[Show abstract][Hide abstract] ABSTRACT: The actin cytoskeleton has an important role in the organization and function of the immune synapse during antigen recognition. Dynamic rearrangement of the actin cytoskeleton in response to T cell receptor (TCR) triggering requires the coordinated activation of Rho family GTPases that cycle between active and inactive conformations. This is controlled by GTPase-activating proteins (GAP), which regulate inactivation of Rho GTPases, and guanine exchange factors, which mediate their activation. Whereas much attention has centered on guanine exchange factors for Rho GTPases in T cell activation, the identity and functional roles of the GAP in this process are largely unknown. We previously reported beta2-chimaerin as a diacylglycerol-regulated Rac-GAP that is expressed in T cells. We now demonstrate Lck-dependent phosphorylation of beta2-chimaerin in response to TCR triggering. We identify Tyr-153 as the Lck-dependent phosphorylation residue and show that its phosphorylation negatively regulates membrane stabilization of beta2-chimaerin, decreasing its GAP activity to Rac. This study establishes the existence of TCR-dependent regulation of beta2-chimaerin and identifies a novel mechanism for its inactivation.
No preview · Article · Mar 2009 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The small GTPase Rac contributes to regulation of cytoskeletal rearrangement during chemokine-induced lymphocyte adhesion and migration in a multi-step process that is very precisely coordinated. Chimaerins are Rac1-specific GTPase-activating proteins of unknown biological function, which have a canonical diacylglycerol C1-binding domain. Here we demonstrate endogenous expression of beta2-chimaerin in T lymphocytes and study the functional role of this protein in phorbol ester and chemokine (CXCL12)-regulated T-cell responses. We used green fluorescent protein-tagged beta2-chimaerin and phorbol ester stimulation to investigate changes in protein localization in living lymphocytes. Our results demonstrate that active Rac cooperates with C1-dependent phorbol ester binding to induce sustained GFP-beta2-chimaerin localization to the membrane. Subcellular distribution of GFP beta2-chimaerin in living cells showed no major changes following CXCL12 stimulation. Nonetheless Rac1-GTP levels were severely inhibited in GFP-beta2-chimaerin-expressing cells, which displayed reduced CXCL12-induced integrin-dependent adhesion and spreading. This effect was dependent on chimaerin GTPase-activating protein function and required diacylglycerol generation. Whereas beta2-chimaerin overexpression decreased static adhesion, it enhanced CXCL12-dependent migration via receptor-dependent diacylglycerol production. These studies demonstrate that beta2-chimaerin provides a novel, diacylglycerol-dependent mechanism for Rac regulation in T cells and suggest a functional role for this protein in Rac-mediated cytoskeletal remodeling.
No preview · Article · Feb 2006 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Protein kinase C (PKC) is the only PKC isoform recruited to the immunological synapse after T cell receptor stimulation, suggesting that its activation mechanism differs from that of the other isoforms. Previous studies have suggested that this selective PKC recruitment may operate via a Vav-regulated, cytoskeletal-dependent mechanism, independent of the classical phospholipase C/diacylglycerol pathway. Here, we demonstrate that, together with tyrosine phosphorylation of PKC in the regulatory domain, binding of phospholipase C-dependent diacylglycerol is required for PKC recruitment to the T cell synapse. In addition, we demonstrate that diacylglycerol kinase alpha-dependent diacylglycerol phosphorylation provides the negative signal required for PKC inactivation, ensuring fine control of the T cell activation response.