A new pathway of CD5 glycoprotein-mediated T cell inhibition dependent on inhibitory phosphorylation of Fyn kinase

Bamberger M, Santos AM, Gonçalves CM, Oliveira MI, James JR, Moreira A, Lozano F, Davis SJ, Carmo AM. (2011), J Biol Chem. 286, 30324-36

Abstract

Triggering of the T cell receptor initiates a signaling cascade resulting in the activation of the T cell. These signals are integrated alongside those resulting from the triggering of other receptors whose function is to modulate the overall response. CD5 is an immunotyrosine-based inhibition motif-bearing receptor that antagonizes the overt T cell receptor activation response by recruiting inhibitory intracellular mediators such as SHP-1, RasGAP, or Cbl. We now propose that the inhibitory effects of CD5 are also mediated by a parallel pathway that functions at the level of inhibition of Fyn, a kinase generally associated with T cell receptor-mediated activation. After CD5 ligation, phosphorylation of the negative regulatory tyrosine (Tyr(531)) of Fyn increases, and this correlates with a substantial reduction in the kinase activity of Fyn and a profound inhibition of ZAP-70 activation. The effect requires the last 23 amino acids of the cytoplasmic domain of the receptor, strongly implying the involvement of a new CD5-interacting signaling or adaptor protein. Furthermore, we show that upon CD5 ligation there is a profound shift in its distribution from the bulk fluid phase to the lipid raft environment, where it associates with Fyn, Lck, and PAG. We suggest that the relocation of CD5, which we also show is capable of forming homodimers, to the proximity of raft-resident molecules enables CD5 to inhibit membrane proximal signaling by controlling the phosphorylation and activity of Fyn, possibly by interfering with the disassembly of C-terminal Src kinase (Csk)-PAG-Fyn complexes during T cell activation.

Key figure: CD5 forms dimers at the cell surface; the cytoplasmic tail of CD5 is not involved in CD5 dimerization or in its translocation to lipid rafts

A, CD2 or CD5 or CD5Ext/CD2Int (a chimera consisting of the extracellular and transmembrane domains of CD5 and the intracellular tail of CD2) was expressed at the surface of 293T cells as BRET pairs (for example by cotransfecting genes encoding CD5_Luc together with CD5_GFP in the same cell or CD2_Luc and CD2_GFP). BRET analysis showed that the CD5 BRET pair (CD5_BP) shows BRET_eff values that can readily be fitted by a dimerization model (34). The CD5Ext/CD2Int chimera follows the same profile as seen for the CD5 wild-type BRET pair, whereas CD2_BP shows low BRET_eff values and independence of the acceptor:donor ratio, characteristics of monomers.B, 2G5/CD5.WT and 2G5/CD5.K384^stop cells were stimulated with anti-CD5 mAbY-2/178 or incubated with isotype-control antibody (NS) for 10 min at 37 °C. Cells were collected, washed, and lysed, and total cell lysates were subjected to sucrose density centrifugation. Equal volumes of each fraction were resolved by SDS-PAGE and blotted onto PVDF filters. Membranes were incubated with CD5 mAb followed by HRP-conjugated rabbit anti-mouse antibody, and proteins were visualized by enhanced chemiluminescence.