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1 Microcluster Dynamics during T Cell Spreading1
*Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, PA 19104;
Division of Oncology Research, Mayo Clinic, Rochester, MN;
Department of Pathology, University of Chicago Medical School, Chicago, IL; and
Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104
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)
1 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 PLC1 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 PLC1 and altered formation of PLC1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLC1 signaling complexes.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grant R01AI065644 to J.K.B., Grant F31AI071385 to E.C., and Grant R01AI065474 to D.D.B. D.D.B. is a Leukemia and Lymphoma Scholar.
2 Address correspondence and reprint requests to Dr. Janis K. Burkhardt, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, 816D Abramson Research Center, 3615 Civic Center Boulevard, Philadelphia, PA 19104. E-mail address: jburkhar{at}mail.med.upenn.edu
3 Abbreviations used in this paper: IS, immunological synapse; ER, endoplasmic reticulum; eYFP, enhanced yellow fluorescent protein; HS1, hematopoietic lineage cell-specific protein 1; IP3, inositol 1,4,5-triphosphate; Itk, IL-2-inducible T cell kinase; MBP, maltose-binding protein; MCC, moth cytochrome c; PLC, phospholipase C; SH, Src homology; CMAC, 7-amino-4-chloromethyl coumarin; CRAC, Ca2+-release activated Ca2+; SLP-76, SHZ-domain containing protein of 76 kDa.
4 The online version of this article contains supplemental material.
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