Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.
We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.
Antonetti
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DA
Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Algenstaedt
P
P
Kahn
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CR
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D005810:Multigene Family; D018482:Muscle, Skeletal / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014162:Transfection; D018909:src Homology Domains
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D007527:Isoenzymes; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)