Coordinate phosphorylation of insulin-receptor kinase and its 175,000-Mr endogenous substrate in rat hepatocytes
- Joslin Diabetes Center, Boston, MA (USA)
To investigate the early events in insulin signal transmission in liver, isolated rat hepatocytes were labeled with {sup 32}P, and proteins phosphorylated in response to insulin were detected by immunoprecipitation with anti-phosphotyrosine and anti-receptor antibodies and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography. In these cells, insulin rapidly stimulated tyrosine phosphorylation of the 95,000-Mr beta-subunit of the insulin receptor and a 175,000-Mr phosphoprotein (pp175). Both proteins were precipitated by anti-phosphotyrosine antibody, whereas only the insulin receptor was recognized with anti-insulin-receptor antibody. In the insulin-stimulated state, both pp175 and the receptor beta-subunit were found to be phosphorylated on tyrosine and serine residues. Based on precipitation by the two antibodies, receptor phosphorylation was biphasic with an initial increase in tyrosine phosphorylation followed by a more gradual increase in serine phosphorylation over the first 30 min of stimulation. The time course of phosphorylation of pp175 was rapid and paralleled that of the beta-subunit of the insulin receptor. The pp175 was clearly distinguished from the insulin receptor, because it was detected only when boiling SDS was used to extract cellular phosphoproteins, whereas the insulin receptor was extracted with either Triton X-100 or SDS. In addition, the tryptic peptide maps of the two proteins were distinct. The dose-response curve for insulin stimulation was shifted slightly to the left of the insulin receptor, suggesting some signal amplification at this step. These data suggest that pp175 is a major endogenous substrate of the insulin receptor in liver and may be a cytoskeletal-associated protein.
- OSTI ID:
- 5604255
- Journal Information:
- Diabetes; (United States), Journal Name: Diabetes; (United States) Vol. 40:1; ISSN 0012-1797; ISSN DIAEA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMAL CELLS
ANIMALS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
HORMONES
HYDROXY ACIDS
INSULIN
ISOTOPES
KINETICS
LIGHT NUCLEI
LIVER CELLS
MAMMALS
MEMBRANE PROTEINS
MOLECULAR WEIGHT
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PEPTIDE HORMONES
PHOSPHATES
PHOSPHOPROTEINS
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
PROTEINS
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
SERINE
SOMATIC CELLS
TYROSINE
VERTEBRATES