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Lipocortins 1 and 2 as substrates for the insulin receptor kinase in rat liver
A Karasik1, R B Pepinsky, S E Shoelson
1Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02215.
Abstract:
Lipocortins 1 and 2 are major substrates for the epidermal growth factor receptor and the pp60v-src tyrosine kinases in transformed cells. In the present study, we have characterized the phosphorylation of lipocortins 1 and 2 by the insulin receptor tyrosine kinase in vitro and in vivo. In vitro, the solubilized insulin receptor, partially purified from rat liver, catalyzed phosphorylation of human recombinant lipocortin 1 and purified bovine lipocortin 2. Phosphorylation of lipocortin 1 was increased 15-fold upon stimulation with 10(-7) M insulin. The apparent Km of the reaction was 3.3 microM and was not affected by insulin stimulation. Insulin stimulated phosphate incorporation into lipocortin 2 by 20-fold (apparent Km greater than 20 microM). Both lipocortins were phosphorylated exclusively on tyrosine residues as judged by phosphoamino acid analysis. Based upon peptide mapping, lipocortin 1 was phosphorylated on Tyr-21, a site phosphorylated by other tyrosine kinases. Polyclonal anti-phosphotyrosine antibodies recognized the tyrosine-phosphorylated lipocortin 2, but not lipocortin 1 in its phosphorylated form. In hepatocytes from normal and dexamethasone-treated rats, lipocortin 1 content was less than 50 ng/10(6) cells. Insulin-induced phosphorylation of lipocortin 1 was detected in intact hepatocytes from corticosteroid-treated animals but not in cells from normal rats. No phosphorylation of lipocortin 2 was found, although its content was approximately 100 ng/10(6) cells from normal animals and increased to approximately 1 microgram/10(6) cells following treatment of rats with dexamethasone for 4 days. Thus, although lipocortins 1 and 2 are in vitro substrates of the insulin receptor kinase, only lipocortin 1 is phosphorylated in an insulin-dependent manner in intact hepatocytes, and this is only observed after dexamethasone treatment of the rats.
Insights
Insulin receptor kinase phosphorylates lipocortins 1 and 2 in vitro. In rat liver cells, only lipocortin 1 shows insulin-dependent phosphorylation, but only after dexamethasone treatment.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Endocrinology
Background:
- Lipocortins 1 and 2 are known substrates for tyrosine kinases like EGFR and Src.
- The role of insulin receptor tyrosine kinase (IRK) in lipocortin phosphorylation is not fully understood.
Purpose of the Study:
- To investigate the phosphorylation of lipocortins 1 and 2 by the insulin receptor tyrosine kinase (IRK) in vitro and in vivo.
- To determine the specific sites and conditions of lipocortin phosphorylation by IRK.
Main Methods:
- In vitro kinase assays using purified rat liver IRK and recombinant/purified lipocortins.
- Phosphoamino acid analysis and peptide mapping to identify phosphorylation sites.
- In vivo studies using isolated rat hepatocytes (normal and dexamethasone-treated) to assess insulin-induced phosphorylation.
Main Results:
- IRK catalyzed phosphorylation of lipocortins 1 and 2 in vitro, with insulin significantly increasing phosphorylation of both.
- Lipocortins were exclusively phosphorylated on tyrosine residues.
- Lipocortin 1 was phosphorylated at Tyr-21, a known site for other tyrosine kinases.
- In intact hepatocytes, insulin-induced lipocortin 1 phosphorylation was observed only in cells from dexamethasone-treated rats.
- Lipocortin 2 was not phosphorylated in intact hepatocytes, despite increased levels after dexamethasone treatment.
Conclusions:
- Lipocortins 1 and 2 are in vitro substrates for the insulin receptor kinase.
- Insulin-dependent phosphorylation of lipocortin 1 occurs in vivo in hepatocytes, but requires prior dexamethasone treatment.
- These findings highlight differential regulation of lipocortin phosphorylation by insulin and corticosteroids.