Our studies on ERM phosphorylation bring together the two threads of this project: cortical organization and basophilic kinases. Several kinases have been reported to be ERM kinase but various lines of evidence indicate that major ERM kinases remain to be identified. We now have identified Lymphocyte-oriented kinase (LOK) as the dominant ERM kinase in resting lymphocytes. Two lines of evidence from our studies converged to make this identification. First, we identified LOK by mass spectrometry which indicated it was the most abundant Ser/Thr kinase in a membrane microvillus-enriched fraction we prepared from lymphocytes. Second, our detailed analysis of protein and peptide specificity of LOK demonstrated that it efficiently phosphorylates the key site in ERM proteins, and does so more efficiently than other previously claimed ERM kinases. Of singular importance, we have obtained LOK-knockout mice and find that ERM phosphorylation in lymphocytes is markedly decreased in knockout mice. This is notable, since it is the first knockout in mammals which confirms the identity of a predicted ERM kinase. Our ongoing investigations of the immune system of these mice indicates defects in polarization and migration of lymphocytes in a manner explained by the alteration in ERM phosphorylation. Our studies of phosphorylation biology mediated by basophilic lymphocyte Ser/Thr kinases has progressed nicely in several respects. We have made major progress in the characterization of two phosphorylation sites on key lymphocyte proteins: SHP-1 S591 and WIP S489. Both of these sites are rich in basic residues and are highly favorable sites for basophilic phosphorylation based on our predictive algorithms. We have phospho-specific antibodies against both sites which facilitate our capacity to productively investigate them (in one case a rabbit polyclonal and in the other case a mouse monoclonal made by collaboration with an Industrial partner). Both sites were inducibly phosphorylated rapidly after T-cell receptor stimulation. SHP-1 S591 phosphorylation was transient and had several important functional effects: it markedly reduced SHP-1 phosphatase activity and altered SHP-1 localization. Of particular note was elegant temporal regulation of SHP-1 by sequential phosphorylation of S591 (which decreases phosphatase activity) was followed by Y536 phosphorylation (previously shown to increase phosphatase activity). Also notable was that our studies did NOT confirm other reports that PKC mediates this phosphorylation and suggest involvement of other basophilic kinases (highlighting the importance of elucidation of other basophilic kinases involved in T-cell activation). Regarding WIP S488 phosphorylation, which is much more sustained after T-cell activation, our studies contradict a previous influential publication indicating that S488 phosphorylation releases WIP from WASP. Instead our studies show in three ways that the WIP/WASP complex mediates TCR-induced NFAT-activation without dissociation. Moreover, we find that the N-terminus of WIP is a highly inhibitory region for TCR-mediated transcriptional activation in which at least two elements contribute: the N-terminal polyproline and the N-terminal actin-bindingWH2 domain, suggesting that WIP, like WASP is subject to auto-inhibition.
Ongoing studies are characterizing the localization and function of additional proteins identified by the proteomic (mass spectrometric) analysis of the membrane microvillus-enriched fraction of lymphocytes. In-vitro/in-cell approaches for analysis include: studies of localization (by staining and transfection); assessing molecular interactions (by yeast-two hybrid and immunoprecipitation), perturbation of function (by dominant negative constructs and shRNA). In vivo approaches emphasize knockout mice.
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