Cyclic Adenosine Monophosphate-dependent Phosphorylation of Specific Fat Cell Membrane Proteins by an Endogenous Membrane-bound Protein Kinase: POSSIBLE INVOLVEMENT IN THE REGULATION OF INSULIN-STIMULATED GLUCOSE TRANSPORT

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Abstract The phosphorylation of specific membrane proteins by an endogenous protein kinase has been studied in purified membrane fractions from rat adipocytes using trichloroacetic acid precipitation and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The endogenous phosphorylation of two specific membrane proteins is completely dependent on the presence of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and magnesium ions. This phosphorylation occurs very rapidly at 24°, reaching maximal levels at 1 min. The number of sites specifically phosphorylated in the presence of cyclic AMP probably does not exceed 50,000 per fat cell, requiring the use of very high specific activity (10 to 50 Ci per mmole) [γ-32P]ATP for these studies. The minimal molecular weights of the two specifically phosphorylated proteins are about 22,000 and 16,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The same two proteins are phosphorylated when intact fat cells are exposed briefly to low concentrations of exogenous ATP, a process which results in the suppression of the insulin-stimulated rates of d-glucose transport. At least 95 % of the cyclic AMP-dependent 32P incorporated into trichloroacetic acid-precipitable protein is in the form of protein-bound phosphoserine. The concentration of cyclic AMP required for maximal stimulation of phosphorylation is about 1.5 µm. Cyclic guanosine 3':5'-monophosphate has no significant effect unless its concentration is increased to 10-4 m. Phosphorylation is inhibited by calcium ions. The cyclic AMP-stimulated phosphorylation is inhibited by phloretin and by 5'-adenylyl-β, γ-methylene triphosphonate. The specific cyclic AMP-dependent membrane phosphorylation is also found in membranes from cells of obese rats, an insulin-resistant animal. This phosphorylation system, however, cannot be detected in membranes from guinea pig fat cells. The lack of phosphorylation of specific membrane proteins in guinea pig fat cell membranes is correlated with the insulin insensitivity of this tissue to glucose transport (but not lipolysis) and with the inability of ATP to inhibit the slight stimulation which insulin exerts on glucose transport. The possibility is considered that this specific cyclic AMP-dependent phosphorylating system is involved in the hormonal regulation of glucose transport and the modulation of insulin sensitivity.