Differential Role of Insulin Receptor Substrate (IRS)-1 and IRS-2 in L6 Skeletal Muscle Cells Expressing the Arg1152 → Gln Insulin Receptor *

In L6 muscle cells expressing the Arg1152 --> Gln insulin receptor (Mut), basal tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was increased by 35% compared with wild-type cells (WT). Upon exposure to insulin, IRS-1 phosphorylation increased by 12-fold in both the Mut and WT cells. IRS-2 was constitutively phosphorylated in Mut cells and not further phosphorylated by insulin. The maximal phosphorylation of IRS-2 in basal Mut cells was paralleled by a 4-fold increased binding of the kinase regulatory loop binding domain of IRS-2 to the Arg1152 --> Gln receptor. Grb2 and phosphatidylinositol 3-kinase association to IRS-1 and IRS-2 reflected the phosphorylation levels of the two IRSs. Mitogen-activated protein kinase activation and [3H]thymidine incorporation closely correlated with IRS-1 phosphorylation in Mut and WT cells, while glycogen synthesis and synthase activity correlated with IRS-2 phosphorylation. The Arg1152 --> Gln mutant did not signal Shc phosphorylation or Shc-Grb2 association in intact L6 cells, while binding Shc in a yeast two-hybrid system and phosphorylating Shc in vitro. Thus, IRS-2 appears to mediate insulin regulation of glucose storage in Mut cells, while insulin-stimulated mitogenesis correlates with the activation of the IRS-1/mitogen-activated protein kinase pathway in these cells. IRS-1 and Shc-mediated mitogenesis may be redundant in muscle cells

PED/PEA-15: an anti-apoptotic molecule that regulates FAS/TNFR1-induced apoptosis

PED/PEA-15 is a recently cloned 15 kDa protein possessing a death effector domain (DED). In MCF-7 and HeLa cells, a fivefold overexpression of PED/PEA-15 blocked FasL and TNFalpha apoptotic effects. This effect of PED overexpression was blocked by inhibition of PKC activity. In MCF-7 and HeLa cell lysates, PED/PEA-15 co-precipitated with both FADD and FLICE. PED/PEA-15-FLICE association was inhibited by overexpression of the wild-type but not of a DED-deletion mutant of FADD. Simultaneous overexpression of PED/PEA-15 with FADD and FLICE inhibited FADD-FLICE co-precipitation by threefold. Based on cleavage of the FLICE substrate PARP, this inhibitory effect was paralleled by a threefold decline in FLICE activation in response to TNF-alpha. TNFalpha, in turn, reduces PED association with the endogenous FADD and FLICE of the cells. Thus, PED/PEA-15 is an endogenous protein inhibiting FAS and TNFR1-mediated apoptosis. At least in part, this function may involve displacement of FADD-FLICE binding through the death effector domain of PED/PEA-15