A novel negative regulatory function of the phosphoprotein associated with glycosphingolipid-enriched microdomains: blocking Ras activation

Abstract

In primary human T cells, anergy induction results in enhanced p59Fyn activity. Because Fyn is the kinase primarily responsible for the phosphorylation of PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains), which negatively regulates Srckinase activity by recruiting Csk (the Cterminal Src kinase) to the membrane, we investigated whether anergy induction also affects PAG. Analysis of anergic T cells revealed that PAG is hyperphosphorylated at the Csk binding site, leading to IntroductionIn the thymus, most T cells undergo negative selection as they bind with high affinity to the self-peptide/MHC complexes presented. However, thymic selection is not a tight process and some autoreactive T cells escape into the periphery. 1 To protect the body from these potentially destructive cells, there must exist mechanisms of peripheral tolerance that prevent autoreactive T cells from becoming activated; one such mechanism is anergy. 2 T cells receiving a strong signal via the T-cell receptor (TCR) [signal 1] in the absence of costimulation [signal 2] enter into an unresponsive state (ie, anergy) in which the cells are alive but fail to display functional responses, such as proliferation in response to full activation [signal 1 ϩ 2]. 3,4 Although the mechanism responsible for the induction of anergy remains unknown, the characterization of anergic T cells has clearly demonstrated that the lack of proliferation is due to a block in interleukin-2 (IL-2) production and that anergic T cells proliferate after addition of exogenous IL-2. The defect in IL-2 production is attributed to a block in Ras activation, 5-7 a small G-protein that activates the mitogen-activated protein kinase cascade, ultimately resulting in activation of the transcription factor activator protein-1 (AP-1). Indeed, the ionomycininduced anergy model shows that activation of nuclear factor of activated T cells in the absence of AP-1 and nuclear factor of the -enhancer in B cells (NF-B) leads to the transcription of genes that induce anergy. 8 Within the TCR signaling cascade, the earliest alteration detected in anergic T cells is the up-regulation of the Src family kinase (SFK) p59Fyn. 9,10 However the mechanism by which Fyn contributes to anergy is unknown. In addition to the TCR, 11 Fyn also binds the phosphoprotein associated with glycosphingolipidenriched microdomains (PAG) 12 (also known as the Csk binding protein [Cbp]). 13 Fyn is the kinase responsible for PAG phosphorylation. 14 PAG, a transmembrane adaptor that localizes within the lipid rafts, negatively regulates Src kinase activity by recruiting the C-terminal Src kinase (Csk) to the membrane. 15 Csk in turn phosphorylates the inhibitory tyrosines conserved within the Src kinases. 16 It is noteworthy that T cells overexpressing PAG are resistant to activation via the TCR, a phenotype similar to anergy. 12,17 Therefore, we chose to investigate whether anergy induction leads to changes within PAG that might contribute to the unresponsiveness of anergic T cells. Material...

Regulation of FynT Function by Dual Domain Docking on PAG/Cbp

“…Recruitment of Csk to PAG pY317 has been shown to enhance C-terminal phosphorylation of FynT (Tyr(P) 528 ) as observed by Smida et al (38). Taken together, we propose the following binding model for formation of the FynT-PAG-Csk complex in resting T-cells (Fig.…”

“…Alternatively, the complex may be stabilized either by additional contacts with Csk bound to PAG at Tyr(P) 317 or by contacts with other proteins yet to be identified. This scenario is likely, since PAG has a large number of potential interaction sites, and the formation of such multiprotein complexes involving PAG has recently been confirmed (38). However, although binding of the SH3 domain to mutated ligand sequences was not abolished completely, FynT kinase activity was greatly reduced in in vitro phosphorylation assays using these ligands.…”

“…The precise mechanism for dephosphorylation of PAG is unknown, and it could involve two phosphatases that may be regulated differently in different settings, as suggested previously (35). Two recent studies have focused on a role for PAG in anergy induction in T-cells (18,38). Anergic T-cells have enhanced FynT activity as well as increased levels of PAG-FynT complexes but not PAG-Csk levels.…”

“…Engagement of the FynT-SH2 domain on PAG, however, ensures that FynT remains enzymatically active and that the inhibition is not effectuated until FynT dissociates from PAG and an inactive "closed" conformation can be formed. A role for the SH2 domain in "priming for inhibition" by Csk has been considered for Lck (46) and recently also for FynT (38).…”

Cooperative immunoregulatory function of the transmembrane adaptor proteins SIT and LAX

“…Similarly, although PAG/Cbp Ϫ/Ϫ mice do not show an obvious phenotype [11,12], small interference RNA studies demonstrate that PAG is an important negative regulatory protein in T cells [30]. Thus, it appears as if the strong, positive regulatory function of LAT is balanced by a plethora of negative regulatory TRAPs that help to maintain homeostasis within the immune system.…”