Phosphorylation of Tyrosine 340 in the Pleckstrin Homology Domain of RIAM Is Required for Inside-Out Activation of LFA-1 and LFA-1: ICAM-1-Mediated Adhesion

Abstract 837

Adhesion of lymphocytes to antigen presenting cells (APCs) is a critical event linking innate and adaptive immunity and a mandatory step for the initiation of T cell immune responses. Control of lymphocyte adhesion to APC is accomplished through the regulation of the principle adhesion molecule on the lymphocyte surface, the β2 integrin designated lymphocyte functional antigen 1 (LFA-1), which binds to intercellular adhesion molecule 1 (ICAM-1) on the surface of APCs. In order to mediate its adhesive function LFA-1 must be activated via a process referred to as inside-out signaling, which results in conformation changes of the receptor that extends the ectodomains of the α and β chains leading to a high affinity state. Because LFA-1-mediated adhesion is a central link between innate and adaptive immunity, understanding the mechanisms implicated in inside-out-mediated LFA-1 activation is a subject of intense investigation. Currently, it is poorly understood how signals originating from the TCR are linked to specific mechanisms that regulate inside-out activation of LFA-1. Among the few signaling molecules that have been implicated in this process in hematopoietic cells are the small GTPase Rap1 and its downstream effector RIAM. RIAM is a multidomain protein that includes a talin binding region, two coiled-coiled regions, a small N-terminus proline-rich region, sequential Ras association (RA) and pleckstrin homology (PH) domains, and a large C-terminus proline-rich region, via which interacts with Ena/VASP family proteins and profilin and is recruited to the sites of actin turnover. Through its C-terminus RIAM also interacts with the SH3-domain of PLC-γ1. The RA and PH domains of RIAM function as an integral unit and as a proximity detector since both are required for translocation of RIAM to the plasma membrane. The RA domain of RIAM has specificity for Rap1-GTP whereas the PH domain binds to the PLC-γ1 substrate PI(4,5)P₂. We determined that the integral RA-PH structural unit of RIAM has also an integral functional role because both the RA and PH regions are required for LFA-1-mediated adhesion. Here we sought to determine how TCR proximal signaling events regulate RIAM function leading to inside-out activation of LFA-1 and LFA-1:ICAM-1 mediated adhesion. Using primary human T lymphocytes and Jurkat T cells we determined that upon TCR/CD3-mediated stimulation, activated Src family kinases Fyn and Lck associate with and induce tyrosine phosphorylation of RIAM. To identify the precise domain(s) of RIAM, which undergo phosphorylation by these kinases, we co-expressed individual truncation constructs of RIAM N-terminus, RA-PH, and C-terminus regions along with the active or inactive form of Fyn or Lck in COS cells. Immunoprecipitations and immunoblot assays revealed that active Fyn and Lck mediated robust and selective tyrosine phosphorylation of the RA-PH structural unit of RIAM. Detailed analysis by mass spectrometry identified that tyrosine 340 (Y340) within the PH domain of RIAM was the specific target. The PH domain of RIAM has specificity for PI(4,5)P₂ and is an integral component of the RA-PH proximity detector. We hypothesized that phosphorylation of tyrosine 340 in the PH domain might have a regulatory role in the function of the RA-PH structural unit in mediating LFA-1 activation. To address this issue we used site directed mutagenesis and we introduced a tyrosine to phenylalanine mutation in site 340 (Y34F) of RIAM, rendering this residue resistant to phosphorylation mediated by Fyn and Lck. Expression of RIAM Y340F in Jurkat T cells abrogated LFA-1 activation and LFA-1-mediated adhesion in response to TCR/CD3 stimulation. Under these conditions, activation of LFA-1 in response to PMA, which bypasses TCR-mediated signaling, remained intact. Thus, besides recruitment of RIAM to the plasma membrane, TCR-mediated activation of Src family kinases and phosphorylation of RIAM PH domain is a mandatory requirement for the function of the RA-PH proximity detector as a regulator of LFA-1 activation. Our results provide a biochemical, molecular and mechanistic link between TCR-mediated signaling and inside-out activation of LFA-1 thereby initiating LFA-1: ICAM-1-mediated adhesion and cross-talk between T cells and APC.