I.

Description of ROA Arrangement A. Nature of Research Responsibility Dr. David B. Stephens will conduct research full-time in the laboratory of Dr. John M. Bush during the duration of the ROA visit. During this time he will be conducting all research activities associated with the proposed project, including but not limited to vector construction of wild-type and mutant Rab11 GST-fusion proteins, cell transformations, cell fractionations, affinity purifications, and probing of isolated Rab11-binding proteins for calmodulin. B. Duration of ROA Visit The duration of the visit will be from June 1st thru August 31st of 2002. C. Nature of Visitors Appointment Instructor of biology and physical science at Pulaski Technical College. D. Visitors Rate of Pay 2/9ths of $25,459, Dr. Stephens pay last year, is $5657.55. E. Miscellaneous Employment Arrangements Description of Research to be Performed A. Research Plan for Summer 2002 Research reported to date indicates a relationship between Rab3A and calmodulin in exocytosis from neurons, and calmodulin/Ca2+ has been shown to bind to Rab3A in a [Ca2+ ]dependent manner.1 Several other neuronal small GTPase types have been shown to be calmodulin binding,2, 3 but no other Rab has been found to associate with calmodulin.1 In Dictyostelium discoidium Rab11,4 calmodulin,5 and the Ca2+ -ATPase PAT16 have all been shown to localize to the contractile vacuole, and it has been suggested that the contractile vacuole is a component of a calcium sequestration and excretion pathway that functions to help maintain calcium homeostasis in this organism.6 Examination of the amino acid sequence of Dictyostelium Rab11 reveals two possible calmodulin binding sequence motifs.7 In the following, underlined segments represent possible calmodulin binding sequences. Bold letters represent amino acids that follow motif patterns; italic letters represent those that do not follow the observed motif. [Ca++]-independent sites: IQxxxRxxxxRxxL MTSKGSQEEYDYLYKIVLIGDSGVGKSNLLSRFTRNEFSLETKSTIGVEFATRTIQTEG KTIKAQVWDTAGQERYRAITSAYYRGAVGALLVYDIAKQATYKSVERWILELRENA DRNIEIMLVGNKSDLRHLREVSTDEAKEFSEKHKLTFIETSALDSSNVELAFQNILTQI YHIMSRPSHSTGPQTTIDSNTETIILPTTSEPPAAKSGCC As can be seen, the first part of this sequence obeys the motif very well, with some variance towards the C-terminus of the sequence. [Ca++]-dependent sites: 1-8-14 motif, +2

II.

 +4 net charge

MTSKGSQEEYDYLYKIVLIGDSGVGKSNLLSRFTRNEFSLETKSTIGVEFATRTIQTEG KTIKAQVWDTAGQERYRAITSAYYRGAVGALLVYDIAKQATYKSVERWILELRENA DRNIEIMLVGNKSDLRHLREVSTDEAKEFSEKHKLTFIETSALDSSNVELAFQNILTQI YHIMSRPSHSTGPQTTIDSNTETIILPTTSEPPAAKSGCC This is an interesting site. The motif is followed, there are three positive amino acids, two negative amino acids, and a histidine, which is neutral or positive based on the microenvironment pH. This sequence could thus have the net +2 charge required if it occurs in a microenvironment with a pH below 7.5; this could thus act as a pH-dependent switch for calmodulin binding Using a glutathione-S-transferase (GST) fusion system8 that we have recently been exploring with Rab2, we propose to use Dictyostelium Rab11 wild type as well as active and inactive mutants (all of which we have on hand) fused to GST to attempt isolation of Rab11 effectors.

