University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Fall 2009 Role and Regulation of the Actin-Regulatory Protein Hs1 in Tcr Signaling Esteban Carrizosa University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, and the Immunology and Infectious Disease Commons Recommended Citation Carrizosa, Esteban, "Role and Regulation of the Actin-Regulatory Protein Hs1 in Tcr Signaling" (2009). Publicly Accessible Penn Dissertations. 91. https://repository.upenn.edu/edissertations/91 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/91 For more information, please contact [email protected]. Role and Regulation of the Actin-Regulatory Protein Hs1 in Tcr Signaling Abstract Numerous aspects of T cell function, including TCR signaling, migration, and execution of effector functions, depend on the actin cytoskeleton. Cytoskeletal rearrangements are driven by the action of actin-regulatory proteins, which promote or antagonize the assembly of actin filaments in esponser to external cues. In this work, we have examined the regulation and function of HS1, a poorly-understood actin regulatory protein, in T cells. This protein, which becomes tyrosine phosphorylated upon T cell activation, is thought to function primarily by stabilizing existing branched actin filaments. Loss of HS1 results in unstable actin responses upon TCR engagement and defective Ca2+ responses, leading to poor activation of the IL2 promoter. TCR engagement leads to phosphorylation of HS1 at Tyr 378 and Tyr 397, creating binding sites for SH2 domain-containing proteins, including Vav1 and Itk. Phosphorylation at these residues is required for Itk-dependent recruitment of HS1 to the IS, Vav1 IS localization, and HS1-dependent actin reorganization and IL2 production. Therefore, in addition to directly interacting with branched actin filaments, HS1 egulationr of Vav1 localization provides another mechanism by which HS1 signals to the actin cytoskeleton. Analysis of Ca2+ responses in HS1-/- T cells reveals that the defect in these cells lies at the level of release from intracellular stores. Phosphorylated HS1 interacts with PLCγ1, and mediates its association with the cytoskeleton and regulates microcluster dynamics. Although HS1 is clearly important for signaling downstream of the TCR, HS1-/- mice exhibit normal T cell development and normal peripheral populations. Surprisingly, HS1 is not required for T cell homing to lymphoid organs, for TCR endocytosis or for CD8+ T cell effector function. However, T cells from HS1-/- mice produce reduced amounts of Ifnγ, and are subsequently less likely to become Ifnγ-producing TH1 effector cells. These data demonstrate that HS1 functions as a cytoskeletal adaptor protein and plays specific olesr downstream of TCR engagement. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Immunology First Advisor Dr. Janis K. Burkhardt Keywords T cells, calcium signaling, HS1, microclusters Subject Categories Cell Biology | Immunology and Infectious Disease This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/91 ROLE AND REGULATION OF THE ACTIN-REGULATORY PROTEIN HS1 IN TCR SIGNALING Esteban Carrizosa A DISSERTATION in Immunology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2009 _____________________ Dr. Janis K. Burkhardt, Associate Professor, Dissertation Advisor _____________________ Dr. Steven L. Reiner, Professor, Graduate Group Chair Dissertation Committtee: Dr. Gary A. Koretzky Dr. Terri M. Laufer Dr. Paula M. Oliver Dr. Jordan S. Orange Role and regulation of the actin-regulatory protein HS1 in TCR signaling COPYRIGHT 2009 Esteban Carrizosa This work was supported in part by NIH F31AI071385 iii Abstract ROLE AND REGULATION OF THE ACTIN-REGULATORY PROTEIN HS1 IN TCR SIGNALING Esteban Carrizosa Advisor: Dr. Janis K. Burkhardt Numerous aspects of T cell function, including TCR signaling, migration, and execution of effector functions, depend on the actin cytoskeleton. Cytoskeletal rearrangements are driven by the action of actin-regulatory proteins, which promote or antagonize the assembly of actin filaments in response to external cues. In this work, we have examined the regulation and function of HS1, a poorly-understood actin regulatory protein, in T cells. This protein, which becomes tyrosine phosphorylated upon T cell activation, is thought to function primarily by stabilizing existing branched actin filaments. Loss of HS1 results in unstable actin responses upon TCR engagement and defective Ca2+ responses, leading to poor activation of the IL2 promoter. TCR engagement leads to phosphorylation of HS1 at Tyr 378 and Tyr 397, creating binding sites for SH2 domain- containing proteins, including Vav1 and Itk. Phosphorylation at these residues is required for Itk-dependent recruitment of HS1 to the IS, Vav1 IS localization, and HS1- dependent actin reorganization and IL2 production. Therefore, in addition to directly interacting with branched actin filaments, HS1 regulation of Vav1 localization provides another mechanism by which HS1 signals to the actin cytoskeleton. Analysis of Ca2+ responses in HS1-/- T cells reveals that the defect in these cells lies at the level of release from intracellular stores. Phosphorylated HS1 interacts with PLCγ1, and mediates its iv association with the cytoskeleton and regulates microcluster dynamics. Although HS1 is clearly important for signaling downstream of the TCR, HS1-/- mice exhibit normal T cell development and normal peripheral populations. Surprisingly, HS1 is not required for T cell homing to lymphoid organs, for TCR endocytosis or for CD8+ T cell effector function. However, T cells from HS1-/- mice produce reduced amounts of Ifnγ, and are subsequently less likely to become Ifnγ-producing TH1 effector cells. These data demonstrate that HS1 functions as a cytoskeletal adaptor protein and plays specific roles downstream of TCR engagement. v Table of Contents Chapter 1: Introduction ........................................................................................................ 1 Interaction with APCs and immunological synapse formation .........................................2 CONTROL OF ACTIN DYNAMICS AT THE IMMUNOLOGICAL SYNAPSE .............................4 Arp2/3 complex‐dependent actin regulatory proteins ..................................................................6 INTERPLAY BETWEEN T CELL SIGNALING AND ACTIN DYNAMICS................................ 25 Costimulation‐dependent actin regulatory pathways..................................................................28 Pathways leading to Ca2+ signaling in T cells....................................................................................28 Chapter 2: HS1 functions as an essential actin­regulatory adapter protein at the Immune Synapse............................................................................................................36 Summary ........................................................................................................................................... 36 Introduction..................................................................................................................................... 36 Results................................................................................................................................................ 38 HS1 regulates the accumulation of F‐actin at the immune synapse.......................................38 HS1‐deficient cells fail to stabilize actin following TCR engagement....................................43 Tyrosine phosphorylation of HS1 is required for actin remodeling ......................................47 Tyrosine phosphorylation of HS1 mediates binding to key signaling molecules.............53 HS1 and Vav1 interact specifically and directly..............................................................................53 HS1 is required for maintaining Vav1 at the IS ...............................................................................54 HS1‐mediated actin stabilization is required for sustained TCR signaling.........................57 HS1 is recruited to the periphery of spreading T cells.................................................................63 HS1 binding to F‐actin is important for actin polymerization at the IS................................63 Discussion......................................................................................................................................... 64 vi Chapter 3: Recruitment of HS1 to the immunological synapse by Itk regulates actin responses and PLCγ1 microcluster dynamics ..................................................73 Summary ........................................................................................................................................... 73 Introduction..................................................................................................................................... 74 Results................................................................................................................................................ 76 Itk‐deficient T cells show unstable lamellipodial protrusions .................................................76 The SH2 domain of Itk mediates