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.
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