Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc ؊/؊ T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.

The mammal Shc locus encodes three overlapping isoforms (46, 52, and 66 kDa) that differ in the length of their N-terminal regions. p46/p52Shc and p66Shc have been implicated, respectively, in the cytoplasmic propagation of growth and apoptogenic signals. Levels of p66Shc expression correlate with life span duration in mice. p46Shc and p52Shc are ubiquitously expressed, whereas p66Shc is expressed in a cell lineage-specific fashion. However, the mechanisms underlying the regulation of Shc protein expression are unknown. Here we report the identification of two alternative promoters, driving the transcription of two mRNAs coding for p46/ p52Shc and p66Shc. We show that treatment with an inhibitor of histone deacetylases or with a demethylating agent results in induction of p66Shc expression in cells that normally do not express this isoform but leaves the levels of the two other isoforms unchanged. Moreover, analysis of the methylation pattern of the p66Shc promoter in a panel of primary and immortalized human cells showed inverse correlation between p66Shc expression and methylation density of its promoter. These results identify histone deacetylation and cytosine methylation as the mechanisms underlying p66Shc silencing in nonexpressing cells.

Signalling by immunoreceptors is orchestrated at specific plasma membrane microdomains, referred to as lipid rafts. Here we present a proteomics approach to the temporal analysis of protein association with lipid rafts following T-cell antigen receptor (TCR) triggering. We show that TCR engagement promotes the temporally regulated recruitment of proteins participating in the TCR signalling cascade to lipid rafts. Furthermore, TCR triggering results in profound modifications in the composition of lipid rafts involving a number of proteins associated either directly or indirectly with both plasma and intracellular membranes. Raft-associated proteins can be clustered according to their temporal profile of raft association. The data identify lipid rafts as highly dynamic structures and reveal a dramatic impact of surface TCR triggering not only on components of the TCR signalling machinery but also on proteins implicated in a number of diverse cellular processes.

Following ligand binding the TCR segregates to plasma membrane microdomains, termed lipid rafts, characterized by a highly ordered lipid structure favoring partitioning of glycosyl phosphatidyl inositol‐linked costimulatory receptors and acylated signaling molecules. Here we show that the inducible association of the TCR and key signaling proteins with lipid rafts is dependent on the actin cytoskeleton through a mechanism involving raft coalescence. Although lipid rafts are required for full activation of the TCR‐dependent tyrosine phosphorylation cascade and sustained signaling, triggering of TCR‐proximal events, including Fyn activation and a first wave of Vav phosphorylation, is independent of lipid rafts, while a second wave of raft‐dependent Vav phosphorylation occurs after raft coalescence, as also supported by the finding that Vav is phosphorylated in response to lipid raft clustering by GM1 aggregation. The constitutive association found between Vav and the CD3ζ chain suggests a model whereby the TCR‐associated signaling machinery initiates raft aggregation by promoting F‐actin reorganization, which permits full activation of the tyrosine phosphorylation cascade, further reorganization of the actin cytoskeleton and sustained signaling, leading to cell activation.

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