We seek to understand the molecular basis of early activation events in T-lymphocytes in response to physiologically important stimuli; the functional responses most important to our research are T-lymphocyte cytoskeletal reorganization, adhesion, migration, and gene transcription. One major project is to understand the spatial reorganization during chemokine-induced polarization of peripheral blood T cells (PBT) and to elucidate the biochemical basis for that reorganization using a combination of microscopic, biochemical and molecular genetic approaches. We discovered a fundamental signaling node in chemokine-induced cytoskeletal remodeling in PBT, namely dephosphorylation of a conserved C-terminal Thr in the ERM family of cytoskeletal-linking proteins. Its significance is shown by our findings: 1) its rapidity, faster than previously known chemokine-induced posttranslational modifications; 2) ERM abundance in microvilli coupled with their potent functional inactivation by dephosphorylation; 3) the defects in microvillus collapse and polarization when ERM dephosphorylation is disabled. We are therefore characterizing the molecular steps in the pathway from chemokine signaling to ERM dephosphorylation. We identified Rac1 activation as an essential upstream mediator of ERM dephosphorylation. As required for this role, Rac1 activation is very rapid (5 sec). We find Rac1 activation is pivotal to the chemokine response overall since its inhibition blocks all other downstream chemokine-induced events we measure. Furthermore, our evidence implicates PI-PLC-mediated DAG production downstream of Rac1 and upstream of a PP1 phosphatase.PBT microvilli are a functionally important organelle whose molecular organization needs elucidation because of its central role in initiating lymphocyte binding to endothelium. We are determining the components of lymphocyte microvilli, emphasizing their relationship to ERM proteins which are major organizers of microvilli. We have identified new partners for ERMs using a yeast 2 hybrid configuration we designed to retain optimal FERM domain function. We have succeeded in purifying lymphocyte microvilli and identifying many component proteins by mass spectrometry (MS). Those results facilitate our investigations of: 1) What partners localize ERM to microvilli? 2) What PP1 isoform and regulatory subunit mediates dephosphorylation? 3) What RacGEF mediates the rapid Rac1 activation? 4) What kinase phosphorylates ERMs in microvilli? Moreover, we are developing transgenic mice to rigorously test the relevance of ERM dephosphorylation in vivo.Protein phosphorylation is a central part of normal cell function as well as to carcinogenesis / metastasis. We have made major progress in characterizing fundamental processes that regulate kinase specificity. Peptide specificity and recruitment are two processes which collaborate to determine which sites on which proteins will be phosphorylated by a kinase. However, peptide specificity is not yet understood well enough to help greatly in predicting which kinases are most likely to be relevant for any particular basic phosphorylation site. Therefore, we have: 1) developed an improved approach to determine kinase peptide specificity; 2) validated that that we can now predict more accurately than previously possible which peptides PKC-delta and -zeta will phosphorylate; and 3) shown our predictions are also highly relevant to PKC's in vivo phosphorylation of proteins. 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.
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