Evidence against insulin-stimulated phosphorylation of calmodulin in 3T3-L1 adipocytes.
To examine the possibility that insulin might stimulate calmodulin phosphorylation in intact cells, we compared autoradiographs of two-dimensional gels of [35S]methionine- and 32P-labeled proteins from 3T3-L1 adipocytes, before and after immunoprecipitation with anti-calmodulin antiserum. Insulin stimulated the phosphorylation of one or two proteins of approximately 22 kDa and pI 4.6; this increased phosphorylation was accompanied by an apparent shift in the position of the analogous [35S]methionine-labeled proteins towards the anode. In contrast, insulin had no effect on the phosphorylation state of another protein of 18-22 kDa and pI 4.6. This protein was very heavily labeled with [35S]methionine, co-migrated exactly with purified calmodulin, reacted specifically with two anti-calmodulin antibodies by Western blotting, and was specifically immunoprecipitated with the anti-calmodulin antiserum. Similar amounts of [35S]methionine-labeled calmodulin were immunoprecipitated from control and insulin-stimulated cells, arguing against the possibility that insulin-stimulated phosphorylation of calmodulin changed its affinity for the antibody. We conclude that calmodulin is phosphorylated to a negligible extent in serum-deprived 3T3-L1 adipocytes and that insulin does not stimulate its phosphorylation under conditions in which it stimulates the phosphorylation of one or more neighboring proteins.
Blackshear
P J
PJ
Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, North Carolina 27710.
Haupt
D M
DM
D000273:Adipose Tissue / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D004492:Edetic Acid / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D010766:Phosphorylation; D011233:Precipitin Tests
D002147:Calmodulin; D007328:Insulin; D004492:Edetic Acid