The long-term goal of our research has been to understand how cytoplasmic protein-tyrosine kinases regulate the growth properties of immune cells in response to extracellular stimuli. Our current focus is on the tyrosine kinase, Syk, which was discovered as part of this work. Through its substrates and binding partners, Syk couples the B cell receptor for antigen (BCR) to multiple intracellular signaling pathways resulting in one of several possible physiological outcomes that can vary from proliferation to apoptosis depending on the repertoire of downstream signals that are sent. Syk has functions not easily explained by its known activities and its known substrates at the site of the clustered BCR complex including an ability to suppress cellular responses to apoptotic stimuli. This function has made Syk an attractive target for anti-cancer therapeutics. The mechanism by which Syk dissociates from the BCR complex and is subsequently targeted to other cellular docking sites will be investigated as will the nature of these alternative binding proteins. The putative substrates that Syk phosphorylates as a consequence of its re-localization, as identified by sophisticated mass spectrometric analyses, will be characterized and the consequences of their phosphorylation analyzed to investigate new pathways that are regulated directly by Syk. These studies will be extended to primary B cells using cells derived from a new mouse model in which the activity of Syk can be unambiguously inhibited using a powerful chemical genetics approach. We plan to accomplish three specific aims: 1) to characterize the role of tyrosine-130 phosphorylation in the modulation of Syk-receptor and Syk-protein interactions, 2) to characterize novel Syk substrates and signaling pathways identified through proteomic and phosphoproteomic screens, and 3) to characterize the role of Syk in signaling in primary B cells through chemical genetics. Methodologies to be employed include 1) biochemical and physical evaluations of protein-peptide and protein- protein interactions, 2) proteomic and phosphoproteomic analyses of interacting proteins and kinase substrates, 3) molecular and cellular studies on protein function and 4) the use of chemical inhibitors to block kinase function in cells derived from genetically manipulated mice expressing an engineered kinase.

Public Health Relevance

This project focuses on an enzyme known as Syk that is absolutely required for the B lymphocyte components of the immune system to recognize and respond to infectious pathogens. Its activation can stimulate the responding B lymphocytes to expand in numbers and develop the ability to make antibodies to ward off the infection. Since, this ability of Syk to trigger a proliferative response can also provide a way for leukemia and lymphoma cells to proliferate, an understanding of Syk's functions is critical for identifying suitable therapeutic agents and drug targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI098132-29A1
Application #
8372345
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Ferguson, Stacy E
Project Start
2012-05-01
Project End
2017-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
29
Fiscal Year
2012
Total Cost
$335,249
Indirect Cost
$110,249
Name
Purdue University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Krisenko, Mariya O; Cartagena, Alexander; Raman, Arvind et al. (2015) Nanomechanical property maps of breast cancer cells as determined by multiharmonic atomic force microscopy reveal Syk-dependent changes in microtubule stability mediated by MAP1B. Biochemistry 54:60-8
Jayasundera, Keerthi B; Iliuk, Anton B; Nguyen, Andrew et al. (2014) Global phosphoproteomics of activated B cells using complementary metal ion functionalized soluble nanopolymers. Anal Chem 86:6363-71
Geahlen, Robert L (2014) Getting Syk: spleen tyrosine kinase as a therapeutic target. Trends Pharmacol Sci 35:414-22
Searleman, Adam C; Iliuk, Anton B; Collier, Timothy S et al. (2014) Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2 positive breast cancer. Electrophoresis 35:3463-9
Wang, Wen-Horng; Childress, Michael O; Geahlen, Robert L (2014) Syk interacts with and phosphorylates nucleolin to stabilize Bcl-x(L) mRNA and promote cell survival. Mol Cell Biol 34:3788-99
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
Hu, Lianghai; Yang, Li; Lipchik, Andrew M et al. (2013) A quantitative proteomics-based competition binding assay to characterize pITAM-protein interactions. Anal Chem 85:5071-7
Huang, Rong; Oh, Hyunju; Arrendale, Allison et al. (2013) Intracellular targets for a phosphotyrosine peptidomimetic include the mitotic kinesin, MCAK. Biochem Pharmacol 86:597-611
Yu, Shuai; Huang, He; Iliuk, Anton et al. (2013) Syk inhibits the activity of protein kinase A by phosphorylating tyrosine 330 of the catalytic subunit. J Biol Chem 288:10870-81