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 interact with a number of receptor tyrosine kinases and signaling molecules. We have recently demonstrated that the Grb10 SH2 domain interacts with both the Raf-1 and MEK1 kinases. Overexpression of Grb10 genes with mutations in their SH2 domains promotes apoptosis in cultured cells, a phenotype that is reversed by concomitant overexpression of the wild type gene. Using immunofluorescence microscopy and subcellular fractionation we now show that most of the Grb10 molecules are peripherally associated with mitochondria. Following 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 cytosol. Two-hybrid analysis shows that the Grb10-binding site on Raf-1 co-localizes with its Ras-binding domain. Finally, we show that the endogenous Grb10 and Raf-1 proteins can be co-immunoprecipitated from a partially purified mitochondrial extract, an interaction that is enhanced following the activation of Raf-1 by ultraviolet radiation. Thus, we infer that Grb10 may regulate signaling between plasma membrane receptors and the apoptosis-inducing machinery on the mitochondrial outer membrane by modulating the anti-apoptotic activity of mitochondrial Raf-1.
Nantel
A
A
Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council, Department of Anatomy, Montreal, H4P 2R2 Quebec, Canada. andre.nantel@nrc.ca
Huber
M
M
Thomas
D Y
DY
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D066246:ErbB Receptors / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D051383:GRB10 Adaptor Protein; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D048369:MAP Kinase Kinase 1; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D011506:Proteins; D011994:Recombinant Proteins; D051383:GRB10 Adaptor Protein; D007334:Insulin-Like Growth Factor I; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases