Insulin and glucagon regulation of protein phosphorylation in isolated hepatocytes. Persistence, reversibility, and concentration dependence of hormonal effect. Evidence for common phosphorylation sites for both hormones on the Mr = 46,000 protein.
1982; Elsevier BV; Volume: 257; Issue: 14 Linguagem: Inglês
10.1016/s0021-9258(18)34342-4
ISSN1083-351X
Autores Tópico(s)Muscle metabolism and nutrition
ResumoThe effects of insulin and glucagon on the phosphorylation of proteins were investigated in hepatocytes isolated from fed rats.Proteins present in total cell lysates prepared from hepatocytes incubated with 32Pi were analyzed by one-and two-dimensional polyacrylamide gel electrophoresis.In agreement with previous observations, glucagon (or 8-bromo-CAMP) was shown to enhance to a variable extent the degree of phosphorylation of proteins with M, = 185,000, 93,000, 61,000, 48,000, 46,000, 38,000, and 34,000, whereas insulin essentially increased the phosphorylation of proteins with M, = 46,000 and 34,000, Regardless of the protein considered, changes caused by insulin and glucagon were maximal after a 10-min exposure of the cells to hormone and remained stable for at least 2 h in the continuous presence of the hormone.Removal of the hormone from the incubation medium caused a rapid dephosphorylation of the hormone-sensitive proteins (tl,z = 15-20 min).Activation of cyclic nucleotide-dependent protein kinase by glucagon displayed the same dependence upon the presence of the hormone.Thus, variations in hormone concentration in cell environment rather than cell desensitization appear to be the main factor controlling the duration of bGth insulin and glucagon effects on protein phosphorylation in hepatocytes.Dose-dependence analysis revealed a high sensitivity for insulin effects on protein phosphorylation, halfmaximal and maximal effects being obtained with 3-5 x M and 10"" M, respectively.The stimulation of protein phosphorylation by glucagon was also very sensitive since half-maximal effects were obtained with 2-5 X 10"' M and maximal stimulation with 1-3 X 10"' M. Protein phosphorylation was maximally stimulated by glucagon under conditions where only 10 to 20% of maximal activation of the cyclic nucleotide-dependent protein kinase was observed, indicating that the two processes were not linearly coupled.Glucagon-specific changes in protein kinase activity and in protein phosphorylation induced by a low hormone concentration were partially antagonized by a high insulin concentration.Conversely, the two hormones increased the phosphorylation of a M, = 46,000
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