title: Hematopoietic lineage cell-specific protein 1 is recruited to the immunological synapse by IL-2-inducible T cell kinase and regulates phospholipase Cgamma1 Microcluster dynamics during T cell spreading.
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)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.
authors.affiliation: Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Esteban
authors.initials: E
authors.lastname: Carrizosa
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Gomez
authors.forename: Christine M
authors.initials: CM
authors.lastname: Labno
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Klos Dehring
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Liu
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Freedman
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Billadeau
authors.forename: Janis K
authors.initials: JK
authors.lastname: Burkhardt
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D020013:Calcium Signaling / Q000276:immunology; D016179:Granulocyte Colony-Stimulating Factor / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D054992:Immunological Synapses / Q000276:immunology* / Q000378:metabolism; D047468:Immunoprecipitation; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D051966:Phospholipase C gamma / Q000276:immunology* / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014162:Transfection
chemical_list: D000199:Actins; D011948:Receptors, Antigen, T-Cell; C524015:hematopoietic lineage cell-specific protein 1, mouse; D016179:Granulocyte Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase; D051966:Phospholipase C gamma