2 was immediately linked to regulation of actin

cytoskeleton (Jacinto et al., 2004; Sarbassov et al., 2004). It was suggested that Rho GTPases
and PKCα are involved, however no molecular mechanisms has been determined yet.

1.2 Integrin signalling

1.2.1 Integrins – modulators of cell adhesion

The integrin family of cell adhesion receptors regulates diverse processes within the cell,
e.g. cell growth, cell survival and cell migration. In addition, a wide variety of integrins
contribute to tumor progression either by deregulation of cell proliferation and survival or by
stimulation of cell motility and invasion (Desgrosellier and Cheresh, 2010).

Integrins are heterodimeric cellular receptors that mediate interaction with extracellular
matrix and immunoglobulin superfamily of receptors. Up to date, 24 distinct integrin
heterodimers were described. Upon integrin binding to the extracellular matrix (ECM) and
clustering, various signalling and adaptor proteins are recruited to signalling complex. The
key element of integrin-dependent signalling is focal adhesion kinase (FAK) which becomes
activated by autophosphorylation at tyrosine 397 (Schaller et al., 1994). This allows binding
of Src family kinases and variety of adaptor proteins, e.g. p130Cas (Fig. 1.2).

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 Figure 1.2: Schematic representation of signalling pathways that are regulated by integrin binding to ECM.
(Guo and Giancotti, 2004).

1.2.2 p130Cas/BCAR1

Crk-associated substrate of 130 kDa (p130Cas, also known as BCAR1) was originally
identified in 1990‟s as a protein that is hyperphosphorylated upon transformation by v-Crk
and v-Src oncogenes (Sakai et al., 1994). Subsequently, human p130Cas - BCAR1 (breast
cancer resistance 1) gene was identified in a retroviral insertion screen for genes that
promote resistance to tamoxifen (Brinkman et al., 2000). Both findings demonstrated clear
implications of p130Cas deregulation for cancer biology, thus attracting even more attention
to the physiological role of this protein.

In mammals, family of the Cas proteins includes four members: p130Cas/BCAR1,

HEF1/NEDD9/Cas-L, EFS/SIN and HEPL/CASS4. Although, Cas proteins do not possess
any enzymatic activity, they undergo extensive posttranslational modification and act as
adaptor proteins by promoting numberless protein-protein interactions. Their conserved
structure consists of four main domains: amino-terminal SH3 domain, substrate domain,

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four-helix bundle domain and C-terminal domain (Fig. 1.3). The amino-terminal domain Src
homology 3 (SH3) domain provides a binding site for proteins containing polyproline helix,
e.g. FAK, proline-rich tyrosine kinase 2 kinase (PYK2), Rap 1 GEF called C3G, protein
phosphatases PTP-PEST and PTP1B and FRNK (FAK-related non-kinase). Centrally located
is a large intrinsically unstructured region containing 15 repeats of YxxP motif – the
substrate domain (SD). Phosphorylation by Src family kinases within the substrate domain
creates binding sites for Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains
of effector proteins such as Crk or Nck. Four-helix bundle domain enables binding of 14-3-3
proteins. The carboxy-terminal part of p130Cas is well conserved and comprises a
proline-rich region (RPLPSPP) that is bound by Src family kinases and contributes to
processive phosphorylation of p130Cas. Moreover, the C-terminal domain includes an
YDYVHL motif that is phosphorylated during cell adhesion and can bind Src family
members and BCAR3/AND-34 protein.

Figure 1.3: Schematic representation of p130Cas that emphasizes major structural features of this protein
(Tikhmyanova et al., 2009).

p130Cas protein undergoes extensive and rapid changes in phosphorylation upon both
intracellular and extracellular cues. Activation of growth factor receptors (both receptor
tyrosine kinases and G-protein coupled receptors) might stimulate p130Cas directly by
phosphorylation of Y331 in SD by epidermal growth factor (Zhang et al., 2005) or indirectly
by Src kinases. Upon integrin stimulation, activated focal adhesion kinase (FAK) binds to
Cas SH3 domain and phosphorylates it at C-terminal, thus recruiting Src family kinases
(Vuori et al., 1996). Subsequently, Src kinases phosphorylate multiple tyrosine residues of
substrate domain, creating binding sites for various proteins, most notably Crk adaptor
protein. As p130Cas can bind independently to both FAK and Src, a complex comprising
p130Cas, Src and FAK can act as profound regulator of various cellular processes upon
integrin and growth factors stimulation. Furthermore, p130Cas phosphorylation is modulated
by a number of phosphatases, e.g. PTP-PEST, PP2A or PTP-LAR, as reviewed in

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 (Tikhmyanova et al., 2009). Besides changes in phosphorylation, p130Cas is also subject of
proteolytic cleavage in a cell (Kook et al., 2000).

Within a cell, majority of p130Cas localizes to focal adhesions (Donato et al., 2010) and
plays an important role in regulation of cell migration and motility (Defilippi et al., 2006).
During cell attachment to extracellular matrix, phosphorylation of substrate domain of
p130Cas recruits adaptor proteins Crk/Crk-L that allow the binding of DOCK180, a
GTP-exchange factor for Rac GTPase. Activated Rac stimulates actin cytoskeleton
remodelling by ARP2/3 complex and p21-activated kinase (PAK) activation as reviewed in
(Chodniewicz and Klemke, 2004). Additionally, p130Cas was shown to interact with zyxin
family of LIM proteins to regulate cell migration (Pratt et al., 2005; Yi et al., 2002) and
might enhance the ability of oncogenic Src to produce matrix metalloproteases (Brabek et
al., 2005), thus confirming the role of p130Cas in cell invasion. Interestingly, p130Cas is
likely to be involved in modulation of cell cycle, because p130Cas/Crk complex was shown
to modulate JNK activation (Dolfi et al., 1998; Oktay et al., 1999).

1.3 Crosstalk of integrin and mTOR pathway

mTOR and integrin dependent signalling regulate distinct cellular processes. Though,
several lines of evidence indicate the existence of direct and indirect (mediated by PI3K/Akt)
crosstalk between both signalling pathways (Fig. 1.4).

1.3.1 mTOR/integrin crosstalk

The first notion implicating crosstalk between integrin signalling and mTOR pathway
proposed a model of S6K activation by integrin signalling (Malik and Parsons, 1996).
Authors suggested that integrin interaction with ECM triggers signalling to mTOR pathway,
thus providing cell survival and proliferation signal. Increased S6K activity (determined by
in vitro kinase assay) was reported in REF52 cells that were cultivated at fibronectin
compared to poly-L-lysine. This activity was inhibited by wortmannin (10 nM; 100 nM),
rapamycin (1 nM;10 nM) and tyrosine kinase inhibitors (herbimycinA, genistein). According
to these experiments, PI3K and mTOR are required to integrin-dependent S6K activation.
The role of FAK remained elusive, because cytochalasin D treatment significantly decreased
FAK tyrosine phosphorylation, but S6K activity remained unaffected. Finally, negative
regulator of FAK called FAK-related non-kinase caused reduction in S6K activity, proposing
at least partial requirement of FAK in S6K activation.

Few years ago, a detailed model explaining FAK effect on S6K was proposed (Gan et al.,
2006). Authors demonstrated that FAK coimmunoprecipitates with TSC2 protein in lysates

11
 from 293T cells cotransfected with both TSC2 encoding vector and FAK encoding vector. A
direct interaction between TSC1 and FAK was not revealed. In addition, FAK was shown to
regulate cell growth and phosphorylation of 4E-BP1 and S6K and this regulation is
dependent on its kinase activity, because cells transfected with FAK kinase dead mutant
exhibited reduced size and phosphorylation of both mTOR substrates, S6K and 4E-BP1. The
role of FAK in S6K phosphorylation was further assessed by RNAi that confirmed that FAK
plays a positive role in adhesion-induced S6K phosphorylation. Taken together, this study
described a possible direct crosstalk of integrin and mTOR signalling.

Interestingly, in cells treated with tumstatin, an inhibitor of angiogenesis, Maeshima and

co-authors observed considerable inhibition of FAK, PI3K, Akt and mTOR phosphorylation
(Maeshima et al., 2002). Tumstatin is a 28-kDa fragment of type IV collagen that binds to
αvβ3 integrin and exhibits antiangiogenic activity. However, tumstatin was shown to target
only vascular/capillary epithelial cells and thus its activity is specific only for endothelial
cells. In human umbilical vein epithelial cells tumstatin significantly inhibited protein
synthesis (up to 50%) and 4E-BP1 phosphorylation that caused enhanced binding of 4E-BP1
to eIF4E. As a result, eIF4E associated with 4E-BP1 becomes unavailable for cap-dependent
protein translation, inducing decrease of protein synthesis. Further experiments demonstrated
that tumstatin has no effect on MAP kinase pathway downstream of FAK (Sudhakar et al.,
2003). To summarize, Maeshima and co-workers revealed an indirect crosstalk (PI3K/Akt
dependent) of integrin and mTOR pathways.

1.3.2 mTOR/integrin crosstalk in cancer

Both the mTOR pathway and integrins have been repeatedly described to participate in
carcinogenic events within the body. Indeed, a number of studies, suggesting a possible
crosstalk between mTOR pathway and integrin signalling in cancer cell lines, emerged
recently (Han et al., 2006; Ta et al., 2008; Vojtechova et al., 2008).

In non-small cell lung carcinoma cells, proliferation was induced upon integrin binding to
fibronectin via the Akt/mTOR/S6K pathway (Han et al., 2006). In this study, authors showed
that fibronectin stimulated Akt/PKB, 4E-BP1 and p70S6K phosphorylation and inhibited
phosphorylation of PTEN (phosphatase and tensin homologue deleted on chromosome 10),
the tumor suppressor that antagonizes PI3K signalling. Moreover, cells plated on fibronectin
(20 μg/ml, 2 hours) exhibited reduced LKB1 protein and mRNA level and also the
phosphorylation of AMPKα, a potent negative regulator of TSC2, was reduced. Taken
together, data collected in this study demonstrate the indirect crosstalk of integrin/mTOR

12
pathway mediated by PI3K/Akt. Unfortunately, the upstream regulation of Akt/PKB was not
examined and integrin-interacting proteins that participate in this pathway remain elusive.

