Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine, glucagon or vasopressin. 32P‐labeled glycogen synthase was purified from extracts of control or hormone‐treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 32P‐labeled polypeptide, apparent Mr 88000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent Mr 14000 (CB‐1) and 28000 (CB‐2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB‐2 phosphorylation was increased by all three hormones while CB‐1 was most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitro by individual protein kinases, it was found that electrophoretically similar CNBr fragments could be obtained. However, neither cyclic‐AMP‐dependent protein kinase nor three different Ca2+ ‐dependent enzymes (phosphorylase kinase, calmodulin‐dependent protein kinase, and protein kinase C) were effective in phosphorylating CB‐2. The protein kinases most effective towards CB‐2 were the Ca2+ and cyclic‐nucleotide‐independent enzymes casein kinase II (PC0.7) and FA/GSK‐3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as FA/GSK‐3, is involved or phosphatase activity is regulated, or both.
|Original language||English (US)|
|Number of pages||10|
|Journal||European Journal of Biochemistry|
|State||Published - Aug 1984|
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