Recognizing Us versus Them
My doctoral studies consisted of three independent projects that produced portions of seven peer-reviewed papers, three of which were first authorship, while four were co-authorships. The first project consisted of the regulation of actin polymerization in T cells by a kinase in a kinase domain-independent mechanism. The second project consisted of a collaboration with Dr. Karsten Sauer at the Scripps Research Institute, elucidating the localization of proteins affected by the secondary messenger IP4. The third project included designing a cell-permeable inhibitor of protein-protein interaction that affects specific cytokine expression in T cells.
The Tec family kinase, Itk, is critical for both the activation and the development of T lymphocytes. Upon mutational analysis of Itk function, it was discovered that Src Homology 2 (SH2) domain mutants of Itk rendered the enzyme inactive. Further, this mutation inhibits the localization of Itk to a T cell receptor (TCR) immunological synapse, and regulates TCR-induced actin-dependent cytoskeletal events. In extension, Itk-/- murine T cells display significant defects in TCR-induced actin polymerization. Additionally, Jurkat T cells deficient in the expression of Linker for Activated T cells (LAT), an adaptor protein critical for Itk activation, display impaired cytoskeletal events, while the expression of Src Homology 3 (SH3) domain mutant Itk transgene reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat T cells has no effect on cytoskeletal events. The molecular mechanism for this effect on the T lymphocyte was then explored and it was found that Itk forms a multimeric protein complex with the adaptor protein SH2 containing Leukocyte Protein of 76 kilodaltons (SLP-76), the actin polymerization organizing molecule Wiskott-Aldrich Syndrome protein (WASp), and the GTPase Vav1. Itk interacts with this complex in an SH2 and SH3 domain cooperative mechanism. Itk’s presence allows the complex to form, its absence causes the complex to fall apart. Collectively, these data suggest that Itk regulates TCR-induced actin-dependent cytoskeletal events, most likely in a kinase independent manner. This work was primarily published in a first author paper in the Journal of Immunology and in portions of other second author papers.
Production of the secondary messengers inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG) by Phospholipase C-gamma 1 (PLCg1) plays a paramount role in signaling from the T cell receptor. The phosphorylation of IP3 to inositol (1,3,4,5)-tetrakisphosphate (IP4) by inositol (1,4,5)-trisphosphate 3-kinase B (ItpkB) generates another inositol-polyphosphate with a novel signaling function. In vitro studies have shown that IP4 acts as a soluble ligand for the pleckstrin homology (PH) domain of the Tec kinase Itk. Molecular and cellular studies shows that ItpkB regulates Itk recruitment, thereby enabling Itk’s full activation, including targeting PLCg1 phosphorylation and subsequent downstream effectors, including the activation of Erk. This regulation affects the positive selection of a developing T cell. This work was published in the journal Science.
In a follow-up series of experiments to address the association between Itk and SLP-76 upon TCR engagement, a polyarginine-modified peptide (R9-QQP) representing a portion of the SLP-76 polyproline-rich region was generated as a competitive inhibitor aimed to disrupt the inducible interaction between Itk and SLP-76 in live cells. R9-QQP was readily taken up by cells as demonstrated by flow cytometric analysis and confocal microscopy of cells incubated with FITC-conjugated peptide. R9-QQP inhibits TCR-induced association of Itk and SLP-76, transphosphorylation of Itk on tyrosine 511, and expression of TH2 cytokines in a dose-dependent manner. The inhibition mediated by R9-QQP is specific in comparison to the non-inhibitory effects by control peptides that represent either the polyarginine vehicle alone (R9) or a scrambled sequence of the polyarginine tagged cargo peptide (R9-SCR). Further evidence of specificity is the lack of inhibition by R9-QQP in the association between SLP-76 and other SLP-76 binding proteins. The effects of R9-QQP on Itk activation and cytokine production are also demonstrated upon intraperitoneal administration of the peptide in vivo. In view of observations implicating Itk as a regulator of TH2 cytokines, the data underscores the significance of Itk as a target for pharmacologic intervention. This work was published in the journal Molecular and Cellular Biology.