In a different study, Vojtechova and co-workers observed that mTOR signalling is reduced
after treatment of H19 cells [Rous sarcoma virus (RSV)-transformed hamster fibroblasts)]
with Src kinase specific inhibitor SU6656 (Vojtechova et al., 2008). Authors reported
reduced Akt (T308, S473), mTOR (S2448), S6 (S235/236) and 4E-BP1 phosphorylation that
was not overcame by expression of constitutively active Akt (although inhibitory
phosphorylation of TSC2 was restored), thus suggesting that mTORC1 is not under control
of Akt/PKB in these cells. Similar results were obtained by expression of Src kinase-dead
double Y416F-K295N mutant that has a dominant negative effect on the endogenous c-Src.
Although, mTORC1 regulation by Src kinase was already reported (Karni et al., 2005; Shah
et al., 2002), this study hypothesizes that mTORC1 might be regulated by Src kinase
independently on TSC1/2 complex. However, the exact mechanism of mTOR regulation by
Src kinase remains to be elucidated, but this study implicates direct regulation of mTOR
pathway by Src kinase.

Recently, p130Cas was implicated in adriamycin resistance (Ta et al., 2008). Authors
demonstrated that p130Cas expression renders MCF-7 breast cancer cells less sensitive to
adriamycin growth-inhibitory and proapoptotic effects and observed enhanced
phosphorylation of ERK 1/2 and Akt/PKB (S473) in MCF-7 cells overexpressing p130Cas
upon adriamycin treatment. Since expression of proapoptotic protein Bak was reduced,
authors assumed that p130Cas activates growth and survival pathways that are regulated by
Src kinase, ERK kinase or Akt/PKB, thus inhibiting mitochondrial-mediated apoptosis in the
presence of adriamycin.

13
Figure 2.4: Schematic representation of integrin signalling and mTOR pathway crosstalk based on recent
publications Corradetti and Guan (2006) and www2.ub.edu, modified.

14
2 Materials and Methods

2.1 Materials

2.1.1 Organisms used

Escherichia coli DH5α

2.1.2 Cell lines used

Rat2 fibroblasts of Rattus norvegicus

MEF S cas-/- embryonic fibroblasts of Mus musculus, transformed with Src

oncoprotein (Src529F)

MEF SC cas-/- embryonic fibroblasts of Mus musculus, transformed with Src

oncoprotein (Src529F), transfected by p130Cas wild type by
retroviral transfection

MEF S15F cas-/- embryonic fibroblasts of Mus musculus, transformed with Src
oncoprotein (Src529F), transfected by p130Cas that has 15 mutations
Y→F in substrate domain by retroviral transfection

MEF SC WT cas-/- embryonic fibroblasts of Mus musculus, transformed with Src

oncoprotein (Src529F), transfected by p130Cas wild type by
lipofection

MEF SC YE cas-/- embryonic fibroblasts of Mus musculus, transformed with Src

oncoprotein (Src529F), transfected by p130Cas bearing point
mutation in position 12 (tyrosin was changed to glutamate) by
lipofection

MEF SC YF cas-/- embryonic fibroblasts of Mus musculus, transformed with Src

oncoprotein (Src529F), transfected by p130Cas bearing point
mutation in position 12 (tyrosin was changed to phenylalanine) by
lipofection

15
2.2 Plasmids constructed and used

Figure 2.1: Vector pEGFP-C1 was used for construction of plasmids with various versions of rheb genes
(adapted from Clontech).

CMV

5000

eGFP

1000
pEFPC1_DN
4000
5270 bps Bgl II 1339

2000
Rheb2 DN
Neo Ssp I 1541
3000

Hin dIII 1748

Eco RI 1898
Kpn I 1914

Ssp I 2200

Ssp I 2753

Figure 2.2. Plasmid pEFPC1_DN was constructed using pEFPC1 vector and rheb versions. All plasmids
used in this study (Table 3.1.) were constructed using pEFPC1 by similar cloning techniques.

16
 Size in
Name Backbone Description Restriction
bp
pEGFPC1_WT pEGFP-C1 5270 Rheb wt-GFP fusion BglII, EcoRI

pEGFPC1_DN pEGFP-C1 5270 Rheb D60V-GFP fusion BglII, EcoRI

pEGFPC1_S16H pEGFP-C1 5270 Rheb S16H-GFP fusion BglII, EcoRI

pEGFPC1_CAA pEGFP-C1 5270 Rheb Q64L S16H-GFP fusion BglII, EcoRI

Table 2.1- Plasmids constructed during this study

2.2.1 List of primary and secondary antibodies used

Anti-p130Cas, mouse IgG1antibody, BD Transduction Laboratories, diluted 1:1000

Anti-S6 Ribosomal Protein mouse (54D2) mAb, mouse antibody, Cell Signaling, 1:1000

Anti-Actin (I-19):sc-1616, goat polyclonal IgG Ab, Santa Cruz Biotechnology, 1:1000

Anti-Akt, rabbit polyclonal Ab, Cell Signaling, 1:1000

Anti-FAK (C-20): sc-558, rabbit polyclonal antibody, Santa Cruz Biotechnology, 1:500

Anti-GFP: sc-8334B, rabbit polyclonal antibody, Santa Cruz Biotechnology, 1:1000

Donkey anti-goat IgG-HRP:sc-2033, HRP conjugated, Santa Cruz Biotechnology, 1:5000

Goat anti-mouse IgG-HRP:sc-2031, HRP conjugated, Santa Cruz Biotechnology, 1:5000

Goat anti-rabbit IgG-HRP:sc-2030, HRP conjugated, Santa Cruz Biotechnology, 1:5000

Phospho-Akt (Ser473) (D9E) XP™ Rabbit mAb, Cell Signaling, diluted 1:1000

Phospho-FAK (Tyr397), rabbit polyclonal antibody, Biosource 1:1000

Phospho-Paxillin (Tyr116) rabbit Ab, Cell Signaling, 1:1000

Phospho-S6 Ribosomal Protein (Ser235/236) (2F9) rabbit mAb, Cell Signaling, 1:1000

17
2.2.2 Solutions for cell cultures handling

Complete DMEM medium DMEM (Dulbecco‟s Modified Eagle‟s

medium) (GIBCO)

10% fetal bovine serum (Sigma-Aldrich)

1% nonessential amino acids (GIBCO)

2% antibiotics and antimycotics (GIBCO)

Cell lysis buffer 1% Triton X-100

50 mM HEPES pH 7,4

5 mM EGTA

protease inhibitor Mix M (SERVA)

10 nM Na3VO4

Cell cultures preservation medium 10% DMSO in FBS

2.2.3 Solutions for protein analysis

SDS-PAGE running buffer 0,1% SDS

25 mM Tris (pH 8,3)

190 mM glycine

1,5x Laemmli buffer 2,5% SDS

0,375 M Tris-HCl

0, 00075% brome phenol blue

10% glycerol

Western blot/transfer buffer 20% methanol

192 mM glycine

25 mM Tris

TBS 20 mM Tris-HCl

500 mM NaCl

TTBS 0,05% Tween v TBS

Ponceau S stain 0,1% Ponceau S in 0,05% acetic acid

Blocking solution 3% bovine serum albumin in TBS

18
 Primary and secondary Ab incubation solution: 1% BSA in TTBS

Mounting medium 6 g of glycerol

2.4 g of mowiol

6 ml deionizied water

12 ml of buffer (60 nM Pipes, 25 mM

HEPES, 10 mM EGTA, 1 mM MgCl2)

Stripping buffer 10% SDS (w/v)

100 mM β-mercaptoethanol

50 mM Tris-HCl pH 6.8

2.2.4 Solutions for DNA manipulation and bacterial cultures handling

TAE buffer 40 mM Tris pH 8.5

20 mM acetic acid

2 mM Na2EDTA

Loading buffer 60% glycerol

60 mM EDTA

0.06% brom phenol blue

2 x LB (Luria-Berthani Broth) 20 g/l Universal peptone M66 (Merck)

5 g/l NaCl

10 g/l Yeast Extract (OXOID)

Nutrient agar plates 4% nutrient agar n.2 (Imuna Pharm a.s.)

kanamycin sulfate 50 μg/ml

2.3 Methods

2.3.1 Cell culture techniques

2.3.1.1 Cell culture maintaining

Cell cultures were cultivated on 60 mm cell culture dishes in 4 ml of complete DMEM

supplemented with 10% FBS, 1% amino acids, 2% antibiotics under standard cultivation
conditions (37°C, humidified 5% CO2 atmosphere). Cell lines were maintained by passaging

19
 when grown to confluence. Upon passaging, cells were washed once with 500 μl of 0.25%
trypsin-EDTA and incubated in 37 °C with another 500 μl of trypsin for 3 minutes. Cells
were rinsed off the dish by complete DMEM and desired number of cells was transferred to
a new dish. Fresh complete DMEM was used to replenish the medium up to 4 ml.

2.3.1.2 Cell cultures preservation

For cell culture preservation, cells were trypsinized as mentioned in 2.3.1.1, transferred to
a 15 ml centrifugation tube and pelleted at 180 x g, room temperature for 3 minutes.
Supernatant was aspired and cells were resuspended in cell cultures preservation medium
(10% DMSO in FBS). Cell suspension in preservation medium was immediately transferred
to cryovials and stored in -70 °C in isopropanol-filled container for 24 hours. After 24 hours,
cryovials were moved to container with liquid nitrogen for long-term storage.

To thaw a cell culture, cryovials were quickly transferred from liquid nitrogen to water
bath (37 °C). After 3 minutes, melted suspension was mixed with 5 ml of complete DMEM
and centrifugated at 180 x g, room temperature for 3 minutes. Supernatant was discarded and
cell pellet was resuspended in 4 ml of fresh DMEM and transferred to a cell culture dish.

2.3.1.3 Transfection procedure and stable cell lines establishment

Cells were cultivated in complete DMEM medium at 60 mm dishes to confluence of 80%.

One day prior to the transfection, cells were washed with 3 ml of fresh DMEM (without
FBS, amino acids and antibiotics) and cultivated overnight. Before the transfection, 4 μg of
plasmid DNA was mixed with 500 μl of DMEM (without FBS, antibiotics, amino acids) and
incubated for 5 minutes at room temperature. At the same time, 12 μl of transfection reagent
(Lipofectamine 2000) was mixed with another 500 μl of DMEM (without FBS, amino acids,
antibiotics) and left for 5 minutes-long incubation. After 5 minutes, both DNA and
transfection reagent were mixed and incubated together for another 20 minutes. Finally, the
mixture of DNA and Lipofectamine was added to cell culture and cells were cultivated under
standard conditions. After 6 hours, cells were washed with and cultivated in fresh DMEM
(without FBS, amino acids, antibiotics) and next day complete DMEM was added.

Two days after the transfection, the transfection efficiency was checked by fluorescence
microscopy and cells were subjected to selection by geneticin (500 μg/ml) for 3 weeks.
Afterwards, fluorescence-activated cell sorting (FACS) was used to sort out cells with high
level of fluorescence. This population was cultivated for another 2 weeks and then re-sorted.

