Signaling Pathways

That Control
Gene Activity

Chapter 14
Molecular Cell Biology, 5th ed.
Jeffrey Jong-Young Yen, Ph.D.
IBMS, Academia Sinica

   
 Signal

• Any cellular stimulus leading to cell growth, 
differentiation, death or cellular function.

Signal Amplification
 From one extracellular stimulus to a
gross biological effect.

   
   Protein Kinase
• Ser/Thr protein kinase: protein that can add a
phosphate group to the Ser or Thr residue of
another protein, e.g. cyclin dependent kinase, Raf
kinase, etc.
• Tyrosine kinase: protein that can add a phosphate
group to the tyrosine residue of another protein,
e.g. EGF receptor, Wee1 kinase, Src kinase, etc.
• Dual-specific protein kinase: protein that has both
Ser/Thr and tyrosine kinase activity, e.g. MAP
kinase kinase (MEK).
   
   Protein Phosphatase
• Protein Ser/Thr phosphatase: protein that
removes phosphate group from the phosphorylated
Ser/Thr residue of another protein.
• Protein tyrosine phosphatase: protein that
removes phosphate group from the phosphorylated
tyrosine residue of another protein.
• Dual specific protein phosphatase: protein that
has both Ser/Thr and tyrosine phosphatase
activity.
   
    Kinase Cascade

A stimulus that turns on a series of 
kinase and target gene expression

   
 Detection of Kinase Activation

• Kinase immunoprecipitated from cellular extracts

+ appropriate substrate + γ-32P-ATP resolved on
SDS-PAGE autoradiography.
• Autophosphorylation: most kinases auto-
phosphorylate itself in vitro.
* auto-tyrosine phosphorylated (anti-pTry)
* auto-Ser/Thr phosphorylated (anti-pSer/Thr)

   
 Adaptor proteins

Proteins that do not have any enzyme

activity but are required for activation
of other downstream proteins.
Usually activated through protein-
protein interaction via the SH2 and/or
SH3 domains.
   
Adapted from Pawson, T, Gish, GD & Nash, P (2001) Trends Cell Biology 11, 504­511. 
   
 SH2 domains
• About 100 aa in length and form a globular
structure with the N- and C-terminal ends coming
close together.
• First identified as regions of homology between
Src and Fps kinases that lie outside the catalytic
domain and that are not required for kinase
activity,
• Bind specifically to phosphorylated tyrosine
residues.
   
 SH2 domain­

binds to phosphorylated
Tyr

   
 SH3 domains
• About 60 residues in length: also form a globular
structure.
• Discovered as a region of homology between v-
Crk, PLCγ and Src.
• Bind short, ten amino acid Pro-rich motifs in a
sequence-specific fashion.

   
   
 PTB domains 

1) Phosphotyrosine binding domain 
2) Structurally differs from SH2 domains 
3) Recognition is determined by amino 
acid residues N­terminal to the 
phospho­tyr and highly dependent on 
its 3D conformation 

   
 Shc, PTB Domain 
   
 PH domains 
1) Pleckstrin homology domains 
2) Overall sequence similarity very low between 
     different PH domains 
3) PH domain proteins tend to be membrane 
     associated and/or in the inositol phosphate 
     signalling pathway 

4) Principal binding partners: 
phosphoinositides, G protein βγ subunits 
5) May be different functional subfamilies 
6) Mediate protein­protein interactions 
7) Mediate protein­lipid interactions 

