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BIOLOGY Elucidation of G12-HSP90 Protein Interactions Necessary for a G12 Dependent Cell Growth Pathway Abstract Heterotrimeric guanine nucleotide binding proteins (G proteins) are important cell signaling proteins that mediate a wide variety of cellular responses via signal transduction across the plasma membrane. They do so by interacting with a number of downstream target proteins to drive various signaling pathways. The G protein G12 governs pathways regulating cell growth, proliferation, and shape, and has been implicated in tumorigenesis and metastasis. My research will focus primarily on the mechanisms and evolutionary origin of protein-protein interactions between G12 and one of its downstream targets, Heat Shock Protein of 90kDa (HSP90), as part of the cell growth related Serum Response Factor (SRF) pathway. Prior screenings of various G12 point mutants against HSP90 have revealed several key amino acid residues that appear important for this protein-protein interaction to occur. Interestingly, substitution of these residues with the corresponding residues of the fruit fly homolog of G12 abolishes its ability to stimulate SRF. My research will consist of two phases. First, I will compare the amino acid sequences of mammalian G12 to its evolutionary homologs across a broad range of vertebrates and invertebrates, and catalog variations at residues critical for HSP90 interaction. In the second phase, I will engineer mutants of mouse G12 that substitute the residues found to vary in the G12 homologs, and test these for HSP90 binding and SRF activation to determine the precise structural features that conferred upon G12 the ability to stimulate this cellular growth pathway. In addition, I will examine variation in HSP90 across a variety of taxa to identify residues for mutation; these studies should facilitate mapping of G12 binding motifs within HSP90. Background G proteins have been studied extensively in order to elucidate their numerous functions in cell signaling and response. Heterotrimeric G proteins are comprised of 2 functional signaling units: an subunit and a heterodimer. When inactive, the subunits are bound together as a heterotrimer at the inner face of the plasma membrane, bound to a G protein coupled receptor (GPCR). Ligand binding at the extracellular face of the GPCR stimulates the G subunit to release a bound guanosine diphosphate (GDP) molecule, allowing for its replacement with a guanosine triphosphate (GTP). This transition causes a conformational shift in the subunit, dissociating it from the subunit. From there, the subunit interacts with a number of downstream proteins to promote various cellular responses (Suzuki et al., 2009). The G12 subunit is ubiquitously expressed in mammalian tissues. Functions specific to it include promoting cell growth and mitosis through a variety of signaling pathways. Due to the nature of its function, G12 has the potential to promote malignant, uncontrolled cell growth and metastasis, classifying it as a proto-oncoprotein. Indeed, analysis of cDNA libraries collected from sarcoma tissue samples has implicated G12 as a strong transforming oncoprotein (Chan et al., 1993). Importantly, G12 need only be over-expressed, not mutated, in order to potentially drive cancerous proliferation through these pathways (Meigs and Lyakhovich, in review). Fromm et al. (1997) demonstrated G12 stimulation of this SRF pathway to promote cell growth, and potentially cancer cell progression. A better understanding of the protein-protein interactions of these pathways may prove important in discovering ways to combat cancer. Previous research done by Dr. Meigs and colleagues, screening G12 point mutants against a number of its downstream targets, has elucidated several regions of G12 important for interaction. My research will focus on the evolutionary emergence of a G12 dependent cell growth pathway, mediated through its downstream target protein Heat Shock Protein of 90kDaltons (HSP90). HSP90 is primarily understood to be a molecular chaperone protein, maintaining the functional shape of signal transduction proteins including G proteins (Suzuki et al., 2009). Vaiskunaite et al. (2001) reported that a G12 driven SRF pathway is dependent on the function of HSP90, however, other G proteins, namely RhoA and G13, are not. In evolutionary terms, this pathway has been conserved in spite of alternative SRFspecific growth pathways. The amino acid sequence of G12 contains key differences that distinguish it from other G proteins. Dr. Meigs has engineered G12 point mutants that replace native amino acids with those found at identical structural positions in other, “ancestral” G protein subfamilies. Assays designed to examine these G12 mutants for HSP90 binding have identified several single amino acid