20
 In that case, two populations from each cell line were prepared – cells with high level of
fluorescence and cells with medium level of fluorescence. During the whole selection
procedure, cells were exposed to 500 μg/ml of geneticin.

2.3.1.4 Cell lysis

Cell cultures were grown to 80% of confluence under standard conditions in complete
DMEM (10% FBS, 1% amino acids, 2% antibiotics). Three types of lysates were obtained
during this project:

- cells were lysed when grown in complete DMEM (10%FBS, amino acids,
antibiotics)

- for serum-starved cell lysates, cells were washed twice with fresh DMEM
(without FBS, amino acids, antibiotics) and cultivated in it for additional 4 hours.
Afterwards, cells were immediately lysed

- for serum-restimulated cell lysates, cells were washed twice with fresh DMEM
(without FBS, amino acids, antibiotics) and cultivated in it for additional 4 hours.
Afterwards, cells were restimulated with 10% FBS for 30 minutes and
immediately lysed

Before the lysis, cell dishes were washed three times with fresh ice-cold PBS that was
carefully aspired after the third wash. In next step, 400 μl of cell lysis buffer was added
[1% Triton X-100; 50 mM HEPES pH 7.4; 5 mM EGTA; protease inhibitor Mix M (SERVA
Electrophoresis - contains AEBSF-HCl, Aprotinine, Bestatine, E-64, Leupeptine, Pepstatine
A); phosphatase inhibitor Mix; 10 nM Na3VO4] and cells were scraped with a rubber spatula
and transferred to a 1.5 ml tube. Then, cellular lysate was six times passed through
a 21-gauge syringe and centrifugated at 4° C for 15 minutes, 13,000 rpm (Eppendorf
5417R). Afterwards, supernatant was aspired and subjected to protein concentration
determination (Bio-Rad DC Protein assay).

2.3.2 DNA manipulation techniques

2.3.2.1 Plasmid DNA purification

For large scale purifications (e.g. for subsequent transfection to mammalian cells), 10 μl of
melted glycerol stock of bacterial culture transfected with appropriate plasmid was mixed

21
 with 100 ml of 2x LB media (Luria-Bertani Broth) with antibiotic and cultivated in 37 ºC
overnight while shaking. Then, plasmid isolation was carried out using Macherey-Nagel
NucleoBond® Xtra Midi kit according to manufacturer‟s instructions. Briefly, the overnight
culture was pelleted by centrifugation at 4,500 x g for 10 minutes at 4°C. Supernatant was
discarded and pellet was resuspended in 8 ml of buffer RES (contains RNase A).
Subsequently, cells were lysed by 8 ml of buffer LYS and the mixture was incubated at room
temperature for 5 minutes. In a next step, 8 ml of buffer NEU was added to the mixture and
the whole suspension was loaded to equilibrated NucleoBond® Xtra Column with filter. The
column filter was washed with 5 ml of buffer EQU and discarded. Then, the column was
washed with 8 ml of buffer WASH. In elution step, DNA was released from the silica resin
by addition of 5 ml of buffer ELU. For DNA precipitation, 3.5 ml of isopropanol was added
to eluted solution and mixture was loaded into provided syringe. While pressing the plunger
to the syringe, DNA was bound to Nucleobond® Finalizer. In next step, finalizer was
washed with 2 ml of 70% ethanol and DNA was eluted with 400 μl of redissolving buffer
Tris. Every purified plasmid was verified using restriction analysis.

For small scale purifications (e.g. for plasmid verification after bacterial transformation),
a 3 ml of 2x LB media (with antibiotic) was inoculated with an individual bacterial colony of
successfully transformed cells and cultivated overnight in 37°C with constant shaking. Next,
plasmid isolation was carried out using Macherey-Nagel Nucleospin Plasmid QuickPure kit
according to manufacturer‟s instructions. Briefly, prepared bacterial culture was pelleted
at 11,000 x g for 30 seconds. Supernatant was aspired and pellet was completely
resuspended in 250 μl of buffer A1. In next step, cells were lysed by addition of 250 μl of A2
buffer followed by 5 minutes-long incubation. To neutralize the lysate, 300 μl of A3 buffer
was added and the mixture is centrifugated at 11,000 x g for 5 minutes at room temperature.
Then, the supernatant was loaded into Nucleospin Column and the column sitting in a 2 ml
collection tube was centrifugated at 11,000 x g for 1 minute. In subsequent washing step,
400 μl of AQ buffer was added to the column and another centrifugation at 11,000 x g for 3
minutes followed. Plasmid DNA was eluted with 50 μl of AE buffer, after 1 minute-long
incubation, by centrifugation at 11,000 x g for 3 minutes.

2.3.2.2 DNA extraction from agarose gels

DNA extraction from an agarose gel was performed using Macherey-Nagel Nucleospin®
Extract II kit according to manufacturer‟s instructions. Briefly, desired DNA fragment was
visualized by UV lamp, excised from an agarose gel and transferred to a 1.5 ml clean tube.
The weight of the gel slice was determined and 200 μl of buffer NT was added for each

22
 100 mg of gel. Samples were incubated at 50 °C until the gel was completely dissolved and
then were loaded to the Nucleospin® Extract II Column placed into a Collection Tube. The
Collection Tube was centrifugated for 1 minute at 11,000 x g and DNA was bind to a silica
membrane. Flow-through was discarded and silica membrane was washed with 600 μl of
buffer NT3. After centrifugation for 1 minute at 11,000 x g, a flow-through was discarded.
By additional centrifugation for 2 minutes at 11,000 x g excessive buffer NT3 was removed
and membrane was dried. To release DNA from the membrane, Nucleospin® column was
placed into a clean 1.5 ml tube and 40 μl of buffer NE was added. After 1 min incubation
at room temperature, DNA was eluted by centrifugation for 1 minute at 11,000 x g.

2.3.2.3 Plasmid restriction

All plasmids used in this study were fragmented at EcoRI and BglII restriction sites. At the
very beginning, plasmid DNA was isolated from bacterial culture as described in 2.3.2.1.
Sufficient amount of desired plasmid was subjected to restriction to obtain both open vector
and fragment to be inserted. In a 0.5 ml tube following mixture was prepared:

plasmid DNA

10x restriction buffer O+ (MBI Fermentas)

1-10 U BglII (MBI Fermentas) / μg DNA

1-10 U EcoRI (MBI Fermentas) / μg DNA

deionized water

Mixture was incubated in 37° C for 2 hours (in case of fragment restriction) or 4 hours (in
case of vector restriction). After the incubation time was over, sample quality was checked
by electrophoresis in agarose gel.

2.3.2.4 Electrophoresis in agarose gel

DNA electrophoresis was performed in SERVA Electrophoresis BlueMarine 100

apparatus. At first, 1% suspension of agarose (SeaKem LE Agarose, Cambrex) in TAE
buffer was melted by heating in microwave and left for polymerization in the apparatus. The
gel was overlaid with TAE buffer and 2 μl of GeneRuler 1kb DNA Ladder (Fermentas)
was loaded to the first well. Desired amount of sample was mixed 5:1 with loading buffer
and samples were loaded to wells. Voltage was set at 80 V and gel was run for 1.5 – 2 hours.

23
 Then, the gel was stained with ethidium bromide solution (0.5 μg/ml) for 10 minutes,
washed in deionized water for 5 minutes and DNA was visualized under UV lamp.

2.3.2.5 DNA ligation

To ligate selected fragments of DNA, following components were mixed in a 0,5 ml tube:

10x ligation buffer (MBI Fermentas), diluted to 1x ligation buffer

0.3 μl of T4 DNA ligase (MBI Fermentas)

DNA of both vector and fragment to be inserted in molar ratio 1:3

Mixture was incubated for 4 hours at room temperature. Immediately after ligation,
recipient bacterial culture was transformed by 4 μl of ligation mixture (5 pg – 0,5 μg of
DNA) by electroporation.

2.3.2.6 Transformation of E.coli by electroporation

Transformation by electroporation was performed using Gene Pulser Apparatus

(BIO-RAD). At the very beginning, 50 μl of competent cells were mixed with 4 μl of
ligation mixture (contains 5 pg – 0.5 μg) of plasmid DNA. In a next step, DNA and cells
were transferred to a cuvette that has been chilled 5 minutes on ice. The cuvette was
carefully wiped and placed into the sample chamber. The electroporation apparatus was set
to 2.5 kV; 25 uF, 200 Ω. After applying the pulse, mixture was resuspended in 1 ml of 2x LB
with 20 mM glucose and transferred to sterile culture tube. Electroporated bacterial culture
was cultivated for 1 hour at 37 °C with shaking. After incubation, 20 and 200 μl of bacterial
culture was plated at nutrient agar containing 50 μg/ml kanamycin.

2.3.3 Protein manipulation techniques

2.3.3.1 Protein concentration determination

Total cellular protein was determined using Bio-Rad DC Protein Assay that is based on
Lowry assay. Standards (final volume was 50 μl) were prepared using bovine serum albumin
(2mg/ml) diluted in cell lysis buffer. Final concentration of standards were 200; 400; 800 and
1500 μg/ml. 25 μl of samples (cell lysates) were diluted in 25 μl of cell lysis buffer. In the
meantime, reagent A‟ was prepared by adding 20 μl of reagent S to each 1 ml of reagent A.
25 μl of each sample and standard was transferred to an 1.5 ml tube and 125 μl of reagent A‟

24
 was added. At the very end, 1 ml of reagent B was also put into every sample or standard and
after 15 minutes absorbance at 750 nm was read. To acquire the same concentration of
samples, denser samples were diluted in cell lysis buffer. Finally, 1.5x Laemmli buffer and
dithiothreitol (DTT; to final concentration 50 nM) were added and samples were boiled for
10 minutes at 100°C.

2.3.3.2 Protein electrophoresis

Tris-glycine sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis

(SDS-PAGE) was performed using BIO-RAD Mini-PROTEAN® III Cell apparatus and
standard Tris-glycine buffer system. First of all, non-gradient gel (10%; 0.75 mm) that
consists of separating and stacking gel was prepared in the casting frame. For separating gel
preparation, following components were mixed and poured between two BIO-RAD plates
located in the casting frame:

4.17 ml of deionized water

3.33 ml of protogel (30% acrylamide + 0.8% N,N‟-methylenbisacrylamide)

2.5 ml of 4xTris/SDS pH 8.8 (1.5M Tris-HCl + 0.4% SDS)

12 μl of N, N, N', N'-tetramethylethylenediamine (TEMED)

60 μl of 10% ammonium persulfate (APS)

The gel was immediately overlaid with deionized water to prevent gel exposure to oxygen.
After successful polymerization of the separating gel (10 minutes), water was removed,
stacking gel was poured and a comb was inserted to create loading wells. Stacking gel
consists of following components:

3.1 ml deionized water

0.65 ml of protogel (30% acrylamide + 0.8% N,N‟-methylenbisacrylamide)

1.25 ml 4x Tris/SDS pH 6.8 (1.5M Tris-HCl + 0.4% SDS)

10 μl of N, N, N', N'-tetramethylethylenediamine (TEMED)

50 μl of 10% ammonium persulfate (APS)

25
 Finally, 10 μg of proteins in cell lysate sample were mixed with DTT and Laemmli buffer
(details in 2.3.3) was loaded to individual loading. In addition, 2 μl of PageRuler™
Prestained Protein Ladder #SM0671 (Fermentas) was loaded to the first well in the gel.