Dictyostelium cytoplasmic and membrane fractions will be separated and subjected to glutathione affinity techniques, and isolated proteins will then be subjected to SDS-PAGE and probed with anti-calmodulin antibodies in a Western blot. This could then lead to future studies addressing the role of Rab 11 calmodulin/Ca2+ interactions in contractile vacuole function and biogenesis, or studies of other Rab effectors using these techniques. B. Future Studies Provided calmodulin is shown to associate with Rab11, mutations similar to those done to Rab3A will be used to probe the basis of binding.1 The two putative calmodulin binding motifs shown above would be the natural initial targets of these studies; one could foresee trying point mutations of the histidine in the putative Ca++ concentration dependent site to either positive or neutral amino acids to be of possible interest, as well as mutations to the other conserved residues. Free peptides of either of these sequences could be used in studies attempting to block calmodulin binding. Calmodulin competition studies using labeled free calmodulin can be performed to estimate the binding coefficient. W-13 (N-(4-Aminobutyl)5-Chloro-2-Naphthalenesulfonamide), a calmodulin antagonist, can be used to study the phenotypic effect of calmodulin-Rab11 binding.9 Interruption of calmodulin-Rab11 interaction may disrupt CV function, render the CV insensitive to calcium concentration, or have no discernable phenotypic effect. If calmodulin-Rab11 association is not observed, we will proceed to the use of 2-D SDS-PAGE and protein matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) to isolate and attempt to identify other effectors. III. Contribution of Research to NSF-supported Project. As has been seen in studies of the function of the Ras GTPase,10 the elucidation of the function of Rab proteins requires the identification and study of the regulatory factor and effectors proteins that associate with Rabs.11 Other studies have shown that such proteins exist and function in various capabilities in the process of Rab mediated vesicular transport.12 It is one of the objectives of the hosts NSF-supported project13 to study Rab effectors and regulatory proteins in D. discoideum and their interaction with both the Rab 4 and Rab 11 GTPases to regulate CV function and vesicular transport. Contractile vacuole associated calmodulin in Dictyostelium is a reasonable target as a Rab effector, and the current study will also allow for development of GST-fusion techniques of possible use in other effector isolations. Contribution of Research to Visitors Future Research Dr. Stephens has considerable experience in protein affinity isolation, purification, and characterization, including the following work: A. Research as a postdoctoral fellow into the solubilization, purification, and reconstitution of the transporter associated with antigen processing (TAP) in the laboratory of Dr. Matthew Androlewicz at the H. Lee Moffitt Cancer Research Center, University of South Florida. TAP is an ATP-hydrolytic peptide pump situated in the endoplasmic reticulum membrane, involved in the peptide charging of the major histocompatibility complex 1. The initial problem was the determination of conditions that would allow for the solubilization of this integral membrane protein without loss of biological activity. Experimental evidence has now been collected which demonstrates peptide binding by TAP solubilized under the proper conditions and reconstituted into proteoliposomes; it was determined that an important factor in maintenance of peptide binding function was the replication of the proportions of different phospholipids found in the ER membrane, leading to the publication of the first paper detailing the reconstitution of peptide-binding activity in solubilized TAP. Skills acquired include mammalian cell culture, radioiodination of proteins, membrane protein solubilization and reconstitution by proteoliposome formation, and autoradiography. B. Production, isolation, and characterization methods for polyclonal catalytic antibodies as a graduate student in the laboratory of Dr. Brent Iverson. This research involved the synthesis of both transition-state-analog haptens and substrates, injection of and blood collection from laboratory animals, isolation of pure IgG from sera, and a variety of techniques for catalytic and immunoassay. Initial work determined that hapten-affinity chromatography cannot purify

IV.