substitutions that disrupt this interaction. Dr. Meigs’s lab also examined the fruit fly version of G12, a protein termed Concertina, for its function in several aspects of cell biology. A surprising finding was that Concertina, despite harboring many properties similar to G12, lacks the ability to drive the SRF pathway in cells. Furthermore, introduction of two single amino acids from Concertina into G12 caused a full loss of its ability to stimulate SRF (T. Meigs, personal communication). In my initial proteomic comparisons of G12 across numerous taxa, I uncovered interesting variations at several of these key amino acid positions in the G12 homologs of the frogs Xenopus laevis and Xenopus tropicalis. These species exhibit the Concertinaspecific amino acid residues similar to Dr. Meigs’s engineered point mutants, yet retain many of the same characteristics as mouse G12. Further investigation led to the discovery that these frog species exhibited a divergent sequence for HSP90 as well. Methods My primary focus will be to create fusion proteins of HSP90 and the Xenopus variant and screen them against G12 point mutants engineered to harbor key residues found in G12 homologues. Specifically, I will be interacting a constitutively active mutant of G12—continuously bound to GTP—termed G12QL against the target protein in a series of “pulldown” assays. A fusion of the target protein HSP90 to the protein Glutathione-S-Transferase (GST) will be bound to heavy sepharose beads coated in glutathione. HSP90-GST fusion proteins will be achieved via transformation of bacterial cells with plasmid vectors engineered to encode the HSP90-GST fusion. HSP90 protein is quite large at 90kDa, making fusion of the whole protein difficult and impractical. Previous work in Dr. Meigs’s lab has shown that G12 binds at the C-terminus of HSP90. Therefore a GST fusion of that specific HSP90 region should be sufficient for performing these experiments. The immobilized HSP90-GST fusion proteins will be mixed with cell lysates containing the G12QL point mutants. Any interaction with HSP90 will effectively coprecipitate the G12 proteins along with the sepharose beads. Subsequently, gel electrophoresis and G12-specific immunoblotting will be used to detect these interactions. Conclusion The purpose of this research is to better understand the mechanism of interaction between G12 and HSP90 as part of a potentially cancerous cell growth pathway. Understanding specifically how these interactions occur should improve our ability to design and discover new methods for the treatment of various types of cancer associated with these pathways. References Suzuki N, Hajicek N, Kozasa T. (2009) Regulation and physiological functions of G12/13-mediated signaling pathways. Neurosignals 17: 55-70. Chan A.M., Fleming T.P., McGovern E.S., Chedid M., Miki T, Aaronson S.A. (1993) Expression cDNA cloning of a transforming gene encoding the wild-type G12 gene product. Mol. Cell. Biol. 13: 762-768. Fromm C., Coso O., Montaner S., Xu N., Gutkind J.S. (1997) The small GTP-binding protein Rho links G protein coupled receptors and G12 to the serum response element and to cellular transformation. Proc. Natl. Acad. Sci. USA 94: 10098-10103. Meigs T., Lyakovich A. G protein alpha 12. In review; Springer Publishing. Vaiskunate R., Kozasa T., Voyno-Yasenetskaya T.A. (2001) Interaction between the G subunit of the heterotrimeric G12 protein and Hsp90 is required for G12 signaling. J. Biol. Chem. 276: 46088-46093. Budget and Justification Student stipend for 8 weeks of full-time summer research $ 1500.00 Supplies/Reagents for Cell Growth, DNA Mutagenesis, and Protein Analysis Acrylamide solution Gel-loading micropipet tips $ 57.60 $ 54.23 Falcon tubes, sterile (for bacterial transformations) $ 109.46 Anti-G{alpha}12 polyclonal antibody $ 259.00 Wizard PCR/gel cleanup kit $ 129.60 T4 DNA ligase $ 34.58 JM109 competent bacterial cells $ 222.76 Benchmark Protein Standards $ 133.00 _____________________________________________________________ TOTAL: $ 2500.23 Time Period 6/6/2011 – 7/6/2011: Structural comparisons of G12 and its homologs using BLAST program. Polymerase Chain Reaction based engineering of constructs encoding HSP90GST fusion proteins, and verification by DNA sequencing. Transformation of bacterial cells to express the protein constructs. 7/7/2011 – 7/30/2011: Bacterial expression and affinity purification of protein products. Interaction assays between HSP90-GST fusions and G12. Computer-assisted quantification of protein binding. Publication Outlet This work will be submitted to the UNC-Asheville Journal and also presented at the Undergraduate Symposium on Research and Creativity. Also, it is our anticipation that data from this research ultimately will comprise part of a manuscript Dr. Meigs will submit for publication in a peer-reviewed journal.