Caspase-8 and c-FLIPL Associate in Lipid Rafts with NF-κB Adaptors during T Cell Activation

Ravi S Misra,Jennifer Q Russell,Andreas Koenig,Jennifer A Hinshaw-Makepeace,Renren Wen,Demin Wang,Hairong Huo,Dan R Littman,Uta Ferch,Jurgen Ruland,Margot Thome,Ralph C Budd

doi:10.1074/jbc.m610610200

Abstract

Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells.

Highlights

It was subsequently shown that a non-functional mutation in the caspase-8 gene in humans [5, 6] or the deletion of caspase-8 in murine T cells [7] resulted in an immunodeficiency syndrome characterized by markedly decreased production of IL-2 and proliferative capacity of T cells. It remained uncertain from these studies how caspase activation was linked to T cell antigen receptor (TCR) ligation, what were the regulatory protein(s) and substrate(s) of caspase activity, and whether this required the presence of the death receptor Fas (CD95/APO1)
Recent reports indicate that the para-caspase, mucosa-associated lymphoid tissue translocation protein 1 (MALT1), B cell lymphoma protein 10 (Bcl-10), and caspase-recruitment domain membrane-associated guanylate kinase protein 1 (CARMA1) form a complex that is required for TCR-mediated NF-␬B activation [17,18,19,20,21,22]
Because CARMA1, Bcl-10, and MALT1 localize to lipid rafts upon TCR stimulation, we considered that the active caspase complex we had previously defined in activated T cells [15] might form in lipid rafts and be connected with CARMA1, Bcl-10, and MALT1

Summary

Introduction

It remained uncertain from these studies how caspase activation was linked to T cell antigen receptor (TCR) ligation, what were the regulatory protein(s) and substrate(s) of caspase activity, and whether this required the presence of the death receptor Fas (CD95/APO1). Examination of fresh wild-type T cells revealed that essentially all caspase-8 and c-FLIPL were localized to the non-raft c-FLIPL co-precipitated with the active caspase complex in lipid rafts, the majority of which was processed to p43 FLIP despite the reverse ratio in the whole cell lysate (Fig. 1B, lower panel).

Results

Conclusion