Samples were separated at 10 mA for each gel in the stacking gel and 20 mA for each gel
in the separating gel.

2.3.3.3 Western blot

Western blot was performed using BIO-RAD Mini Trans-blot® Electrophoretic Transfer
Cell. After the electrophoresis, separating gel, filter paper and nitrocellulose membrane
(Trans-blot Transfer Medium; 0,45 um; BIO-RAD) were soaked in western blot/transfer
buffer (20% methanol, 192 mM glycine, 25 mM Tris) for 10 seconds. Separating gel was
immediately transferred to soaked filter paper and nitrocellulose membrane was put onto the
gel with another filter paper covering the other side of the membrane. This “gel sandwich”
was placed into the gel holder cassette BIO-RAD and cooling unit was added to the tank.
Finally, the tank was completely filled with western blot/transfer buffer and subjected to
100 V for 75 minutes with constant cooling.

2.3.3.4 Immunodetection

Nitrocellulose membrane with transferred proteins was at first stained in Ponceau S

solution (10 minutes, RT) to confirm the even concentration of samples and to check the
overall quality of the separation and transfer. Ponceau S stain was washed off by 100 ml of
TTBS (10 minutes for every wash, shaking, RT, three times repeated) and incubated
in blocking buffer (4% BSA in TBS) in 37ºC for 45 minutes. Then, nitrocellulose membrane
was transferred to primary antibody incubation solution (1% BSA in TTBS, details in section
2.2.3) and primary antibody was added (list of antibodies used can be found in section
2.2.1.). Incubation in primary antibody was performed overnight.

Next day, the membrane was washed three times in 100 ml of TTBS and transferred to
secondary antibody incubation solution (1% BSA in TTBS) with secondary antibody added.
During this study, only horseradish peroxidase-conjugated secondary antibodies were used.
Incubation in secondary antibody was performed for 1 hour at room temperature.
Subsequently, membrane was washed three times with 100 ml of TTBS and once with
100 ml of TBS. Signal visualization was performed using SuperSignal® West Pico
Chemiluminiscent Substrate (Thermo Scientific). Membrane was incubated in the substrate

26
 Working Solution (mixture of luminol/enhancer solution and peroxide solution; 1:1) for
5 minutes and the chemoluminiscence was detected using LAS 4000 detection system.

Alternatively, nitrocellulose membrane was reprobed for another primary antibody after
the first detection at LAS-4000. Membrane was incubated in stripping buffer [10% SDS
(w/v),50 mM Tris/HCl, 100mM β-mercaptoethanol] for 30 minutes at 50°C and then washed
three times in TTBS for 10 minutes. Then, the membrane was blocked in blocking solution
as usually and standard immunodetection protocol was followed.

2.3.3.5 Cytometry

Cells were grown on cell culture plates to confluence of 60%. One day prior the
experiment, cells were trypsinized and 106 cells were transferred to a 15 ml centrifugation
tube. After centrifugation at 250 x g and 4ºC for 5 minutes (Eppendorf 5804R) cells were
washed twice with 1 ml of ice-cold PBS and resuspended in 500 μl of PBS. Subsequently,
500 μl of ice-cold 2% paraformaldehyde was added. After 1 hour of incubation at 4ºC, cells
were washed with 1 ml of ice-cold PBS and centrifugated at 4ºC, 250 x g for 5 minutes.
In next step, cells were fixed in 1 ml of precooled 70% ethanol (added drop wise under
consant stirring) and the suspension was left at 4ºC overnight. Next day, cells were pelleted
by centrifugation at 4ºC, 250 x g for 5 minutes and ethanol was aspired. Cells were
resuspended in 1 ml of propidium iodide staining solution (100 nM RNase, 40 μg/ml PI in
PBS). Control samples contained only 100 nM RNase in PBS. All samples were incubated
for 30 minutes in 37ºC in dark before the cytometric analysis was performed.

Cells were processed as described above and analyzed on LSRII (Becton Dickinson).
Analysis of data was performed utilizing the BD FACSDiva Software version 6.1.2 (Becton
Dickinson).

2.3.3.6 Microscopy

During this study, cells were stained by following fluorophores: DAPI, phalloidine.

Sterile glass cover slips were covered with 50 μl of fibronectin (10 μg/ml; diluted in PBS)
in advance. Cells were cultivated on glass cover slips covered with fibronectin overnight and
then carefully washed twice with PBS. For fixation, cover slips were submerged to
4% paraformaldehyde for 15 minutes and then twice washed with PBS. After that, cells were
incubated in 100 μl of phalloidin solution (AlexaFluor phalloidin 594 nm; Invitrogen; diluted
1:100 in 1% BSA) for 15 minutes and washed four times with PBS to remove excessive

27
 fluorophore. During the final part of mounting procedure, DABCO [(1,4-diazabicyclo
(2.2.2.) octane)] and 50 μg/ml of DAPI was added to 100 μl of the mounting medium.
Images were acquired by Nikon Eclipse TE2000-S using 20×/0.45 objective.

2.3.3.7 Statistical analyses

Two-tailed Student‟s t tests were used for the pair-wise comparison of experimental
groups.

2.3.3.8 Densitometry

The densitometric analysis of immunoblots was performed using Image Quant TL 5.0.
software. Images with detected chemiluminiscence signal acquired by LAS-4000 were
analyzed for pixel intensities, and the background calculated using rolling ball method was
subtracted.

28
3 Results

3.1 The role of p130Cas in mTOR signalling

In order to assess the role of p130Cas in mTOR signalling we employed cell lines with
different level of p130Cas expression, transfected with activated version of mouse Src
protein (SrcF529). All lines used in this part were created and further characterized by Dr
Brabek during another study (Brabek et al., 2004; Brabek et al., 2005). In this study,
following versions of mouse embryonic fibroblasts (MEF) were used:

MEF S cas-/- MEF, SrcF529 transfected

MEF S15F cas-/- MEF expressing p130Cas that has dysfunctional substrate domain
(all 15 tyrosines in substrate domain were mutated to phenylalanines),
created by retroviral infection, SrcF529 transfected

MEF SC cas-/- MEF transfected with mouse wild type p130Cas by retroviral
infection, SrcF529 transfected

These cells lines were described to possess distinct metastatic and invasive properties,
including matrix metalloproteinase-2 activation and actin organization. p130Cas was
revealed as an important regulator of mentioned processes within the cell (Brabek et al.,
2004; Brabek et al., 2005). The substrate domain of p130Cas was found critical for
formation of large podosome structures (Fig.3.1), Matrigel invasion and phosphorylation of
Src substrates.

Figure 3.1: MEF expressing p130Cas variants stained for F-actin and phosphorylated cortactin (Brabek et
al., 2005). A. A cell lacking p130Cas (cell line MEF S) or expressing p130Cas Y15F-all tyrosines in
substrate domain of p130Cas changed for phenylalanines (cell line MEF S15F) has rather small and
punctuate podosomes. B. Example of a cell expressing wild type of p130Cas (cell line MEF SC). In these
cells rather large podosomes are observed.

29
3.1.1 The role of p130Cas in mTORC1 signalling

First of all, we explored the level of activation of mTORC1 in cells lacking p130Cas
(MEF S), overexpressing wt p130Cas (MEF SC) and overexpressing p130Cas with mutated
substrate domain (MEF S15F). As a readout of mTORC1 activity, phosphorylation of S6
protein at serines 235 and 236 was chosen. Since S6 protein was reported to be
phosphorylated by S6K (Flotow and Thomas, 1992) at mentioned serines, S6
phosphorylation status reflects the activity of mTORC1. To obtain comparable data, cell
lines were cultivated to similar confluence and lysed after 4 hours of serum starvation. In
addition, cell lysates from serum restimulated cells were also made. Restimulated cells were
serum starving for 4 hours and then 10% FBS was added for 30 minutes. Immediate cell
lysis followed as described in 2.3.1.4. After separation by SDS-PAGE, proteins were
transferred to nitrocellulose membrane and phosphorylation status of serines 235 and 236
was examined by immunodetection.

Data obtained by the immunodetection (Fig.3.2A) clearly showed us enhanced

phosphorylation of S6 protein (at serines 235 and 236) in MEF SC compared to MEF S cells.
Immunoblots were quantified using Image Quant TL 5.0 (Fig. 3.2B) and densitometrical
analysis confirmed that phosphorylation of S6 was significantly increased in MEF SC
compared to MEF S in both serum starved (p=0.018) and serum restimulated cells (p=0.018).
These findings lead us to the notion that activity of mTORC1 is regulated by p130Cas.

Moreover, in both MEF S and MEF SC we noticed an increase in phosphorylation of S6

protein at serines 235 and 236 in response to serum stimulation. Surprisingly, in cell lysates
made from MEF S15F, after stimulation with FBS no remarkable elevation of S6
phosphorylation was detected.

To assess the role of substrate domain of p130Cas in the regulation of mTORC1, we

analyzed phosphorylation of S6 in MEF S15F cells. This cell line contains p130Cas with
mutated substrate domain, all 15 tyrosines were exchanged for phenylalanines. The ability of
mutated p130Cas to bind certain interacting partners, e.g. protein Crk is limited due to the
fact that phenylalanines cannot be phosphorylated by Src kinase. We found that the level of
S6 protein phosphorylation in MEF S15F is not statistically different from control MEF S
cells, but significantly lower when compared to MEF SC (p=0.001). Surprisingly, we also
found that MEF S15F cells are refractory to serum induced stimulation of S6 protein
phosphorylation. Taken together, these observations suggest that substrate domain of
p130Cas is responsible for the effect of p130Cas on mTORC1 signalling.

