Protein-tyrosine kinases regulate the growth properties of cells in response to extracellular mitogens. The long-term objective of this study has been to understand how cytoplasmic protein-tyrosine kinases regulate the responses of cells to extracellular promoters of cell growth using lymphocyte activation as a model system. These studies are important for understanding the role of protein-tyrosine kinases in human malignancies and in diseases of the immune system. In many hematopoietic cells, the actions of activating antigens are mediated by a cytoplasmic protein-tyrosine kinase known as Syk (p72-syk) that is functionally and physically coupled to antigen receptors. How Syk, once activated by receptor ligation, interacts with the components of the downstream biochemical pathways that are stimulated by receptor ligation is the question that will be pursed in this proposal.
Three specific aims are to be investigated: (1) to determine the role of phosphorylation on the interactions of Syk with downstream signaling molecules in B cells; (2) to characterize the interactions of Syk with tubulin and to explore the effects of the Syk-catalyzed phosphorylation of tubulin on its participation in protein/protein interactions; and (3) to identify by genetic screens novel Syk interacting proteins. Methodologies to be used in achieving these objectives include (1) analysis of sites of protein phosphorylation, mutagenesis of tyrosine phosphorylation sites and reconstitution of signal transduction in Syk knockout cells, (2) analysis of the effects of phosphorylation on tubulin assembly, (3) structure determination by NMR of protein/protein interaction sties and (4) cloning of cDNAs for Syk-interacting proteins by expression screening and yeast two-hybrid screening. An analysis of the role of Syk in B cell receptor signaling will serve as a general model for understanding receptor-mediated signaling pathways in a variety of cell types.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA037372-15
Application #
2653991
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Mccarthy, Susan A
Project Start
1984-03-15
Project End
2002-01-31
Budget Start
1998-02-01
Budget End
1999-01-31
Support Year
15
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Purdue University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Fei, Bei; Yu, Shuai; Geahlen, Robert L (2013) Modulation by Syk of Bcl-2, calcium and the calpain-calpastatin proteolytic system in human breast cancer cells. Biochim Biophys Acta 1833:2153-64
Xue, Liang; Geahlen, Robert L; Tao, W Andy (2013) Identification of direct tyrosine kinase substrates based on protein kinase assay-linked phosphoproteomics. Mol Cell Proteomics 12:2969-80
Lipchik, Andrew M; Killins, Renee L; Geahlen, Robert L et al. (2012) A peptide-based biosensor assay to detect intracellular Syk kinase activation and inhibition. Biochemistry 51:7515-24
Arrendale, Allison; Kim, Keunho; Choi, Jun Young et al. (2012) Synthesis of a phosphoserine mimetic prodrug with potent 14-3-3 protein inhibitory activity. Chem Biol 19:764-71
Moon, Kyung D; Zhang, Xiaoying; Zhou, Qing et al. (2012) The protein-tyrosine kinase Syk interacts with the C-terminal region of tensin2. Biochim Biophys Acta 1823:199-205
Xue, Liang; Wang, Wen-Horng; Iliuk, Anton et al. (2012) Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates. Proc Natl Acad Sci U S A 109:5615-20
Martin, Victoria A; Wang, Wen-Horng; Lipchik, Andrew M et al. (2012) Akt2 inhibits the activation of NFAT in lymphocytes by modulating calcium release from intracellular stores. Cell Signal 24:1064-73
Galan, Jacob A; Paris, Leela L; Zhang, Hua-jie et al. (2011) Proteomic studies of Syk-interacting proteins using a novel amine-specific isotope tag and GFP nanotrap. J Am Soc Mass Spectrom 22:319-28
Chen, Chih-Hong; Martin, Victoria A; Gorenstein, Nina M et al. (2011) Two closely spaced tyrosines regulate NFAT signaling in B cells via Syk association with Vav. Mol Cell Biol 31:2984-96
Paris, Leela L; Hu, Jianjie; Galan, Jacob et al. (2010) Regulation of Syk by phosphorylation on serine in the linker insert. J Biol Chem 285:39844-54

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