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.

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.

Statins are widely used hypocholesterolemic drugs that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme of the mevalonate pathway whose biosynthetic end product is cholesterol. In addition to lowering circulating cholesterol, statins perturb the composition of cell membranes, resulting in disruption of lipid rafts, which function as signaling platforms in immunoreceptor signaling. Furthermore, by inhibiting protein prenylation, a process also dependent on mevalonate, statins block membrane targeting and hence activity of small GTPases, which control multiple pathways triggered by these receptors. T-cell activation is crucially dependent on Ras, Rho and Rab GTPases. Furthermore TCR signaling is orchestrated at lipid rafts, identifying T-cells as potential cellular targets of statins. Here we report that simvastatin suppresses T-cell activation and proliferation as the result of its capacity to inhibit HMG-CoA reductase. T-cell treatment with simvastatin does not affect intracellular cholesterol levels or raft integrity nor, accordingly, the initial tyrosine phosphorylation-dependent cascade. Conversely, inhibition of protein prenylation by simvastatin results in a dramatic impairment in the pathways regulated by small GTPases, including the Ras/MAP kinase pathway, the Rac/stress kinase pathway, and the Rab-dependent pathway of receptor endocytosis. The results identify Ras superfamily GTPases as strategic molecular targets in T-cell immunosuppression by statins.

Initially identified as components of the signaling pathways triggered by receptor tyrosine kinases and leading to Ras activation, Shc proteins have been more recently implicated in the regulation of signals controlling not only cell proliferation, but also cell survival and apoptosis. Here we briefly review the current understanding of Shc proteins as promoters of apoptosis. Specifically, we focus on the 66 kDa isoform of ShcA, whose paramount importance in the regulation of oxidative stress responses leading to cell apoptosis and ageing has been by now firmly established.

The ShcA locus encodes 3 protein isoforms that differ in tissue specificity, subcellular localization, and function. Among these, p66Shc inhibits TCR coupling to the Ras/MAPK pathway and primes T cells to undergo apoptotic death. We have investigated the outcome of p66Shc deficiency on lymphocyte development and homeostasis. We show that p66Shc ؊/؊ mice develop an age-related lupus-like autoimmune disease characterized by spontaneous peripheral T-and B-cell activation and proliferation, autoantibody production, and immune complex deposition in kidney and skin, resulting in autoimmune glomerulonephritis and alopecia. IntroductionThe Shc protein family includes 4 members, ShcA, ShcB, ShcC, and RaLP. 1,2 ShcA is expressed as 3 isoforms of 52, 46, and 66 kDa, which display the PTB-CH1-SH2 Shc family signature, preceded in p66Shc by a CH2 domain containing a phosphorylatable serine (Ser36) 3 and a cytochrome c-binding region within the CH2-PTB domains. 4 In addition to structural differences, p52Shc/p46Shc differ from p66Shc in expression and function. The shorter isoforms are constitutively and ubiquitously expressed, whereas p66Shc expression is regulated by an alternative promoter 5 and is tissue restricted. 6 ShcA isoforms differ also in their subcellular localization and function. p52Shc is a cytosolic protein acting as adaptor in pathways triggered by surface receptors controlling proliferation, chemotaxis, and survival. 7,8 p46Shc localizes to mitochondria, where it subserves an unknown function. 9 On the other hand, p66Shc is expressed as 2 pools, one cytosolic and the other mitochondrial, and is endowed with antimitogenic and proapoptotic activities. Indeed, p66Shc inhibits activation of the Ras/MAPK pathway by tyrosine kinase receptors and the T-cell antigen receptor (TCR) by competing with p52Shc. Furthermore, in fibroblasts, p66Shc participates in oxidative stress-induced apoptosis by triggering the mitochondrial pathway as a p53 target. 10 p66Shc-mediated apoptosis is dependent on Ser36 phosphorylation, which is mediated by PKC␤ and required for p66Shc translocation from the cytosol to mitochondria by the prolylisomerase Pin1. 11 In mitochondria, p66Shc is maintained in an inactive state within a high-molecular-mass complex, which includes the TIM-TOM import complex and Hsp70. Following proapoptotic stimulation, p66Shc is released and acquires the capacity to oxidize cytochrome c and catalyze H 2 O 2 production, leading to mitochondrial dysfunction, opening of the permeability transition pore (PTP), and apoptosis. 4,12 Moreover, p66Shc modulates the levels of reactive oxygen species (ROSs) by suppressing activation of FKHRL1, a forkhead transcription factor that controls catalase expression and is implicated as such in H 2 O 2 scavenging. 13 Alterations in oxidative metabolism in p66Shc Ϫ/Ϫ fibroblasts, characterized by decreased mitochondriadependent energy generation and increased aerobic glycolysis, 14 also contribute to the reduction in ROS levels observed in these cells. In agreement with the capac...