p56Lck Tyrosine Kinase Enhances the Assembly of Death-inducing Signaling Complex during Fas-mediated Apoptosis

Abstract

Although the death-inducing signaling complex (DISC) is rapidly assembled, several lines of evidence suggest that formation of this complex is not the first consequence of cell surface CD95 (Fas) stimulation but rather a later step in this process. Activation of Fas triggers a cascade of signaling events that culminate in cellular apoptosis. Tyrosine kinases are critical effectors in T cell activation. However, their functional involvement in death receptor-mediated apoptosis is unknown. Here, we used p56Lck -deficient cells to show that CD95-induced cell death is highly dependent on p56Lck activity and its localization within plasma membrane. We found that p56 Lck Lck activation and localization.In T lymphocytes activation-induced cell death results from repeated stimulation through the T cell receptor (TCR) 3 (1). Activation-induced cell death plays a crucial role in regulating peripheral homeostasis and self-tolerance by eliminating autoreactive and activated T cells. Members of the "death receptor" subgroup of the tumor necrosis receptor family, characterized by their intracellular "death domain" motifs, have been identified as important mediators of apoptosis (2). Like all death receptors, ligation of CD95 (Fas/APO-1) by its ligand (FasL) or agonistic antibodies triggers death-inducing signaling complex (DISC) formation (3), which typically induces caspase activation (4). DISC formation occurs through the rapid recruitment of a set of proteins including Fas-associated death domain protein (FADD), procaspase-8, procaspase-10, and the caspase 8/10 regulator c-FLIP to the cytoplasmic domain of CD95 (5, 6). This oligomerization of procaspase-8 results in its autocatalytic cleavage and release from the DISC as an active heterotetramer containing two p18 and two p10 subunits (7-10). Although the DISC is rapidly assembled, several lines of evidence suggest that formation of this complex is not the first consequence of Fas stimulation but rather a later step in the conversion of signaling "inefficient" Fas complexes into "activated" receptor complexes (11,12). In particular, formation of Fas microclusters (12) and high stability supramolecular clusters via receptor palmitoylation (13, 14), actin reorganization (12), inducible or constitutive association with detergent-resistant microdomains known as lipid rafts (15)(16)(17)(18)(19)(20)(21)(22), and the production of acid sphingomyelinase-mediated ceramide (11) have been proposed as important intermediate steps preceding robust DISC formation.These studies clearly suggest that Fas-induced apoptosis can be controlled at the level of the cytoplasmic membrane. Although specific interactions between Fas, FADD, and procaspase-8/10 via their death domains and death effector domains are well documented, little is known about the mechanism(s) that regulates the recruitment of these proteins to CD95 after anti-Fas treatment. Cross-linking of CD95 has been shown to trigger several signal transduction pathways that tightly regulate apoptosis (23). Kinases such as p38 MAPK an...

Involvement of tyrosine phosphatase CD45 in apoptosis

“…Those results confirms the possible involvement of p56lck and/or downstream kinases in our model. Other studies have demonstrated that the tyrosine kinase p56lck is involved in the regulation of apoptosis induced by the mitochondrial pathway [114][115][116]. In our study, nuclear apoptosis was induced in p56lck-deficient cells exposed to TBT, suggesting that p56lck does not have a direct role in CD45-cells resistant to nuclear apoptosis [98].…”

T-cell receptor complex is essential for Fas signal transduction

“…PLC-γ1 interaction with the Fas receptor was transient, and reflected its activation pattern in response to Fas receptor stimulation. As shown by others, Lck was found to be preassociated with the Fas signaling complex in untreated cells (17). Interestingly, Fas ligand stimulation rapidly decreased the amount of total and activated Lck copurifying with the Fas receptor (Fig.…”

p66Shc-dependent apoptosis requires Lck and CamKII activity

“…While the kinase activity of Lck has been reported to be required for Jurkat T-cell apoptosis in response to some apoptogenic stimuli [13,14,20,40], other stimuli are known to induce apoptosis independently of Lck activity [16,21]. To address the requirement for the catalytic activity of Lck in A23187-dependent apoptosis of Jurkat cells, we used Jurkat and JCaM1 cells stably expressing a constitutively active Lck mutant (Y505F) (Fig.…”

“…However, the mechanisms underlying T-cell priming to apoptosis by p66Shc have yet to be fully understood. We investigated the potential involvement of Lck, a Src family kinase which has been implicated in lymphocyte apoptosis induced by various stimuli [11][12][13][14][15][16][19][20][21], in the proapoptotic activity of p66Shc in Jurkat T cells. To specifically dissect the link between p66Shc and Lck we used JCaM1.6 cells, which lack expression of both Lck and p66Shc, stably transfected with either empty vector (JCaM1) or a vector encoding wild-type p66Shc (JCaM1 p66 ) (Fig.…”

“…The implication of Src kinases in apoptosis is not restricted to T cells, as shown for Btk and Lyn in the regulation of B-cell apoptosis triggered by ionizing radiations [17] and UV [18]. Despite the clear involvement of Lck in T-cell apoptosis, the role of its catalytic activity is as yet controversial [13][14][15][16][19][20][21].…”