DISRUPTION OF A PUTATIVE SH3 DOMAIN AND THE PROLINE-RICH MOTIFS IN THE 53-KDA SUBSTRATE OF THE INSULIN-RECEPTOR KINASE DOES NOT ALTER ITS SUBCELLULAR-LOCALIZATION OR ABILITY TO SERVE AS A SUBSTRATE

The recently identified 53-kDa substrate of the insulin receptor famil y was further characterized in several retroviral-generated stable cel l lines overexpressing the wild type and various mutant forms of the p rotein. To facilitate the study of its subcellular localization in NIH 3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous pr otein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosi ne phosphorylated in insulin-stimulated cells. Immunofluorescence stud ies showed for the first time that a fraction of the 53-kDa protein wa s localized to the plasma membrane. Confocal microscopy of cells doubl e-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the leve l of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and t wo proline-rich regions, putative binding sites for SH3 and WW domains . Disruption of these three motifs by the introduction of previously c haracterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insuli n receptor, or its colocalization with the insulin receptor, suggestin g these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent si gnaling proteins. (C) 1998 Wiley-Liss, Inc.