30
 Figure 3.2: Phosphorylation of S6 protein (S235/236) is affected by p130Cas. Cells were cultivated in
DMEM supplemented with 10% FBS, 1% amino acids and 2% antibiotics overnight. Next day, dishes were
washed three times with fresh DMEM (without FBS and amino acids and antibiotics) and cells were further
cultivated in 4 ml of DMEM (without FBS and amino acids, antibiotics) for 4 hours. Serum starved cells
(- FBS) were lysed immediately after the incubation was finished. 10% FBS was added to serum
restimulated cells (+ FBS) after 4 hours of starvation and cells were lysed after 30 minutes of cultivation
with FBS. A. A representative immunoblot of S6 protein phosphorylation in indicated cell lines and control
immunoblots for p130Cas and actin are shown. B. Densitometric quantification of S6 protein
phosphorylation. The densitometric analysis was performed using Image Quant TL 5.0. software. The level
of S6 protein phosphorylation is shown as fold increase of S6 protein phosphorylation relative to
serum-starved MEF S cells. The data obtained from at least three independent experiments are presented as
mean +/- standard deviation. Statistical confidence between marked cell lines is based on pair-wise
Student‟s t-test.

In addition, we decided to explore the role of SH3 domain of p130Cas in mTORC1

signalling. Cell lines expressing p130Cas with mutant versions of SH3 domain were
employed for this purpose (constructed and kindly provided by R. Janoštiak). Cell lines were
created from MEF S by lipofection of plasmid DNA coding p130Cas wt (SC WT) or
p130Cas with mutated SH3 domain (SC YE; SC YF). Both cell lines expressing p130Cas
with mutated SH3 domain have a single amino acid exchange in tyrosine 12. Exchange of
tyrosine to glutamate (SC YE) sterically mimics phosphorylation of residue 12 and disrupts
interaction with SH3 binding proteins (R. Janoštiak, unpublished results). Exchange of
tyrosine to phenylalanine in p130Cas (SC YF) creates unphosphorylable residue.

31
 We prepared cell lysates from MEF SC WT, MEF SC YE and MEF SC YF (as described
in 2.3.1.4) from both serum starved and serum restimulated cells. All cell lines analyzed
showed slightly increased S6 phosphorylation after serum restimulation (Fig. 3.3). On the
other hand, no significant difference in S6 phosphorylation among analyzed cell lines was
detected when compared to MEF S cells. This result indicates that SH3 domain of p130Cas
is not important for mTORC1 regulation.

Figure 3.3: Phosphorylation of S6 protein (S235/236) is not affected by mutation in SH3 domain of
p130Cas. Cells were cultivated in DMEM supplemented with 10% FBS, 1% amino acids and
2% antibiotics overnight. Next day, cells were starved for serum for 4 hours (- FBS) and lysed. 10% FBS
for 30 min was added to serum restimulated cells (+ FBS) after 4 hours of starvation and cells were lysed.
A. A representative immunoblot of S6 protein phosphorylation in indicated cell lines and control
immunoblots for S6 protein are shown. B. Densitometric quantification of S6 protein phosphorylation. The
densitometric analysis was performed using Image Quant TL 5.0. software. The level of S6 protein
phosphorylation is shown as fold increase of S6 protein phosphorylation relative to serum-starved MEF S
cells. The data obtained from at least three independent experiments are presented as mean +/- standard
deviation.

32
3.1.2 The role of p130Cas in mTORC2 signalling

Having explored the role of p130Cas in mTORC1 signalling, we further decided to

examine the role of p130Cas also in mTORC2 signalling. Similarly, we employed cell lines
expressing distinct variants of p130Cas (MEF S, MEF S15F, MEF SC) and exposed them to
conditions with no serum and serum readdition after starvation. In this case, phosphorylation
of Akt/PKB at serine 473 was chosen as readout of mTORC2, because mTORC2 directly
phosphorylates S473 of Akt/PKB (Sarbassov et al., 2005).

When comparing phosphorylation of Akt/PKB (S473) in p130Cas deficient cells (MEF S)

and p130Cas reexpressing cells (MEF SC) (Fig.3.4), we observed significant enhancement
of PKB (S473) phosphorylation in cells containing p130Cas under serum starvation (-FBS),
(p=0.0064).

Figure 3.4: Phosphorylation of Akt/PKB at serine 473 is enhanced in p130Cas expressing cells compared to
p130Cas deficient cells. Cells were cultivated and lysed according to 2.3.1.4. A. Representative
immunoblot of Akt/PKB phosphorylation in indicated cell lines and control immunoblots for p130Cas and
actin are shown. B. Densitometric quantification of Akt/PKB phosphorylation. The densitometric analysis
was performed using Image Quant TL 5.0. software. The level of Akt/PKB phosphorylation is shown as
fold increase of Akt/PKB phosphorylation relative to serum-starved MEF S cells. The data obtained from at
least three independent experiments are presented as mean +/- standard deviation. Statistical confidence
between marked cell lines is based on pair-wise Student‟s t-test.

33
 Hence, after serum restimulation (+FBS), the difference in Akt/PKB phosphorylation
(S473) between MEF SC and MEF S was even more pronounced (p=0.00005). Taken
together, we proposed that p130Cas can affect mTORC2, as absence of p130Cas caused
significant decrease in mTORC2 activity under both serum starvation and serum readdition
conditions.

In addition, we tried to elucidate the role of p130Cas substrate domain in the regulation of
mTORC2 signalling. We found that basal level of Akt/PKB phosphorylation on S473 in
MEF S is not affected by expression of the mutated p130CAS, as the level of the
phosphorylation under serum starved condition was not statistically different in MEF S15F
cells when compared to MEF S cells. Expression of p130Cas with mutated substrate domain
was not able to enhance mTORC2 activity to the level observed in MEF SC cells.

Notably, we found that MEF S15F cells are refractory to serum induced stimulation of
Akt/PKB phosphorylation on S473. Taken together, our data suggest that the substrate
domain of p130CAS is responsible for the effect of p130CAS protein on both mTORC1 and
mTORC2 signalling.

34
3.2 Rheb protein and its effects on mTOR signalling, integrin
signalling and cell cycle progression

In a second part of this study, we explored the effect of mTOR signalling deregulation on
integrin signalling. For mTOR pathway deregulation, Rheb protein manipulation was
chosen, mainly because of its upstream regulatory position in mTOR pathway and large
number of mutants already described. In this study, stable cell lines expressing distinct
variants of Rheb protein were established and changes in integrin signalling, cellular
morphology and cell cycle were examined.

rheb (Ras-homolog enriched in brain) was first described in 1994 in a screen for genes
that are rapidly induced by synaptic activity in brain neurons (Yamagata et al., 1994). It
belongs to Ras superfamily of small G-proteins and further studies revealed that rheb gene is
conserved from yeasts to mammals (Urano et al., 2001). In yeasts, Rheb was implicated in
arginine uptake (Urano et al., 2001) and possible cell cycle regulation (Mach et al., 2000),
whereas in Drosophila melanogaster Rheb was shown to regulate cell cycle progression and
cellular growth (Patel et al., 2003).

Figure 3.5: Schematic representation of Rheb protein regulation (Hennessy et al., 2005). Rheb belongs to
Ras superfamily of GTPases. GTPases in general bind guanine nucleotides and serve as a molecular switch
to regulate a number of cellular processes (Bourne et al., 1990). Rheb activity is regulated by TSC2 protein
that possess GTPase-activating activity towards Rheb. GTP-bound Rheb stimulates mTORC1 signalling by
direct binding to mTOR kinase (Long et al., 2005) or by interaction with endogenous inhibitor of mTOR,
FKBP38 that displaces FKBP38 from mTOR, relieving the inhibition of mTOR kinase domain (Ma et al.,
2008).

In mammals, genetic and biochemical studies placed Rheb upstream of mTOR kinase in
mTOR signalling pathway and Rheb was revealed as an activator of mTOR under the control
of TSC1/2 complex. TSC1/2 complex acts as a GTPase-activating protein (GAP) that

35
enhances hydrolysis of GTP-Rheb (Garami et al., 2003; Tee et al., 2003), (Fig.3.5). Up to
date, no guanine nucleotide exchange factor (GEF) for Rheb was revealed. Unlike other
Ras-related G-proteins, Rheb has a low intrinsic GTPase activity and preferentially occurs in
GTP-bound state (Im et al., 2002).

In last decade, a wide variety of Rheb activity mutants were prepared (Fig. 3.6). Of these,
we used a mutant that possesses a single amino acid mutation in residue 60; asparagine was
changed to valine (D60V). Expression of this version of Rheb caused inhibition of mTORC1
signalling and exhibited dominant negative (DN) phenotype when expressed in yeasts and in
mammals (Tabancay et al., 2003). Next, constitutively active (CA) Rheb mutant with single
amino acid mutation S16H (serine 16 for histidine) was chosen. At endogenous levels of
expression S16H Rheb exhibits increased GTP loading in vivo and is resistant to TSC1/2
GAP activity in vitro (Yan et al., 2006), (Tab. 2).

Name Mutation Function % GTP bound % GTP bound

in the absence in the presence
of TSC1/2 of TSC1/2
Rheb WT - 80% 2%

Rheb DN D60V high GDP loading DNF DNF

high GTP loading,
Rheb CA S16H 90% 88%
resistant to GAP
Rheb GTP Q64L high GTP loading 88% 66%
S16H
Rheb CAA ? ? ?
Q64L

Table 2: Characteristics of Rheb protein versions in light of GTP/GDP loading. Percentage of Rheb
molecules that are GTP-loaded is mentioned. DNF – data not found

In addition, another constitutively active version of Rheb protein (CAA) was used in this
study. Two separate activating mutations were introduced into Rheb CAA: S16H and Q64L.
Combination of these mutations has not been described yet. Both mutations are expected to
confer Rheb constitutively active properties when introduced separately (Jiang and Vogt,
2008; Yan et al., 2006). We hoped that Rheb harbouring both constitutively active mutations
will stimulate mTORC1 activity even more than individual mutations.

36
 Figure 3.6: A model of Rheb GTPase structure in a complex with GTP (PDB id:1XTS ). Positions of
individual mutations that were introduced to the molecule are highlighted in yellow.

