Phosphorylation of Tyrosine 340 in the Plekstrin Homology Domain of RIAM Is Required for Translocation of RIAM to the Plasma Membrane, Phosphorylation of RIAM-Associated PLC-g1 and LFA-1 Activation

Adhesion of lymphocytes to antigen presenting cells (APCs) is a critical step linking innate and adaptive immunity. Lymphocyte-APC adhesion is accomplished through 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. LFA-1 must be activated via a process referred to as inside-out signaling, which results in conformation changes leading to a high affinity state. Among the few signaling molecules implicated in inside-out signaling 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. Through its C-terminus, RIAM constitutively interacts with PLC-γ1. The RA domain of RIAM has specificity for Rap1-GTP whereas the PH domain binds to the PLC-γ1 substrate PI(4,5)P₂. The RA-PH domain region of RIAM functions as an integral unit and as a proximity detector, and both RA and PH are required for translocation of RIAM to the plasma membrane. Using primary human T lymphocytes and Jurkat T cells we determined previously that RIAM undergoes tyrosine phosphorylation by Src family kinases upon TCR stimulation. In the present study we sought to determine the role of tyrosine phosphorylation in RIAM function. To identify the precise region(s) of RIAM, which undergo phosphorylation by these kinases, we co-expressed individual truncation constructs of RIAM N-terminus, RA-PH or 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. Tandem mass spectrometry (LC-MS/MS) identified that tyrosine 340 (Y340) within the PH domain was the specific target. Because this tyrosine is localized within the RA-PH integral unit, we examined whether phosphorylation of Y340 in the PH domain might have an active role in the function of RIAM. Using site directed mutagenesis, we introduced a tyrosine-to-phenylalanine mutation (Y340F) rendering this residue resistant to phosphorylation, and FLAG-tagged RIAM-WT or FLAG-tagged RIAM-Y340F constructs were expressed in Jurkat T cells. Anti-FLAG immunoprecipitation followed by immunoblot showed that RIAM-WT and RIAM-Y340F displayed comparable interaction with PLC-γ1. However, phosphorylation of PLC-γ1 associated with RIAM-Y340F was impaired. Because Src family kinases and Itk, which are involved in PLC-γ1 phosphorylation and activation localize at the lipid rafts upon T cell stimulation, we examined whether RIAM-Y340F might display differential translocation to the plasma membrane thereby altering the ability of RIAM-associated PLC-γ1 to undergo activating phosphorylation. Isolation of membranous and cytosolic fractions by nitrogen cavitation revealed that in contrast to RIAM-WT, which rapidly translocated to the membrane fraction upon T cell stimulation, RIAM-Y340F remained exclusively in the cytosolic fraction. To investigate whether RIAM-Y340F displayed altered plasma membrane localization in vivo, we used mCherry-RIAM-WT or mCherry-RIAM-Y340F and live cell imaging. Although RIAM-WT readily translocated to the plasma membrane and colocalized with Rap1-GTP, RIAM-Y340F was unable to translocate to the plasma membrane and was detectable only in the cytoplasm. Because RIAM translocation to the plasma membrane and PLC-γ1 activation are required for inside-out activation of LFA-1, we examined the effects of RIAM-Y340F on LFA-1 activation. We determined that expression of RIAM Y340F abrogated LFA-1 activation and LFA-1-mediated adhesion in response to TCR/CD3 stimulation. Thus, TCR-mediated phosphorylation of Y340 in RIAM PH domain by Src family kinases is a mandatory requirement for activation of RIAM-associated PLC-γ1 and LFA-1 activation. Our results provide a 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.