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

Abstract

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins.

Fetuin-A, a Hepatocyte-Specific Protein That BindsPlasmodium bergheiThrombospondin-Related Adhesive Protein: a Potential Role in Infectivity

“…Regarding its major physiological function, fetuin-A has, however, been unequivocally shown to inhibit pathological calcification at the systemic level (35) and to regulate bone mineralization (39). As previously reported, fetuin-A is the human homologue of bovine fetuin, with an approximate molecular weight of 60 kDa (1,21,24,42). Human fetuin-A has a two-chain form whose N-terminal heavy chain is disulfide bonded to the C-terminal light chain.…”

Novel Isoform of Insulin Receptor Substrate p53/p58 Is Generated by Alternative Splicing in the CRIB/SH3-binding Region

“…1). The partial or variant CRIB domain has been characterized functionally (6,7), and the SH3-binding domain has been noted previously (14), but we wish to note two observations. First, the CRIB domain and the SH3-binding site overlap and may represent two functional modes of the same site.…”

Cdc42hs Facilitates Cytoskeletal Reorganization and Neurite Outgrowth by Localizing the 58-Kd Insulin Receptor Substrate to Filamentous Actin

“…(A) The Bgl2 fragment of IRS-58 isolated from the two-hybrid screen was cloned into the Bluescript vector and sequenced on both strands with an automated ABI sequencer. The derived protein sequence is shown with three putative protein-protein interaction domains as reported previously (Yeh et al, 1996(Yeh et al, , 1998. SH3-binding domain is in bold type and underlined; SH3 domain is underlined; and the WW-binding domain is in bold.…”