Insulin signaling in the yeast Saccharomyces cerevisiae. 3. Induction of protein phosphorylation by human insulin.
A low affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae has been identified recently (Müller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W., preceding paper in this issue). Since the mammalian insulin receptor functions as a tyrosine kinase with autophosphorylation capacity, kinase studies were performed with the partially purified insulin-binding protein preparation. Incubation with [gamma-32P]ATP in vitro led to phosphorylation of the 53-kDa insulin-binding protein on serine but not on tyrosine residues. In addition, a 70-kDa polypeptide, copurified with the insulin-binding protein preparation, was tyrosine-phosphorylated under the same conditions. Phosphorylation of both proteins was enhanced by human insulin. These results obtained by immunoprecipitation and immunoblotting using specific anti-phosphoserine/threonine/tyrosine antibodies were confirmed by phosphoamino acid analysis of the individual immunoprecipitated and gel-purified 32P-labeled phosphoproteins. During gel filtration, the 53-kDa protein coeluted as a 300-kDa complex together with the 70-kDa phosphotyrosine-containing protein and was coimmunoprecipitated with the latter using an anti-phosphotyrosine antibody, strongly arguing for complex formation between the two proteins. The data presented raise the possibility that stimulation of glycogen synthesis by insulin in yeast is mediated by a 53-kDa insulin-binding protein and a 70-kDa phosphotyrosine-containing protein which are organized in a large plasma membrane-bound signaling complex. Elucidation of the function and molecular mode of interaction of these components in yeast may help to understand metabolic insulin signaling in mammalian cells.
Müller
G
G
Hoechst Marion Roussel Deutschland GmbH, D-65926 Frankfurt am Main, Germany. Guenter.Mueller@hmrag.com
Rouveyre
N
N
Upshon
C
C
Bandlow
W
W
D005656:Fungal Proteins / Q000378:metabolism*; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology*; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D005656:Fungal Proteins; D007004:Hypoglycemic Agents; D007328:Insulin; D011972:Receptor, Insulin