Protein-tyrosine kinases regulate the growth properties of cells in response to extracellular mitogens. The long-term objective of this project has been to understand, on a molecular basis, how cytoplasmic protein-tyrosine kinases function to couple receptors that lack enzymatic activity to the activation of intracellular pathways that lead to cell proliferation or differentiation. To accomplish this goal, we have focused our studies on the tyrosine kinases involved in the activation of B lymphocytes through the engagement of cell surface antigen receptors. Our major focus is on the tyrosine kinase, Syk, which is a required component of the signaling machinery in B cells. We hypothesize that the covalent modification of Syk on amino acid residues located with the linker region that separates the N-terminal SH2 domains from the C-terminal catalytic domain regulate multiple aspects of Syk's function in B cells. Preliminary studies indicate that these covalent modifications of both serine and tyrosine residues regulate the ability of Syk to couple the BCR to different intracellular pathways, the export of Syk from the nucleus, and the ability of Syk to function in general following receptor cross-linking. Consequently, we hypothesize that the effects of inhibitory or stimulatory co- receptors that regulate B cell responsiveness arise, in part, through alterations in the state of phosphorylation of the Syk linker region. To explore these hypotheses, we plan to accomplish three specific aims: 1) to investigate the role of the tyrosine-phosphorylation of Syk in the physiological outcome of BCR-stimulated signaling in B lymphocytes, 2) to investigate the role of the serine-phosphorylation of Syk in the regulation of Syk function and nuclear export and 3) to characterize proteins that interact with Syk as defined by yeast two-hybrid analyses. Methodologies to be used include 1) generation of transgenic mice, 2) structure determination of protein-protein interactions by high resolution NMR, 3) analyses of protein phosphorylation and signal transduction in cellular model systems, and 4) biochemical and genetic measurements of protein-protein interactions.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA037372-19
Application #
6450502
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Mccarthy, Susan A
Project Start
1984-03-15
Project End
2007-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
19
Fiscal Year
2002
Total Cost
$249,375
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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