At the very beginning of this part of study, we had chosen pEGFPC1 plasmid for
vector/recipient plasmid in which rheb variants were cloned. Cloning of genes in appropriate
reading frame into a multiple cloning site of this vector leads to expression of fusion protein
with EGFP at the N-terminus of the fusion protein. The N-terminal EGFP fusion was chosen
to prevent interference with C-terminal farnesylation signal of Rheb (Hancock, 2003;
Yamagata et al., 1994). Direct fusion of Rheb variants with EGPF was chosen in order to
allow us convenient sorting of transfected cells with defined expression levels of Rheb.

pEGFPC1 vector for was used for construction of following plasmids:

 pEGFPC1 WT coding Rheb wild type

 pEGFPC1 DN coding Rheb possessing dominant negative properties – D60V

 pEGFPC1 CA coding Rheb possessing constitutively active properties – S16H

 pEGFPC1 CAA coding Rheb variant expected to exhibit increased

constitutively active properties – S16H +Q64L mutations

Various rheb genes, both wild type and mutated were excised from Rh2-IRES-puro
plasmids. These plasmids were prepared by Dr. Rösel. We employed BglII and EcoRI
restriction endonucleases to excise rheb genes. The restriction mixture contained 2 μl of O+
buffer, 0.4 μl of BglII, 0.4 μl of EcoRI, 12 μl of plasmid DNA and 5.2 μl of deionized water.
After incubation (2 hours), the whole mixture was transferred to 1% agarose gel and agarose

37
gel electrophoresis was performed. In a next step, fragments were isolated from the gel using
a gel extraction kit (details in session 2.3.2.2). Meanwhile, pEGFPC1 was opened in BglII
and EcoRI sites using similar restriction mixture (total volume was 100 μl, 35 μl of which
was plasmid DNA), separated on agarose gel and extracted from the gel. Upon visualization
in UV light we expected DNA restriction fragments of 509 bp and 4761 bp for rheb gene
variants and opened pEGFPC1 vector, respectively (Fig. 3.7A).

In consequential ligation step, vector pEGFPC1 and rheb fragments (WT, DN, CA, CAA)
were ligated together under following conditions: 6 μl of vector DNA, 3 μl of fragment
DNA, 0.3 μl of T4 DNA ligase, 1 μl of ligation buffer were mixed and incubated at RT for 4
hours. Then, electrocompetent E. coli DH5α cells were transformed with 4 μl of ligation
mixture by electroporation. Thereafter, 20 μl and 200 μl of transformed cells were plated at
nutrition agars with kanamycin antibiotic at final concentration of 50 μg/ml and incubated at
37 ºC overnight.

Figure 3.7: Visualization of plasmid restriction. A. Both fragment containing rheb and vector pEGFPC1
were cut by BglII and EcoRI and separated by agarose electrophoresis. B. Bacterial clones that were
transformed by ligated rheb-containing pEFPC1 plasmid were used for plasmid isolation, plasmids were cut
by BglII, EcoRI and separated by agarose electrophoresis. In both cases (DN and WT plasmid), only clone3
contained rheb gene fragment and thus was used for further experiments.

Next day, 4 colonies from each rheb mutant were collected and each colony was used to
inoculation of 4 ml of 2x LB medium. After 16 hours, cells were centrifugated and plasmid
DNA was isolated using Nucleospin Plasmid kit (as described in 2.3.2.1). Isolated DNA was
fragmented using restriction endolucleases EcoRI and SalI to check the plasmid accuracy.

38
Restriction mixture was separated by agarose gel electrophoresis and clone with properly
inserted rheb was chosen (Fig. 3.7B). Selected bacterial clones were inoculated into 100 ml
of 2x LB and large-scale plasmid isolation was performed using MN Nucleobond kit
(described in session 2.3.2.1).Concentration of isolated plasmid DNA – variants wild type
Rheb (WT), dominant negative Rheb (DN), constitutively active Rheb (CA) and double
constitutively active Rheb (CAA) was determined by spectrometry. Plasmid pEGFPC1
without rheb (but coding EGFP) was also isolated again and used as control DNA for
transfection and all further analysis (v0 – empty vector). For subsequent transfection by
lipofection, 4 μg of plasmid DNA and 12 μl of transfecting reagent Lipofectamine 2000 were
transferred to Rat2 fibroblasts (as described in 2.3.1.3). Since the third day after transfection,
cells were selected by antibiotic geneticin at final concentration 600 μg/ml. Cell lines were
cultivated with geneticin for three weeks and then, green fluorescent protein content was
determined by fluorescence-analyzed cell sorting (FACS).

In all cell lines derived from Rat2 fibroblasts, Rheb was fused with green fluorescent
protein and thus, GFP expression reflected Rheb expression directly. We wished to prepare
Rat2 derived cell lines with stable expression of Rheb protein variants. To achieve this we
employed fluorescence-activated cell sorting (FACS). During the first round of sorting, all
GFP-positive cells were chosen and these were further cultivated for 2-3 weeks in media
containing geneticin. Then, second sorting was performed. This time, cells were sorted
according to the green fluorescence protein amount into two groups: cells with high level of
GFP (GFP high) and cells with rather moderate level of GFP expression (GFP low).
(Fig. 3.8). Interestingly, only 1000 GFP-positive cells were collected in case of dominant
negative mutant DN (Fig. 3.8B). These cells were placed into 96-well plate and carefully
cultivated under selection of geneticin in final concentration of 1200 μg/ml. Unfortunately,
DN cells lost their fluorescence in few weeks, so this cell line was not further analyzed.

Taken together, we obtained four cell lines: v0 (empty vector), WT (wild type Rheb), CA
(constitutively active Rheb) and CAA (double constitutively active Rheb). In addition, for
every cell line two different sub-lines were prepared: line with high expression of GFP-Rheb
(high GFP) and line with low expression of GFP-Rheb (low GFP).

39
 Figure 3.8: Dot plot describes fluorescence intensity (FITC) and inner complexity (SSC) of cell population
during second round of sorting. Cells were sorted into two populations – cells expressing GFP (low GFP)
and cells expressing high levels of GFP (high GFP). Percentage of cells in each population is marked in the
middle of each population. A. Rat2 transfected with vector coding constitutively active Rheb (CA). B. Rat2
transfected with vector coding Rheb DN. GFP-expressing population is makes only 0.041% of original
population that was obtained in the first round of sorting.

To confirm Rheb expression, cell lines were subjected to immunodetection of Rheb and
GFP (Fig. 3.9). Cell lines that were marked as high GFP really exhibited higher GFP and
Rheb levels. Next, we performed preliminary testing of Rheb mutants‟ activity. As a readout
of mTORC1 activity we have chosen phosphorylation of p70S6 kinase (T389) and 4E-BP1
(T37/46), both direct targets of mTORC1 kinase. As a readout of integrin signalling
phosphorylation of Paxillin on tyrosine 112 was chosen. We found that, constitutively active
mutants exhibit higher phosphorylation of p70S6K, 4E-BP1 and also Paxillin in GFP low
population. These results suggested that mTOR signalling could affect integrin signalling.

Surprisingly, we found that cells expressing high levels of activated Rheb (GFP high) do
exhibit neither increased activation of mTORC1 kinase nor increased levels of
phosphorylated Paxillin when compared to non-mutated Rheb WT. We hypothesize that
during long term cultivation that was necessary for two rounds of cell sorting, cells somehow
compensated the overactivation of mTORC1 signalling. In further experiments we focused
only on cell lines expressing low levels of GFP/Rheb.

40
Picture 3.9: Stable cell lines that were transfected with various rheb variants were subjected to
immunodetection to confirm Rheb expression and its effect on mTORC1 and integrin signalling. As
mentioned above, two cell lines (GFP low and GFP high) were obtained after the sorting. Cells were
cultivated in DMEM up to confluence of 80% and then lysed as described in 2.3.1.4. Immunodetection of
GFP, Rheb and phosphorylated S6K, 4E-BP1 and Paxillin was performed. Similar results were obtained
in at least two experiments (once in case of Phospho-Paxillin), representative immunoblots are shown.

41
3.2.1 Characterization of Rheb variants in context of mTORC1 signalling

At first, we examined the effect of Rheb varieties (WT, CA, CAA) on mTORC1 signalling
under serum deprivation (-FBS) and serum readdition conditions (+FBS). As mentioned
before, phosphorylation of ribosomal protein S6 was considered as a readout of mTORC1
signalling. All data were compared to control cells v0 - Rat2 fibroblasts transfected with
empty pEGFPC1 plasmid only.

Bearing in mind recent publications, we expected higher phosphorylation in cell lines

expressing constitutively active Rheb versions. Interestingly, we observed (Fig. 3.10) this
effect only under conditions of serum deprivation (4 hours of deprivation as mentioned in
2.3.1.4).

Figure 3.10: Constitutively active variants of Rheb stimulate S6 protein phosphorylation (S235/236). Serum
starved (4 hours) and serum restimulated cells (4 hours of starving, 30 min of restimulation by FBS) were
lysed as described in 2.3.1.4. A. Representative immunoblot of S6 phosphorylation in indicated cell lines
and control immunoblots for S6 protein and GFP are shown. B. Densitometric quantification of S6
phosphorylation. The densitometric analysis was performed using Image Quant TL 5.0. software. The level
of S6 phosphorylation is shown as fold increase of S6 phosphorylation relative to serum starved control v0
cells. The data obtained from at least three independent experiments are presented as mean +/- standard
deviation. Statistical confidence between marked cell lines based on pair wise Student‟s t-test.

42
 In cellular lysates from serum starved cells, Rheb CAA version (mutations S16H+Q64L)
exhibited two times higher phosphorylation of S6 protein when compared to v0 cells
(p=0.034). In case of constitutively active Rheb CA (S16H), S6 phosphorylation was also
significantly higher (p=0.011) than S6 phosphorylation in control cells. Importantly,
GFP-Rheb expression in Rat2 Rheb CA cells was the lowest among all three mutants (Fig.
3.10A). Thus, Rheb CA would have probably caused higher phosphorylation compared to
WT and CAA versions if the Rheb expression was similar. Rheb WT overexpression haven‟t
caused significant increase of S6 phosphorylation (S235/236) when compared to control
cells.

At serum restimulated cells, two interesting phenomena were detected. First, no increase
of S6 phosphorylation was observed in cells overexpressing Rheb variants when compared
to control cell line. All four cell lines (control v0, WT, CAA, CA) exhibited similar level of
phoshorylation (Rheb CA expressing cell line probably has a little higher S6 phosphorylation
due to the lowest GFP-Rheb expression). Secondly, at serum restimulated cells, a decrease of
S6 protein expression was detected (Fig. 3.10A). We hypothesize that decrease of S6 protein
expression after readdition of serum reflects higher precipitation of the S6 protein after cell
lysis and is artificially caused by manipulation.

3.2.2 mTORC2 is not influenced by Rheb versions overexpression

Although no changes in mTORC2 signalling caused by mutations in Rheb protein were

ever reported, we were curious to explore whether expression of our Rheb mutants will
affect mTORC2 signalling. Similarly to previous chapter, cell lines expressing various Rheb
protein mutants were used for immunodetection. In case of mTORC2, phosphorylation of
Akt/PKB at serine 473 was examined under both serum starvation and serum readdition
conditions (Fig. 3.11).

In case of Akt/PKB phosphorylation (S473) in cell lines that express various versions of
Rheb protein (WT, CAA, CA), no significant differences were observed under both serum
starvation and serum readdition conditions. Overexpression of Rheb WT exhibited rather
similar effect on Akt/PKB phosphorylation (S473) as Rheb CAA expression. Rheb CA
expression induced lower phosphorylation of Akt/PKB, probably due to lower GFP-Rheb
expression in S16H cells compared to Rheb WT and Rheb CAA expression.

