Functional characterization of autophosphorylation sites of the activated insulin receptor-tyrosine kinase

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6079975

Flores-Riveros, J R; Lane, M D

Insulin receptor, solubilized from 3T3-L1 cellular membranes and then purified, was autophosphorylated with (..gamma..-/sup 32/P)ATP in the absence or presence of insulin. Specific phosphopeptides generated by trypsin digestion of the /sup 32/P-labeled ..beta..-subunit were identified and separated by reverse phase HPLC. In the absence of insulin, radioactivity of the phosphopeptides is evenly distributed among four major peaks designated as sites I, II, III and IV, according to their order of elution. This pattern is maintained for at least the first 30 min of autophosphorylation. When the reaction is carried out in the presence of insulin, > 50% of the total /sup 32/P radioactivity is found in site I and the rate of /sup 32/P incorporation into this site is markedly higher than into sites II, III and IV. Maximal activation of tyrosine kinase activity, as estimated by substrate phosphorylation, is coincident with the nearly complete phosphorylation of site I. Delayed activation of previously autophosphorylated receptor by insulin, but not by EGF or IGF-I, produced a similar pattern where phosphorylated site I predominates. These observations indicate that one major insulin-regulated autophosphorylation site in the ..beta..-subunit is responsible for activation of the insulin receptor tyrosine kinase. The isolation of this phosphopeptide on a preparative scale and its characterization are now in progress.

• Research Organization: Johns Hopkins Univ. School of Medicine, Baltimore, MD
• OSTI ID: 6079975
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ENZYME ACTIVITY
ENZYMES
GROWTH FACTORS
HORMONES
HYDROLASES
HYDROXY ACIDS
INSULIN
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
MEMBRANE PROTEINS
MITOGENS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PEPTIDE HYDROLASES
PEPTIDES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
SEPARATION PROCESSES
SERINE PROTEINASES
TRACER TECHNIQUES
TRANSFERASES
TRYPSIN
TYROSINE