catalytic antibodies from whole IgG samples, a stumbling block to earlier attempts; both catalytic monoclonal and polyclonal antibodies failed to elute from a hapten-functionalized column under a wide variety of conditions. Whole IgG fractions were thus used, and hapten titrations of catalytic reactions were used to estimate catalytic fraction. Studies of both the maturation of the catalytic immune response and variation of response between individuals were performed. Mathematical models predicting the behavior of different distributions of pooled heterogeneous catalysts were constructed. Subsequently, a new separatory technique utilizing a substrate column for catalytic elution of abzymes was successfully developed, allowing for significant enrichment of the catalytic fraction in the IgG samples. Skills acquired include polyclonal and monoclonal antibody production, NMR spectroscopy, affinity FPLC, reverse-phase HPLC, quantitative UV-Visible spectrophotometry, enzyme-linked immunosorbent assay, acrylamide electrophoresis, protein affinity purification, enzyme kinetics, and phosphoramidite and phosphonium synthetic techniques. Although Dr. Stephens has done quite a bit of work with protein techniques, he has not had much opportunity to work with genetic techniques. The proposed project will allow him to learn a host of DNA manipulation (vector design and construction, ligation, PCR, et cetera) and cell transformation techniques, broadening his repertory of research abilities to keep pace with the current level of research in the biological and biochemical arena. This will be essential in Dr. Stephens ultimate career goal of gaining a faculty position at a university with competitive molecular and cellular biology research activity. V. Contribution of Research to Visitors Home Organization As an instructor in the physical and biological sciences at Pulaski Technical College, Dr. Stephens is involved in training students who are either gaining core credits for transfer to four-year universities or seeking two-year degrees in health-related professions. Pulaski Technical College is one of the largest and fastest-growing two-year colleges in Arkansas, with the Spring 2001 enrollment reaching 4,486 students. By fall 2002, it is projected to top 5,500. The college serves an additional 4,500 students annually in continuing education, customized training for business and industry, and adult education. Pulaski Tech offers more than 40 Associate of Applied Science degree and certificate programs in business, information technology, health, technical and industrial fields. An accredited two-year college, it offers a comprehensive Associate of Arts degree program for students who plan to transfer to four-year colleges and universities to complete bachelor's degrees, a role that has expanded greatly in recent years due to its lower cost, making Pulaski Tech an attractive entry for the economically-disadvantaged into a university education. Pulaski Technical College offers credit classes and programs designed for university transfer or leading to the Associate of Applied Science degree or technical certificate. Pulaski Technical College broke ground April 23 for construction of a new Allied Health Education Center; when completed, the 33,300 square-foot, two-story structure will house the colleges programs in practical nursing, respiratory therapy and dental assisting, as well as multipurpose classrooms and office space. Participation in this study would allow Dr. Stephens to pass along current real-world experience of research techniques and activities in lectures and teaching labs, broadening his students understanding of scientific methods. Budget for ROA Visit Visitors Biographical Sketch A. Professional Preparation University of Arkansas at Little Rock University of Arkansas at Little Rock University of Texas at Austin University of South Florida at Tampa B. Appointments Biology Chemistry Biochemistry Cell Biology B.S. 1990 B.S. 1990 Ph.D. 1996 1996-1997

VI. VII.

Instructor at Pulaski Technical College, teaching physics, chemistry, and biology. This position has been held from July 2000 up to the present. C. Publications Stephens, D.B.; Iverson, B.L. (1993) "Catalytic Polyclonal Antibodies" Biochemical and Biophysical Research Communications 192:1439-1444. Stephens, D.B.; Wilmore, B.H.; Iverson, B.L. (1994) "Polyclonal Antibodies and Catalysis" Bioorganic and Medicinal Chemistry 2:1-6. Stephens, D.B., and Androlewicz, M.J. (1997) "Reconstitution of Peptide-binding Activity by TAP in Proteoliposomes", FEBS Letters 416:353-358. Stephens, D.B., Thomas, R.E., Stanton, J.F., and Iverson, B.L. (1998) Polyclonal Antibody Catalytic Variability, Biochemical Journal 332:127-134. D. Synergistic Activities 1. Volunteer chemistry teacher for high school students at the Marywood Children and Family Services Center in Austin, Texas during the 1993-1994 school year, helping students stay current with their high school education to ease reentry into the regular school system. Development of a substrate chromatography method for polyclonal catalytic antibody separation from the hapten-specific population of antibodies during graduate student research. Catalytic antibodies would bind matrix-bound substrate at a pH of 8, but catalysis would only occur at a pH of 6, cleaving substrate from the column and eluting the catalytic species. This made possible the hitherto unachieved goal of affinity purification of catalytic antibodies. Development of establishment of relative phospholipid subpopulations in liposomes to effect reconstitution of activity in transmembrane proteins active in peptide transport during postdoctoral research. It was found necessary to replicate the relative proportions of different phospholipids found in the endoplasmic reticulum to retain peptide-binding activity in the transporter for antigen processing. Operation of a chemistry consulting firm, Event Horizons Development, offering free phone advice to the general public on topics such as science fare project development, environmental chemical toxicity questions, et cetera. Consulting activities with Attorney Hubert Alexander in a total of six cases, aiding in assessing the clients defense from a chemistry standpoint.

