Carrizosa, Esteban

Abstract Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We have shown that the actin regulatory protein HS1 is required for the stabilization of F-actin and Vav1 at the immunological synapse (IS) and for efficient Ca2+ responses. The Tec family kinase Itk also regulates actin responses, in part by facilitating the localization of Vav1 at the IS, suggesting that these proteins act in the same pathway. Using video microscopy, we found that T cells lacking Itk or HS1 behaved similarly, extending unstable actin-rich protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk’s SH2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, HS1 failed to localize to the IS. Since Itk is required for PLCγ1 phosphorylation and Ca2+ store release, we examined the Ca2+ signaling pathway in HS1 knockout T cells in greater detail. T cells lacking HS1 exhibited abnormal Ca2+ store release. These cells showed normal phosphorylation but altered localization of PLCγ1. We conclude that Itk-dependent recruitment of HS1 is required for actin stabilization at the IS, and directs the localization of PLCγ1. Supported by NIH F31-AI071385 to EC and NIH R01-A1065644 to JKB

Abstract Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk’s Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)γ1 phosphorylation and calcium store release, we examined the calcium signaling pathway in HS1−/− T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished calcium store release, but TCR-dependent PLCγ1 phosphorylation was intact, indicating that HS1’s role in calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLCγ1 and altered formation of PLCγ1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCγ1 signaling complexes.