   
 PH domain of phospholipase C delta 1
   
 PDZ domains 

1) ~90 amino acids 
2) Bind to consensus carboxyl­terminal 
motif of target protein  (­ [Ser/Thr]­X­
Val) 
3) specificity conferred by the ­2 and 
­4  positions relative to the carboxy­
terminus 
4) Play an important role in the spatial 
organisation of voltage­ and  ligand­
gated  ion channels at  synapses 
5) proteins with up to 7 PDZ domains 
have been characterised 
   
 hCASK (type II) PDZ domain 

Adapted from atb.slac.stanford.edu/.../coords.php?viewcoord=8 
   
 Figure 1. PDZ domain structures. (A) PDZ3 of PSD­95 (cyan), complexed 
with the C­terminal pentapeptide of CRIPT (KQTSV, yellow). (B) The PDZ 
domain of a­1 syntrophin (green), complexed with the PDZ domain of 
nNOS (blue). (C) Homodimer of Grip1 PDZ6 (pink and purple), complexed 
with the C­terminal octapeptide of Liprin (ATVRTYSC, yellow). 
Adapted from www.cgr.harvard.edu/macbeath/research/pdz.html 
   
   
   
TGFβ­Smad
Signaling pathway

   
 TGFβ signaling and Human tumors 

• Deletion of smad4 (human pancreatic 
cancers)
• Loss of type I or II TGFβ receptor 
(retinoblastoma, colon and gastric cancer, 
hepatoma, some T and B­cell malignancies)
• Mutations in Smad2

   
   
Ligand­induced receptor dimerization
   
   
   
   
   
 Figure 2. Comparison of the receptor complexes for Epo and IL­6.
   
   
 EpoR and Jak2 – required for development of erythrocytes

E13
   
 Figure 1. Erythropoietin (Epo) binds
its receptor (Epo R) on the surface of
red blood cell progenitors in the bone
marrow causing proliferation,
maturation, and differentiation, there
preventing or correcting anemia. Epo
may also bind Epo R expressed on the
surface of cancer cells and may elicit
tumor growth via cell proliferation,
protection from apoptosis, and/or
angiogenesis. [Note: figure adapted
from Brower, V. (2003) Nat. Med.
9:1439.]

   
   
 Negative regulation of cytokine signaling

   
SOCS­2: negative regulator of GH receptor 

SOCS­2 deficient mice: significantly larger than the WT littermates

EpoR mutant (C­terminal truncation): defective in binding to SHP1

­prolonged intracellular signaling
­more erythrocytes produced (hematocrit 60 v.s. 45­47%)

   
RTK            activation of Ras

   
   
   
   
   
   
 Pathways that involve signal­
induced protein cleavage

   
   
 F
i
g
u
r
e
 
1
:
 
Figure 1: Notch receptors are expressed on the cell surface as heterodimeric
proteins. They are composed of an extracellular domain andM an intracellular

a
domain containing multiple protein-protein interaction domains as well as a
transactivation domain. Notch signaling is triggered upon ligand-receptor
m
interaction which induces two sequential proteolytic cleavagesby
metalloproteases of the ADAM family, and by a γ‑secretase activity of
presenilins (PS). This second cleavage allows the release andmtranslocation of
the intracellular domain of Notch (NICD) into the nucleus where it associates
  CSL. Binding of NICD to CSL leads to
with a
  transcriptional activation.
l
 A family of transcription factors called sterol 
regulatory element­binding proteins 
(SREBPs) that control cholesterol and 
fatty acid synthesis. Unlike other transcription
 factors, the SREBPs are synthesized as 
intrinsic membrane­bound proteins of the 
endoplasmic reticulum (ER). In lipid­depleted 
cells, SCAP facilitates the incorporation of 
SREBPs into vesicles that bud from the ER and 
move to the Golgi apparatus, where the 
SREBPs are processed sequentially by two 
membrane­bound proteases. The second 
protease cuts the SREBPs within a membrane
­spanning helix in a process called Regulated 
Intramembrane Proteolysis (RIP). This 
liberates the active transcription factor 
domain of the SREBPs so that they can 
enter the nucleus and activate genes 
encoding enzymes required for synthesis of 
cholesterol and fatty acids. 
   
 Figure 2:
Classical IL­6 Signaling
   
 Figure 3:
IL­6 Transsignaling