2.

3.

4. 5.

E. Collaborators and Other Affiliations 1. Collaborators Brent L. Iverson, Ph.D. Department of Chemistry and Biochemistry, University of Texas at Austin. John F. Stanton, Ph.D. Department of Chemistry and Biochemistry, University of Texas at Austin. Richard E. Thomas, M.S., United States Navy Graduate and Postdoctoral Advisors Matthew J. Androlewicz, Ph.D. H. Lee Moffitt Cancer Research Center, University of South Florida, Tampa, Florida John M. Bush, Ph.D. Biology Department, University of Arkansas at Little Rock. Brent L. Iverson, Ph.D. Department of Chemistry and Biochemistry, University of Texas at Austin.

2.

Coppola, T., Perret-Menoud, V., Luthi, S., Farnsworth, C.C., Glomset, J.A., and Regazzi, R. (1999) Disruption of Rab3-Calmodulin Interaction, but not Other Effector Interactions, Prevents Rab3 Inhibition of Exocytosis. The EMBO Journal. 18:5885-5891.
2

Sahyoun, N., McDonald, O.B., Farrell, F., and Lapetina, G. (1991) Phosphorylation of a Ras-related Protein, Rap-1b, by a Neuronal Ca2+ /Calmodulin-dependent Protein Kinase, CaM Kinase Gr. Proc. Natl. Acad. Sci USA. 88:2643-2647.
3

Wes, P.D., Yu, M., and Montell, C. (1996) RIC, a Calmodulin-binding Ras-like GTPase. The EMBO Journal. 15:5839-5848.
4

Harris, E., Yoshida, K., Cardelli, J., and Bush, J. (2001) Rab11-like GTPase Associates with and Regulates the Structure and Function of the Contractile Vacuole System in Dictyostelium discoidium. Journal of Cell Science. 114:3035-3045.
5

Zhu, Q., Liu, T., and Clarke, M. (1993) Calmodulin and the Contractile Vacuole Complex in Mitotic Cells of Dictyostelium discoidium. Journal of Cell Science. 104:1119-1127.
6

Moniakis, J., Coukell, M.B., and Janiec, A. (1999) Involvement of the Ca2+ -ATPase PAT1 and the Contractile Vacuole in Calcium Regulation in Dictyostelium discoidium. Journal of Cell Science. 112:405-414.
7

Rhoads, A.R., and Friedberg, F. (1997) Sequence Motifs for Calmodulin Recognition. The FASEB Journal. 11:331-340.
8

Ohta, Y., Suzuki, N., Nakamura, S., Hartwig, J.H., and Stossel, T.P. (1999) The Small GTPase RalA Targets Filamin to Induce Filopodia. Proc. Natl. Acad. Sci. USA. 96:2122-2128.
9

Apodaca, G., Enrich, C., and Mostov, K.E. (1994) The Calmodulin Antagonist, W-13, Alters Transcytosis, Recycling, and the Morphology of the Endocytic Pathway in Madin-Darby Canine Kidney Cells. Journal of Biological Chemistry. 269:19005-19013.
10

Wittinghofer, A., and Valencia, A., (1995). Three Dimensional Structure of RAS and RAS-related Proteins Reviewed in the Guidebook to Small GTPases, Pages 20-29. Edited by Zerial, M., and Huber, L., Oxford University Press. Oxford, England.
11

Zerial, M., (1995). Rab Protein Reviewed in the Guidebook to Small GTPases page 295-306. Edited by Zerial, M., and Huber, L. Oxford University Press. Oxford, England.
12

Loomis, W., Kuspa, A., and Shaulsky, G. (1994). Gene Discovery in Dictyostelium. Genetic Engineering. 16: 49-64.
13

Bush, J.M. The Role of Vesicular Transport on the Function and Biogenesis of the Contractile Vacuole in Dictyostelium discoidium. NSF Career Award Abstract #9734093.