Localization of endogenous Grb10 to the mitochondria and its interaction with the mitochondrial-associated Raf-1 pool

Grb10 belongs to a small family of adapter proteins that are known to inter act with a number of receptor tyrosine kinases and signaling molecules. We have recently demonstrated that the Grb10 SH2 domain interacts with both th e Raf-1 and MEK1 kinases. Overexpression of Grb10 genes with mutations in t heir SH2 domains promotes apoptosis in cultured cells, a phenotype that is reversed by concomitant overexpression of the wild type gene. Using immunof luorescence microscopy and subcellular fractionation we now show that most of the Grb10 molecules are peripherally associated with mitochondria. Follo wing insulin-like growth factor I or serum treatment, small pools of Grb10 can also be found at the plasma membrane and in actin-rich membrane ruffles , whereas overexpression of Grb10 leads to its mislocalization to the cytos ol. Two-hybrid analysis shows that the Grb10-binding site on Raf-1 co-local izes with its Ras-binding domain. Finally, we show that the endogenous Grb1 0 and Raf-1 proteins can be co-immunoprecipitated from a partially purified mitochondrial extract, an interaction that is enhanced following the activ ation of Raf-1 by ultraviolet radiation. Thus, we infer that Grb10 may regu late signaling between plasma membrane receptors and the apoptosis-inducing machinery on the mitochondrial outer membrane by modulating the anti-apopt otic activity of mitochondrial Raf-1.