43
Figure 3.11: Expression of constitutively active Rheb protein variants has no significant effect on Akt/PKB
phosphorylation. Cell lines were cultivated and lysed as described in 2.3.1.4 A. Representative immunoblot
of Akt/PKB phosphorylation in indicated cell lines and control immunoblots for Akt/PKB protein and GFP
are shown. B. Densitometric quantification of Akt/PKB phosphorylation. The densitometric analysis was
performed using Image Quant TL 5.0. software. The level of Akt/PKB phosphorylation is shown as fold
increase of Akt/PKB phosphorylation relative to serum-starved control v0 cells. The data obtained from
at least three independent experiments are presented as mean +/- standard deviation.

44
3.2.3 Expression of Rheb variants has no effect on cellular morphology

Having all Rheb variants tagged with GFP enabled us to visualize the localization of Rheb
within the cells and examine the effect of Rheb variants expression on cellular morphology.
Cells were seeded on fibronectin coated plates and stained for filamentary actin and DNA
(as described in 2.3.3.6). No changes in filamentary actin were observed among cell lines,
not even when compared to control cells transfected with empty vector (Fig. 3.12). All Rheb
variants also exhibited similar localization within the cells. The GFP signal was enriched
in close proximity of nuclei and in cytoplasm. This localization is in agreement with
published data suggesting that Rheb localizes to endoplasmatic reticulum and then to Golgi
membranes (Buerger et al., 2006).

Figure 3.12: Cell lines were placed on glass cover slips covered with fibronectin and stained for filamentary
actin (red pseudocolor) and DNA (blue pseudocolor). GFP fluorescence was also visualized (green
pseudocolor). Images were acquired by Nikon Eclipse TE2000-S using 20×/0.45 objective. Scale bar,
20 um. A. Control cells (empty vector v0). B. Rat2 transfected with vector encoding Rheb WT. C. Cells
transfected with vector coding Rheb CAA. D. Cells transfected with vector coding Rheb CA.

45
3.2.4 Rheb affects integrin signalling

As we aimed to focus on crosstalk of mTOR and integrin signalling, we were interested in

changes of integrin signalling-connected proteins (e.g. FAK, Paxillin) in cell lines
transfected with various rheb versions (WT, CAA, CA). At first, we decided to explore
phosphorylation of Paxillin at tyrosine 118 in both serum starved and serum restimulated
cells (Fig. 3.14).

Figure 3.14: Phosphorylation of Paxillin at tyrosine 118 in cell lines transfected with rheb variants. Cell
lines were cultivated and lysed as described in 2.3.1.4 A. Representative immunoblot of Paxillin
phosphorylation in indicated cell lines and control immunoblot for GFP is shown. B. Densitometric
quantification of Paxillin phosphorylation. The densitometric analysis was performed using Image Quant
TL 5.0. software. The level of Paxillin phosphorylation is shown as fold increase of Paxillin
phosphorylation relative to serum-starved control v0 cells. The data obtained from at least three
independent experiments and presented as mean +/- standard deviation. * Statistical confidence between
marked cell lines based on pair-wise Student‟s t-test.

In serum starved cells, constitutively active cell line CAA exhibited the highest rate
of Paxillin phosphorylation (p=0.045) when compared to control v0. Slightly higher
phosphorylation of Paxillin (Y118) was detected also in cells overexpressing Rheb WT, but
the enhancement of phosphorylation was not significant when compared to control cell line
v0. Phosphorylation of Paxillin (Y118) in Rheb CA line was less pronounced than in Rheb
CAA line, probably because Rheb-GFP expression in Rheb CA (S16H) variant was the

46
lowest. Under conditions of serum readdition, all cell lines showed similar level of Paxillin
(Y118) phosphorylation that does not differ from the level of phosphorylation in control cells
v0.

Having observed an increase in Paxillin signalling, we were also curious to explore

phosphorylation of another integrin signalling-connected protein - focal adhesion kinase –
at tyrosine 397 (autophosphorylation site) in Rheb expressing cell lines (Fig. 3.15). In both
serum starved cells (- FBS) and serum restimulated cells (+ FBS), neither changes in FAK
phosphorylation (Y397) nor FAK expression were detected.

Figure 3.15: Phosphorylation of focal adhesion kinase at tyrosine 397 in cell lines transfected with different
rheb versions. Cell lines were cultivated and lysed as described in 2.3.1.4 A. Experiment was repeated two
times and a representative immunoblot of FAK (Y397) phosphorylation in indicated cell lines and control
immunoblot for FAK is shown.

47
3.2.5 Role of p130Cas in cell cycle progression

Rheb protein belongs to important regulators of mTORC1 signalling due to its ability to
activate mTOR kinase activity (Garami et al., 2003; Inoki et al., 2003). Although the
mechanisms of mTOR activation by Rheb is not completely known (Avruch et al., 2009),
this connection confers Rheb an important role in cell growth regulation. Thus, we decided
to explore the role of Rheb in cell cycle progression.

Cell lines transfected with distinct versions of Rheb protein (WT Rheb, constitutively
active Rheb CA: S16H and double constitutively active Rheb CAA: S16H + Q64L) or
control cell line v0 were fixed and stained with propidium iodide to allow us detection of
DNA content within the cells. Because of the fact that every cell line contains only 30% or
less of GFP positive cells, lines were divided into two subpopulations – GFP negative
population and GFP positive population. Only GFP-positive population was considered to be
still expressing Rheb protein versions, GFP-negative population was used as control.

Taken together, we observed a significant increase of G2/M cells in case of Rheb WT

overexpressing cell line and also in both lines expressing constitutively active mutants when
compared to control line v0 or GFP negative populations (Figure 3.16). This result suggests
that Rheb could play an important role in regulation of cell cycle progression.

In addition, we were interested in the question, weather Rheb expression increases volume
of cells. Such observation was made in case of cells transfected by constitutively active
Rheb:Q64L that had up to 25% greater volume than cells expressing Rheb wt (Jiang and
Vogt, 2008). Cells of all cell lines (v0, WT, CA, CAA) were analyzed by cytometry via
forward scatter (FSC) parameter that reflects cell size. To summarize, we found out that cell
size of Rheb WT and Rheb CAA cells is only slightly increased (5%) when compared to
control v0 (Fig. 3.17). Rheb CA cells showed no difference in cell size when compared to
control v0 cells.

48
Figure 3.16: Cells transfected with distinct variants of Rheb protein are stuck in G2/M phase of the cell
cycle. Cell lines were fixed and stained with propidium iodide as described in 2.3.3.5. Then, cell lines were
analyzed by BD FACSDiva Software while the cytometry was performed at BD LSRII. In each cell line,
GFP-positive and GFP-negative population was gated and cell cycle histogram of each of them was
calculated. Each population comprises at least 1500 cells. Only single cells and cells with sufficient inner
complexity were analyzed. Similar results were obtained in three independent experiments.

49
Figure 3.17: Cells transfected with distinct variants of Rheb protein differ in cellular volume. Cell lines
were fixed and stained with propidium iodide as described in 2.3.3.5. Then, cell lines were analyzed by BD
FACSDiva Software while cytometry was performed at BD LSRII. In each cell line, GFP-positive (green)
and GFP-negative (blue) population was gated and forward scatter of at least 1500 cells was measured.
Only single cells and cells with sufficient inner complexity were analyzed. Similar results were obtained in
three independent experiments. A. A histogram shows cellular volume (FSC-A, x-axis) of all cells of both
populations. A median value for each population was determined. B. Data are presented as relative cell size
compared to v0-GFP positive population +/- standard deviation.

50
4 Discussion
p130Cas was first described as a major tyrosine-phosphorylated protein in cells
transformed by viral oncogenes v-crk (Mayer et al., 1988) and v-src (Kanner et al., 1990). In
last decade, its role in cell adhesion, motility, survival in both Src-transformed and
untransformed cells was confirmed (Cary et al., 1998; Vuori et al., 1996). Importantly,
p130Cas is also implicated in cellular pathology, mainly by its ability to stimulate cell
invasion and metastasis induction (Brabek et al., 2005).

Function of p130Cas is tightly coupled to both Src kinase and focal adhesion kinase
(FAK) that phosphorylate substrate domain of p130Cas (Fonseca et al., 2004; Polte and
Hanks, 1995), thus creating binding sites for numberless interaction partners. Src kinase
activation was documented in significant number (up to 50%) of human cancers derived
from colon, liver, lung, breast and pancreas [reviewed in (Dehm and Bonham, 2004)] and
this fact confers Src important role in human pathogenesis. Elucidating the role of p130Cas
for regulation of mTOR pathway – the main regulator of protein synthesis in cell – in
Src-transformed cells may contribute to our overall understanding of the process of cellular
transformation.

At first part of the study, we observed significant increase of S6 protein phosphorylation

(S235/236) in Src-transformed cells transfected with wild type p130Cas, when compared to
basal S6 phosphorylation in p130Cas-deficient cells (Fig. 4.2). Furthermore, we addressed
the role of substrate domain of p130Cas in mTORC1 signalling using cell line with mutated
substrate domain (SD). These cells exhibited similar level of basal S6 phosphorylation to
control p130Cas-deficient cells (Fig. 4.2) suggesting that substrate domain of p130Cas is
indispensable for stimulation of basal S6 phosphorylation. In addition, cells with mutated SD
failed to respond to serum by activation of mTORC1, emphasizing the role of p130Cas and
its substrate domain in mTOR signalling. In order to get a complex view, we also analyzed
cells with mutated SH3 domain of p130Cas and confirmed that SH3 domain was not
important for mTOR signalling (Fig. 4.3). Altogether, we have shown that p130Cas regulates
the phosphorylation status of S6 protein independently of serum stimulation. Our results are
suggestive of p130Cas localization upstream of mTORC1. Bearing in mind that several
research groups reported activation of components of mTOR pathway by Src kinase (Shah et
al., 2002; Vojtechova et al., 2008), p130Cas may easily contribute to signal transduction
from Src to mTOR.

In the next part of the study, we employed cell lines expressing p130Cas versions from
plasmid-based vector. These cell lines express p130Cas 10-15 times less when compared to

51
cell lines discussed in previous paragraph that were prepared by retroviral transfection. It is
noteworthy that no increase in S6 phosphorylation (S235/236) was observed in
p130Cas-reexpressing cells (plasmid) compared to p130Cas-deficient cells (Fig. 4.3). This
result is in contradiction with data from p130Cas-overexpressing cells (retroviral) that
exhibited significantly increase in basal S6 phosphorylation when compared to
p130Cas-deficient cells (Fig. 4.2). We hypothesize that such a discrepancy between both
cases may be caused by different level of p130Cas expression. To summarize, p130Cas
affects mTOR signalling probably only under high expression level of p130Cas which
targets this signalling pathway to pathogenic conditions when p130Cas is overexpressed.
BCAR1/p130Cas overexpression is a common feature of tamoxifen-resistant cells of primary
breast carcinoma and correlates with poor prognosis and rapid recurrence of the disease (van
der Flier et al., 2000).

