Immunophilins control T lymphocyte adhesion and migration by regulating C3G binding to CrkII. Crk adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII, the two alternative-spliced forms of CRK, possess an N-terminal SH2 domain, followed by an SH3 domain, while CrkII possesses in addition a C-terminal linker region plus an SH3 domain, which operate as regulatory moieties. Here, we investigated the ability of immunophilins, that function as peptidyl-prolyl isomerases (PPIases), to regulate Crk proteins in human T lymphocytes. We found that endogenous CrkII, but not CrkI, associates with the immunophilins, cyclophilin A (CypA) and FKBP12, in resting human Jurkat T cells. In addition, CypA increased C3G binding to CrkII, while inhibitors of immunophilins, such as cyclosporine A (CsA) and FK506, inhibited CrkII, but not CrkI association with C3G. Expression in Jurkat T cells of PICCHUx, a plasmid encoding the human CrkII1-236 sandwiched between CFP and YFP, demonstrated a basal level of FRET, which increased in response to cell treatment with CsA and FK506, reflecting increased trans-to-cis conversion of CrkII. Crk-C3G complexes are known to play an important role in integrin-mediated cell adhesion and migration. We found that overexpression of CrkI or CrkII increased adhesion and migration of Jurkat T cells. However, immunophilin inhibitors suppressed the ability of CrkII-, but not CrkI-overexpressing cells to adhere to fibronectin-coated surfaces and migrate towards the SDF1? chemokine. The present data demonstrate that immunophilins regulate CrkII, but not CrkI activity in T cells and suggest that CsA and FK506 inhibit selected effector T cell functions via a CrkII-dependent mechanism.EndFragment
A novel motif in the V3 domain of PKC-theta determines its immunological synapse localization and functions in T cells via association with CD28. Protein kinase C-theta (PKC-theta) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. We found that the PKC-theta V3 domain is necessary and sufficient for IS localization mediated by Lck-dependent association with CD28. We identified a conserved proline-rich motif in V3 required for CD28 association and IS localization. CD28 association was essential for PKC-theta-mediated downstream signaling and TH2 and TH17, but not TH1, differentiation. Ectopic V3 expression sequestered PKC-theta from the IS and interfered with its functions. These results identify a unique mode of CD28 signaling, establish a molecular basis for the IS localization of PKC-theta, and implicate V3-based “decoys” as therapeutic modalities for T cell-mediated inflammatory diseases.
Development of unique antibodies directed against each of the six different phosphotyrosine residues within the T cell receptor CD3zeta chain. Signal transduction from the T cell antigen receptor (TCR)/CD3 complex involves six different immunoreceptor tyrosine-based activation motifs (ITAM) located within the cytoplasmic tails of the CD3 chains. Each ITAM possesses two conserved tyrosine residues that can undergo phosphorylation upon TCR/CD3 crosslinking and become a docking site for SH2-containing effector molecules. Specificity of the SH2 domains is determined by their ability to bind a phosphorylated tyrosine in the context of a longer peptide motif within the target protein. As a result, phosphorylation of different tyrosines within the CD3 cytoplasmic tails creates docking sites for distinct SH2-containing signaling proteins that differentially impact on the quality of the T cell response. In the present study, we prepared antibodies specific for each of the six different phosphotyrosines of the mouse CD3zeta chain. The antibodies were characterized with respect to their cross-reactivity, ability to recognize the phosphorylated versus non-phosphorylated forms of tyrosine-containing motifs, and cross-reactivity with the homologous phospho-motifs on the human CD3zeta protein. The antibodies were found to be specific and selective for phospho-CD3zeta. They can serve as useful tools for distinguishing between the six potential tyrosine phosphorylation sites on the CD3zeta chain, and for correlating the phosphorylation of specific CD3zeta tyrosine residues with activation of signaling pathways that dictate T cell differentiation into responding, anergic, or apoptotic cells.
Hodgkin's lymphoma cells exhibit high expression levels of the PICOT protein. PICOT was originally discovered as a protein kinase C (PKC) binding protein in human Jurkat T-lymphocytes in which it was found to modulate PKCtheta-dependent functions. In addition, RT-PCR analysis suggested the expression of PICOT in a wide range of organs and cell types, including cells that are devoid of PKCtheta. We aimed at analyzing the expression of the PICOT protein in mouse lymphoid organs, and to compare them with those of Jurkat T-lymphocytes and other cell lines. We also analyzed whether PICOT expression in T-lymphocytes is dependent on the presence of PKCtheta, and whether it correlates with cell growth rate. Western blot analyses demonstrated PICOT expression in all lymphoid organs and cell lines tested. In addition, similar expression levels were observed in lymphoid organs of wild-type and PKCtheta-null mice, suggesting that PICOT expression in T-lymphocytes is independent of PKCtheta. However, PICOT expression levels were higher in Jurkat T-lymphocytes and other lymphoma cell lines compared to freshly isolated lymphocytes, while T-lymphocyte mitogens, such as concanavalin A, increased PICOT expression concomitantly with the induction of a faster T-lymphocyte growth rate. Finally, immunohistochemistry of freshly-isolated lymph nodes from Hodgkin's lymphoma patients revealed significantly higher levels of PICOT in Hodgkin's cells, compared to the normal surrounding lymphocytes. The present results show a direct correlation between PICOT expression levels and increased cell growth, both in vitro and in vivo, and suggest that immunostaining of PICOT might be useful for in situ identification of transformed cells, such as those of Hodgkin's lymphoma.