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Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations
1Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Abstract:
Insulin stimulates autophosphorylation of the beta subunit of its receptor and activates the associated tyrosine kinase. This kinase, in turn, phosphorylates a number of specific protein substrates; however, the functional and structural identity of these substrates is largely unknown. In this study, we demonstrate that insulin also stimulates the phosphorylation of calmodulin by rat hepatocyte insulin receptors partially purified by wheat germ agglutinin affinity chromatography. Phosphorylation occurred predominantly on tyrosine residues and had an absolute requirement for insulin receptors, divalent cations, and certain basic proteins. Maximal 32P incorporation was observed at an insulin concentration of 5 X 10(-9) M, and the K0.5 for insulin was approximately 4 X 10(-10) M. Phosphorylation of calmodulin was dependent upon ATP, saturating at 100 microM ATP with a K0.5 of 30 microM. Insulin-stimulated phosphorylation of calmodulin was also dependent upon Mg2+ or Mn2+, but was approximately 12-fold greater in the presence of Mg2+. Maximal phosphorylation was observed in the absence of Ca2+ and was inhibited at Ca2+:EGTA ratios greater than 0.8 (0.16 microM free Ca2+). Certain basic proteins, such as polylysine, histone Hf2b, and protamine sulfate, were necessary to observe insulin-stimulated phosphorylation of calmodulin. The relative amount of insulin-stimulated phosphorylation of calmodulin observed in the presence of each of these proteins differed. Maximal insulin-stimulated phosphorylation was observed in the presence of polylysine. These data suggest that both Ca2+ and calmodulin may participate in the early post-receptor events in the cellular mechanism of insulin action in hepatocytes.
Insights
Insulin signaling involves the tyrosine kinase activity of insulin receptors, which can phosphorylate calmodulin. This insulin-stimulated calmodulin phosphorylation, dependent on specific conditions, suggests its role in early insulin action mechanisms in hepatocytes.
Area of Science:
- Biochemistry
- Cell Biology
- Endocrinology
Background:
- Insulin receptor activation initiates intracellular signaling cascades.
- Tyrosine kinase activity of the insulin receptor phosphorylates various substrates.
- The identity and function of many insulin receptor substrates remain largely unknown.
Purpose of the Study:
- To investigate whether insulin receptors can phosphorylate calmodulin.
- To characterize the conditions required for insulin-stimulated calmodulin phosphorylation.
- To explore the potential role of calmodulin in insulin signaling pathways.
Main Methods:
- Partial purification of rat hepatocyte insulin receptors using wheat germ agglutinin affinity chromatography.
- Assaying insulin-stimulated phosphorylation of calmodulin using radiolabeled phosphate (32P).
- Varying concentrations of insulin, ATP, divalent cations (Mg2+, Mn2+), and Ca2+ to determine optimal phosphorylation conditions.
Main Results:
- Insulin receptors partially purified from rat hepatocytes stimulate the tyrosine phosphorylation of calmodulin.
- Insulin-stimulated calmodulin phosphorylation requires insulin receptors, divalent cations (Mg2+ preferred over Mn2+), and basic proteins (e.g., polylysine).
- Optimal insulin concentration was 5 X 10(-9) M, with a K0.5 of 4 X 10(-10) M; ATP K0.5 was 30 microM.
- Phosphorylation was maximal in the absence of Ca2+ and inhibited by higher Ca2+ concentrations.
Conclusions:
- Calmodulin is a novel substrate for the insulin receptor tyrosine kinase.
- Insulin-stimulated calmodulin phosphorylation occurs under specific ionic and protein conditions.
- These findings suggest a potential role for Ca2+ and calmodulin in early post-receptor insulin signaling events in hepatocytes.