In the next part of the study we explored whether p130Cas affects also mTORC2 (Fig.
4.4). Src-transformed cells reexpressing p130Cas exhibited significantly higher
phosphorylation of Akt/PKB at serine 473 than p130Cas-deficient Src-transformed cells and
this effect was serum independent. These results suggested that p130Cas might participate in
mTORC2 regulation. Conversely, another study reported elevated phosphorylation of
Akt/PKB (S473) in p130Cas overexpressing cells upon adriamycin treatment, but no effect
on Akt/PKB phosphorylation was observed without adriamycin added (Ta et al., 2008).
These data precludes a straightforward interpretation, but we might speculate about the
importance of Src activation for signalling amplification and successive crosstalk of p130Cas
and mTOR signalling. Besides mTORC1, the substrate domain of p130Cas is indispensable
for effect of p130Cas on mTORC2 signalling triggered by serum readdition (Fig. 4.4).

To summarize, we observed that overexpression of p130Cas affects both mTOR

complexes in Src-transformed cells. The crosstalk of integrin and mTOR pathway might be
realized upstream of mTOR kinase and independent of Rheb protein, because Rheb protein
is believed not to influence mTORC2 (Yang et al., 2006b). In order to address hypothetical
locations of crosstalk of p130Cas/mTOR pathway, both indirect and direct connections were
taken into account. Indirect connection comprises p130Cas interacting with PI3K,
transmitting the signal downstream to Akt/PKB and TSC1/2. This indirect pathway is likely
to function in vivo, as PI3K was already shown to interact with p130Cas in Src-dependent
manner (Riggins et al., 2003). Direct connection of p130Cas and mTOR pathway is PI3K
and Akt/PKB independent. Up to date, a few suggestions describing the character of such a
connection appeared (Gan et al., 2006; Vojtechova et al., 2008), but they focus only on
mTORC1. Gan and co-workers described that FAK associates with TSC1/2 and stimulates

52
mTOR signalling upon integrin binding to ECM, but p130Cas was not discussed in this
context up to date. In case that both mTORC1 and mTORC2 are regulated by p130Cas
simultaneously, the crosstalk should be realized on the level of TSC1/2 or Akt/PKB (Fig
4.1). Further downstream mTOR pathway divides and separate regulation of both mTOR
complexes by p130Cas is considered rather improbable. However, to understand the
crosstalk precisely detailed position of p130Cas within the mTOR cascade remained to be
established.

Figure 4.1: Possible crosstalk of mTOR pathway and integrin signalling (only p130Cas analyzed in this
study) can be mediated in two independent ways. Direct crosstalk comprises p130Cas interaction with any
of mTOR pathway components, e.g. TSC1/2 or Akt/PKB. Indirect crosstalk includes interaction of p130Cas
with PI3K, then signalling to Akt/PKB, TSC1/2 and mTOR.

In the second part of this study, we decided to explore integrin/mTOR signalling from
different point of view. By employing constitutively active variants of Rheb protein we
explored whether mTOR pathway affects integrin signalling in Rat2 fibroblasts.

Rheb protein has emerged a key player downstream of TSC1/2 in activating signalling to
mTORC1 effectors, e.g. S6K and 4E-BP1. Recently, Rheb was repeatedly shown to induce
oncogenic transformation (Mavrakis et al., 2008; Nardella et al., 2008). Most recently, Rheb
was described to be overexpressed in human carcinomas and its expression in murine basal
keratinocytes of transgenic mice lead to diffuse skin hyperplasia (Lu et al., 2010). In
summary, elucidation of processes connected to Rheb overexpression and constitutive
activation may bring new insight into understanding of certain malignant processes.

53
 In order to assess the role of Rheb in integrin signalling, we established stable cell lines
expressing distinct versions of Rheb protein: wild type Rheb, constitutively active Rheb with
mutation in S16H and Rheb mutant comprising mutations Q64L and S16H whose function
and effects on downstream signalling has not been described yet. Moreover, we tried to
establish dominant negative cell line (mutation D60V), but we were not able to create stable
line transfected with pEGFPC1 DN plasmid. Although the transfection procedure was
successful, cells lost the plasmid quickly and within two weeks of cultivation with geneticin,
transfected cells disappeared from the culture. Studies on Rheb in Schizosaccharomyces
pombe have shown that expression of Rheb D60V results in growth inhibition and G1 arrest
(Mach et al., 2000). In case that Rheb D60V causes G1 arrest in mammalian cells as well,
transfected cells would be easily overgrown by cells that do not express D60V. In order to
avoid long-term manipulation with cell lines, we tried to establish transiently transfected cell
lines expressing dominant negative Rheb. Unfortunately, we were not able to obtain cells
with sufficient Rheb expression in HEK293 (data not presented in this study). To conclude,
Rheb DN version was not examined during this study.

First of all, we characterized Rheb expressing cell lines in the light of mTOR signalling.
The hallmark of constitutively active Rheb mutants is their ability to activate downstream
signalling of the mTOR pathway. In detail, phosphorylation status of S6K, 4E-BP1 and S6
protein was monitored as Rheb was shown to regulate only mTORC1 (Yang et al., 2006b).
All cell lines overexpressing Rheb protein exhibited increased phosphorylation of mTORC1
substrates-S6K and 4E-BP1 compared to basal phosphorylation in v0 (Fig.4.8). By
densitometrical analysis, we quantified that cell lines expressing Rheb WT, CA and CAA
exhibited at least two times higher phosphorylation of S6 (S235/236) compared to basal
phosphorylation in control v0 cell line.

When comparing the phosphorylation of S6 protein (Fig. 4.9) in Rheb overexpressing cell
lines (WT; CA:S16H and CAA:S16H+Q64L), we get the highest phosphorylation in
constitutively active mutant CAA comprising mutations S16H and Q64L. In our study, no
significant enhancement in phosphorylation of S6 protein in constitutively active CA or
CAA cells when compared to cells transfected with wild type Rheb was detected. In a
different study, mutation S16H increased phosphorylation of S6K (T389) at least two times
compared to Rheb wt (Yan et al., 2006). We can only hypothesize about the real activity of
our Rheb CA:S16H version, because the level of Rheb-GFP expression in cell line
transfected with rheb CA:S16H variant was reduced compared to the other rheb-transfected
cell lines. Thus, the real activity of Rheb CA:S16H might be underestimated. In addition,
Jiang and co-workers focused on cells transfected with Rheb harbouring mutation Q64L

54
(Jiang and Vogt, 2008). Their analysis showed at least 5-times increase in S6K
phosphorylation in cells transfected with Rheb Q64L compared to Rheb wt. Noteworthy, our
Rheb version harbouring both mutation Q64L and S16H did not activate mTORC1
signalling to such intensity, but rather resembles S16H Rheb version in its activation
potential.

Altogether, we did not observe significantly enhanced activation of mTOR in

constitutively active Rheb-transfected cells when compared to wild type Rheb
overexpressing cells. Remarkably, cell lines expressing Rheb variants failed to respond to
serum addition at mTOR level. As we assume that signalling triggered by serum is mediated
by Rheb and mTORC1, this result brings us to the notion that high GTP loading of Rheb
variants cannot be further significantly attenuated by serum readdition. In next part, we
confirmed that mTORC2 signalling is not under control of Rheb protein which is in
agreement with already published data (Yang et al., 2006b).

In the final part of this study, we returned to the characterization of integrin/mTOR

crosstalk. We focused on Paxillin phosphorylation (Y118) in Rheb-transfected cells (WT,
CA, CAA, v0), because this phosphorylation is performed by FAK upon integrin
stimulation. Importantly, constitutively active double mutant CAA exhibited the highest
level of Paxillin phosphorylation when compared to control cells v0 (Fig. 4.12). Taken
together, our results suggest that Rheb protein influence Paxillin phosphorylation, although
the direct mechanism has not been described yet. However, FAK phosphorylation was
affected by neither Rheb overexpression nor expression of constitutively active Rheb
mutants (Fig. 4.13). Therefore, enhanced Paxillin phosphorylation stimulated by Rheb is
probably not mediated by FAK. Acquired data showing Paxillin regulation by Rheb possibly
revealed a novel negative feedback loop connecting mTORC1 and integrin signalling or a
novel function of Rheb independent of mTOR pathway.

To complete the characterization of Rheb mutants in Rat2 cells, we performed cytometric

analysis of transfected cell lines (WT, CA, CAA, control v0). In all cell lines overexpressing
Rheb, we observed that large number of cells reside in G2/M phase (Fig. 4.14). Similar result
was observed in Drosophila melanogaster after transfection of Rheb wt (Reis and Edgar,
2004). Acquired data suggest that Rheb induces a faster G1/S transition and consequential
accumulation of cells in G2/M as observed by flow cytometry. While cultivating transfected
cells, no changes in proliferation rate within Rheb overexpressing cell lines and control line
v0 were noticed. In such a deregulation of cell cycle progression, the overall rate of cellular
division remains constant.

55
5 Summary

 We reported that overexpression of p130Cas affects mTOR signalling in

Src-transformed cells. Since both mTORC1 and mTORC2 signalling were
stimulated by p130Cas overexpression, we suggest that p130Cas affects mTOR
pathway upstream of Rheb protein.

 Substrate domain of p130Cas is critical for activation of mTOR pathway induced

by p130Cas overexpression.

 Cell lines expressing various versions of Rheb protein were established. All cell
lines overexpressing Rheb protein versions exhibited increased phosphorylation of
mTORC1 substrates - S6K, S6 protein and 4E-BP1 compared to basal
phosphorylation of the mTORC1 substrates in control cell line.

 Unique cell line expressing constitutively active Rheb CAA harbouring mutations
S16H and Q64L was characterized. Rheb CAA expressing cell line showed
similar potential to stimulate mTORC1, integrin signalling and cell cycle
progression as Rheb CA bearing single mutation S16H.

 We observed that overexpression of Rheb CAA affects integrin signalling.

Paxillin but not FAK phosphorylation was elevated in cell lines expressing Rheb
CAA. This finding suggests crosstalk of mTOR and integrin signalling
independent of FAK.

 Cell lines overexpressing Rheb variants failed to respond to serum readdition at

mTOR level. High GTP loading of these Rheb proteins cannot be further
attenuated by serum readdition.

56
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