Our studies of protein kinase specificity have given rise to important general principles. Our studies highlight contributions of disfavored residues in peptide specificity. Notably, we find that Pro at the P+1 position functions as a """"""""veto"""""""" residue in substrate recognition by AGC and CAMK kinases; this assures a very high degree of reciprocal specificity between basophilic kinases and Pro-directed kinases. Knowing the structural basis for kinase function is important for immunology and cancer biology. Therefore, we analyzed the structural basis for patterns of Arg preference (P-2 or P-5) amongst 15 AGC, CAMK and STE kinases. We found they are due to a single pair of acidic residues in the catalytic cleft and that PAK kinases are unique in their dominance of that P-2 Arg preference. Moreover, our computational and mutational studies provide a structural explanation for PKC-delta's unusual independence of activation loop phosphorylation, and reveal unexpected product inhibition and restrictions in function in vivo for the non-phosphorylated PKC-delta. We are now extending these analyses to an even broader range of poorly characterized Ser/Thr kinases and elucidating new specificity patterns. A prior report implicated WIP phosphorylation by PKC-theta as a key event in TCR signaling. We have independently tested this concept and conclude that WIP phosphorylation is unlikely to be a key mechanism of action of PKC-theta. We are making major progress in characterizing the ERM phosphatase as PP1c by a combination of membrane fraction purification, mass spectrometry, Western blot analysis, pulldowns, and in vitro reconstituted systems using recombinant purified proteins. The dephosphorylation is regulated in multiple ways by biologically relevant parameters. We are also identified a major ERM kinase in lymphocytes by a combination of mass spectrometry, detailed analysis of peptide specificity, in vitro protein phosphorylation analysis, and transfection analysis. That ERM kinase is previously poorly-studied kinase. We are investigating the functional impact of its absence in a relevant knockout mouse. We have succeeded in purifying lymphocyte microvilli and identifying many component proteins by mass spectrometry (MS). Amongst the myriad of proteins identified, we have chosen several proteins to characterize extensively for their functional involvement in regulating the unique normal architecture of peripheral blood T-cells. Those proteins are being studied by approaches such as yeast two-hybrid analysis, immunofluorescence analysis of localization of the endogenous proteins, fluorescence microscopic analysis of localization of GFP tagged constructs (and mutants thereof) expressed in hematopoietic cells, and siRNA/RNAi alteration of expression. For one of the genes that is especially promising, we are generating a knockout mouse.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC009257-31
Application #
7338139
Study Section
(EIB)
Project Start
Project End
Budget Start
Budget End
Support Year
31
Fiscal Year
2006
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Hao, Jian-Jiang; Wang, Guanghui; Pisitkun, Trairak et al. (2008) Enrichment of distinct microfilament-associated and GTP-binding-proteins in membrane/microvilli fractions from lymphoid cells. J Proteome Res 7:2911-27
Dong, Xiaoyun; Patino-Lopez, Genaro; Candotti, Fabio et al. (2007) Structure-function analysis of the WIP role in T cell receptor-stimulated NFAT activation: evidence that WIP-WASP dissociation is not required and that the WIP NH2 terminus is inhibitory. J Biol Chem 282:30303-10
Liu, Yin; Kruhlak, Michael J; Hao, Jian-Jiang et al. (2007) Rapid T cell receptor-mediated SHP-1 S591 phosphorylation regulates SHP-1 cellular localization and phosphatase activity. J Leukoc Biol 82:742-51
Liu, Yin; Belkina, Natalya V; Graham, Caroline et al. (2006) Independence of protein kinase C-delta activity from activation loop phosphorylation: structural basis and altered functions in cells. J Biol Chem 281:12102-11
Zhu, Guozhi; Fujii, Koichi; Liu, Yin et al. (2005) A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families. J Biol Chem 280:36372-9
Sarkar, Kakali; Kruhlak, Michael J; Erlandsen, Stanley L et al. (2005) Selective inhibition by rottlerin of macropinocytosis in monocyte-derived dendritic cells. Immunology 116:513-24
Zhu, Guozhi; Fujii, Koichi; Belkina, Natalya et al. (2005) Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases. J Biol Chem 280:10743-8
Zhu, Guozhi; Liu, Yin; Shaw, Stephen (2005) Protein kinase specificity. A strategic collaboration between kinase peptide specificity and substrate recruitment. Cell Cycle 4:52-6
Anderson, Arthur O; Shaw, Stephen (2005) Conduit for privileged communications in the lymph node. Immunity 22:3-5
Salganik, M P; Milford, E L; Hardie, D L et al. (2005) Classifying antibodies using flow cytometry data: class prediction and class discovery. Biom J 47:740-54

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