ANTIAPOPTOTIC SIGNALING BY THE INSULIN-RECEPTOR IN CHINESE-HAMSTER OVARY CELLS

We have sought to determine whether insulin can promote cell survival and protect Chinese hamster ovary (CHO) cells from apoptosis induced b y serum starvation. Low concentrations of insulin were antiapoptotic f or cells overexpressing wild-type insulin receptors but not in cells t ransfected with kinase-defective insulin receptor mutants that lacked a functional ATP binding site. However, treatment with orthovanadate ( 50 mu M), a widely used tyrosine phosphatase inhibitor, led a dramatic reduction in internucleosomal DNA fragmentation in both cell lines. C ells transfected with truncated receptor mutants in either the juxtame mbrane or C-terminal domain were as responsive as cells overexpressing wild-type receptors in mediating insulin antiapoptotic protection. Th e mechanisms underlying insulin antiapoptotic protection were investig ated using a variety of pharmacological tools known to inhibit distinc t signaling pathways. The phosphatidylinositol-3' kinase inhibitors wo rtmannin and LY294002 had only a modest influence whereas blocking pro tein farnesylation with manumycin severely disrupted the antiapoptotic capacity of the insulin receptor. Of interest, cells gained antiapopt otic potential following inhibition of extracellular signal-regulated kinase activation with the pharmacological agent PD98059. Insulin indu ced MKK3/MKK6 phosphorylation and activation of p38 MAP kinase whose a ctivity was inhibited with SB203580. However, the inhibition of p38 MA P kinase had no effect on the protection offered by insulin. We conclu de that the antiapoptotic function of the insulin receptor requires in tact receptor kinase activity and implicates a farnesylation-dependent pathway. Increase in cellular phosphotyrosine content, however, trigg ers antiapoptotic signal that may converge downstream of the insulin r eceptor.