Abstract
Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.
Highlights
Actin cytoskeleton dynamics critically regulate T cell activation
We have investigated the involvement during T cell activation of the cytoplasmic adaptor protein HIP-55, a member of the drebrin/Abp1 class of cellular signal-regulated kinase; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; GCK, germinal center kinase; GFP, green fluorescent protein; HA, hemagglutinin A; HPK1, hematopoietic progenitor kinase 1; IL, interleukin; IS, immunological synapse; mAb, monoclonal antibody; NFAT, nuclear factor of activated T cells; OVA, ovalbumin-derived; PMA, phorbol 12-myristate 13acetate; Staphylococcal enterotoxin E (SEE), staphylococcal enterotoxin E; SH, Src homology; siRNA, small interfering RNA; TRITC, tetramethylrhodamine isothiocyanate; WASP, Wiskott-Aldrich syndrome protein
HIP-55 Is Recruited to the T cell-antigen-presenting cell (APC) Contact Site in Activated T Cells—To investigate HIP-55 involvement in T cell activation, we first studied, using confocal microscopy, its distribution during the formation of conjugates between DO11.10 OVA-specific T cell receptor (TCR)-transgenic CD4ϩ T cells and A20 B cells pulsed with the OVA peptide
Summary
Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP55, a Src/Syk-kinases substrate and member of the drebrin/Abp family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Several actin-binding proteins have been involved in the process of T cell activation mediated by APCs. One of the best studied examples is the complex formed by the Wiskott-Aldrich syndrome protein (WASP) with the actin-related protein Arp2/3 [11]. We have investigated the involvement during T cell activation of the cytoplasmic adaptor protein HIP-55 ( called SH3P7 or mAbp1), a member of the drebrin/Abp class of cellular signal-regulated kinase; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; GCK, germinal center kinase; GFP, green fluorescent protein; HA, hemagglutinin A; HPK1, hematopoietic progenitor kinase 1; IL, interleukin; IS, immunological synapse; mAb, monoclonal antibody; NFAT, nuclear factor of activated T cells; OVA, ovalbumin-derived (peptide); PMA, phorbol 12-myristate 13acetate; SEE, staphylococcal enterotoxin E; SH, Src homology; siRNA, small interfering RNA; TRITC, tetramethylrhodamine isothiocyanate; WASP, Wiskott-Aldrich syndrome protein
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have