In response to signal initiation at a number of different extracellular receptors, phospholipase C (PLC) hydrolyzes phosphatidyl inositol (4,5)bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). In turn, IP3 and DAG control calcium flux, PKC activation and activation of the Ras-Raf-MEK-ERK pathway. In spite of the importance of the PLC enzymes, there is a significant gap in our knowledge regarding the molecular determinants that control PLC function. The two aims in this revsed application focus on the regulation of a single isoform of the phospholipases, PLC?1, in the context of signaling downstream of the T cell receptor. The PLC?1 isoform contains a number of Src homolgy domains that have been implicated in regulation of PLC?1 activity. The precise mechanism of regulation is not known. In our preliminary data we present a number of new findings that provide exciting insights into how the Src homolgy 2 (SH2) domains of PLC?1 function to control phospholipase activity. Following T cell receptor stimulation, PLC?1 is phosphorylated by the Tec family kinase Itk. We describe an unprecedented substrate docking mechanism that involves a direct interaction between the kinase domain of Itk and the carboxy-terminal SH2 domain of PLC?1. This protein-protein interaction is required for phosphorylation of Y783 in PLC?1 by Itk. Quite interesting is the fact that the interaction occurs in a phosphotyrosine-independent fashion;not the expected result for an SH2 mediated binding event. Further preliminary data show another noncanonical SH2 interaction whithin PLC?1 that inhibits the substrate docking interaction with Itk. The structural details of these regulatory complexes will be elucidated during the course of the proposed work and the results transferred into functional assays to develop a molecular level understanding of PLC?1 function. Given the non-canonical nature of the observed SH2 mediated interactions we expect that completion of the proposed aims will be particularly useful in properly dissecting the role of PLC?1 in T cell signaling. To date, biochemical and cell biological probes into the role of the SH2 domains in controlling PLC?1 mediated signaling have been limited to traditional 'loss-of-funtion'mutations that only disrupt canonical phosphotyrosine recognition.
This proposal aims to understand specific molecular events leading to activation of the immune response. The molecules that will be studied are prime therapeutic targets for modulating an immune response in the context of disease. Thus, the public health relevance of the project relates to developing new ways to either limit or enhance the immune response in the face of autoimmunity, immunosuppression or immunological diseases.
|Joseph, Raji E; Kleino, Iivari; Wales, Thomas E et al. (2013) Activation loop dynamics determine the different catalytic efficiencies of B cell- and T cell-specific tec kinases. Sci Signal 6:ra76|
|Xie, Qian; Joseph, Raji E; Fulton, D Bruce et al. (2013) Substrate recognition of PLC?1 via a specific docking surface on Itk. J Mol Biol 425:683-96|
|Joseph, Raji E; Ginder, Nathaniel D; Hoy, Julie A et al. (2012) Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:145-53|
|Joseph, Raji E; Ginder, Nathaniel D; Hoy, Julie A et al. (2011) Purification, crystallization and preliminary crystallographic analysis of the SH2 domain of IL-2-inducible T-cell kinase. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:269-73|
|Min, Lie; Wu, Wenfang; Joseph, Raji E et al. (2010) Disrupting the intermolecular self-association of Itk enhances T cell signaling. J Immunol 184:4228-35|
|Joseph, Raji E; Xie, Qian; Andreotti, Amy H (2010) Identification of an allosteric signaling network within Tec family kinases. J Mol Biol 403:231-42|
|Andreotti, Amy H; Schwartzberg, Pamela L; Joseph, Raji E et al. (2010) T-cell signaling regulated by the Tec family kinase, Itk. Cold Spring Harb Perspect Biol 2:a002287|
|Min, Lie; Joseph, Raji E; Fulton, D Bruce et al. (2009) Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLCgamma1. Proc Natl Acad Sci U S A 106:21143-8|
|Joseph, Raji E; Andreotti, Amy H (2009) Conformational snapshots of Tec kinases during signaling. Immunol Rev 228:74-92|