The purpose of this research proposal is to determine the roles of protein-protein and protein-lipid interactions mediated by pleckstrin homology (PH) domains in signal transduction. Results of this study are expected to shed light on new aspects of mast cell physiology. The investigator's recent study identified protein kinase C (PKC) as a PH domain-binding protein and showed that PKC bound to Btk, a Tec family tyrosine kinase, phosphorylates Btk and inhibits its enzymatic activity. 1) In order to elucidate the mechanism for the Btk (and its relative Emt) regulation by PKC, (a) the PKC binding site on the PH domain of Btk and (b) the PH domain-binding site on PKC will be mapped. (c) Determination of the PKC phosphorylation site on Btk (and Emt) will be informative for understanding the Btk (and Emt) regulation by PKC. The investigator has evidence that PKC-beta1 in membranes is tyrosine-phosphorylated upon FceRI cross-linking and enzymatically regulated by Btk in vitro. Hence, (d) he will characterize biochemical and biologic effects of PKC-betaI tyrosine phosphorylation. In light of the recent findings that PH domains bind to phosphatidylinositol 4,5bisphosphate (PIP2) and that the binding site of this lipid on the PH domain overlaps that of the tentatively assigned PKC binding site, (e) effects of PIP2 on the PKC-PH domain binding will be analyzed. 2) The investigator's preliminary experiments have identified actin as another PH domain-binding protein. Since actin is the most important cytoskeletal protein in many physiologic aspects of the cell, PH domains will be characterized as an actin-binding module. 3) PKC plays key roles in various aspects of cellular signaling. The C-terminal portions of several PH domains including that of Btk bind to the beta/gamma complex of heterotrimeric G-proteins, complexes involved in many signal transduction systems. Therefore, some interesting possibilities are raised. One is that a single PH domain might interact with both PKC and G-protein beta/gamma and hence PKC might affect the activity of the G-protein beta/gamma or vice versa. Another possibility is that PKC and G-protein beta/gamma compete with each other for the PH domain of Btk. (a) These possibilities will be tested by transfection of heterologous cells with relevant expression vectors. (b) Similarly, interactions between Btk and actin will be characterized. (c) Since there are several PH domain-binding molecules, distinct roles of the individual binding molecules will be investigated using interaction-specific inhibitors or PH domain mutants of Btk that have lost the capacity to interact with one or a subset of the PH domain-binding molecules.
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