The long term objective of this research project is to contribute to a better understanding of the mechanisms by which growth factor act to control growth in mammalian cells. The specific gene to be studied, g33, has been cloned and characterized at the sequence level. This gene has many interesting properties, including widespread tissue distribution (6, and L.A. Balogh, K._L. Lee, and F.T. Kenney, unpublished observations) and inducibility, and the potential for producing two discrete protein products. The function of the g33 proteins has remained elusive. Sequence analysis of the predicted protein sequence revealed that g33 protein contains several regions that are similar to functional or regulatory domains of other known proteins. Using amino acid sequence derived from the PROSITE database we compared g33 protein (PIR entry number S03116) to numerous entries in PROSITE using the PROSIS protein analysis software. We found several potential PKC phosphorylation sites, casein kinase II phosphorylation sites. cAMP/cGMP dependent kinase phosphorylation site, N- myristolation site, tyrosine sulfatation site, a region similar to a DEAD box, and a Class I SH3 domain- binding site. No SH3 or SH2 domains or SH2 domain binding sites we found. The strongest homology to protein sequences in the NCB1 database found using the BLAST algorithm is tot he human non- receptor tyrosine kinase called ACK that inhibits the GTPase activity of p21 cdc42 (23), and the homology is restricted to portions of the C- terminal half of the protein including a Class I SH3 domain binding site, and do not include the kinase catalytic domain, the SH3 domain or the Cdc42Hs binding site. The region in g33 protein where the Class I SH3- domain binding site is located has some homology to the ACK protein sequence, which also possesses a Class I SH3 domain binding site. Other homologies that come out in the protein sequence analysis are to proline-rich protein, but the alignments are poor and scattered and restricted mainly to the proline rich regions. The biological function of the gene 33 protein products cannot be inferred from the amino acid sequence and needs to be elucidated experimentally. G33 cDNAs were re- isolated by two different research groups that study genes whose expression increases independent of de novo protein synthesis during the transition from quiescence to proliferation. Such genes, known as immediate-early genes, are postulated to play regulatory roles in the growth response. G33 sequences were cloned from regenerating rat liver cDNA library and from serum-stimulated NIH 3T3 cells, and was shown to function as an immediate early gene in regenerating liver and in mitogen treated H-35 and Balb/C 3T3 cells. These results suggest that g33 participates in the transition from quiescence to proliferation in many mitogen-treated cells in addition to its previously reported involvement in hormone responses. The widespread and perhaps ubiquitous tissue distribution of g33 suggests that this gene may participate in a number of different responses perhaps as a member of a common signal transduction pathway. The presence of possible post- transcriptional modification sites (such as PKC and cAMP dependent protein kinase sites) and protein-protein interaction domains such as the SH3 domain binding site give strength to these hypotheses and suggest experimental targets for future studies on g33 function. In order to analyze in depth the function and expression of this multihormonally- regulated gene, we will express g33 protein in three different expression vectors in order to prepare polyclonal antibodies for analysis of g33 expression at the protein level; express g33 protein as a green fluorescent protein fusion protein to study the localization of g33 protein within the cell in vivo and if it varies in response to known mRNAg33 regulators; and to determine whether g33 protein interacts with other cellular proteins through its SH3 domain binding site or other regions, and whether protein phosphorylation plays a role in those interactions. The proposed project will enable students to be trained in basic research techniques and to give them an experience in studying the effects of structure on function, which will serve as a catalyst for a future research career in biomedical sciences. The students will involve themselves in all aspects of scientific research.
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