Hematopoietic Lineage Cell-Specific Protein 1 Is Recruited to the Immunological Synapse by IL-2-Inducible T Cell Kinase and Regulates Phospholipase Cγ1 Microcluster Dynamics during T Cell Spreading1
| Autor: | Carrizosa, Esteban, Gomez, Timothy S., Labno, Christine M., Klos Dehring, Deborah A., Liu, Xiaohong, Freedman, Bruce D., Billadeau, Daniel D., Burkhardt, Janis K. |
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| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
Mice
Knockout Immunological Synapses Phospholipase C gamma T-Lymphocytes Blotting Western Receptors Antigen T-Cell Mice Transgenic Protein-Tyrosine Kinases Lymphocyte Activation Transfection Article Actins Mice Inbred C57BL Jurkat Cells Mice Microscopy Fluorescence Granulocyte Colony-Stimulating Factor Animals Humans Immunoprecipitation RNA Interference Calcium Signaling Pseudopodia Phosphorylation |
| Popis: | 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)gamma1 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 PLCgamma1 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 PLCgamma1 and altered formation of PLCgamma1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCgamma1 signaling complexes. |
| Databáze: | OpenAIRE |
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