Glucose-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) in isolated rat pancreatic islets
| Autor: | R, Calle, S, Ganesan, J I, Smallwood, H, Rasmussen |
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| Rok vydání: | 1992 |
| Předmět: |
Male
Intracellular Signaling Peptides and Proteins Fluorescent Antibody Technique Membrane Proteins Proteins Rats Inbred Strains In Vitro Techniques Staurosporine Precipitin Tests Rats Islets of Langerhans Alkaloids Glucose Insulin Secretion Animals Autoradiography Insulin Electrophoresis Polyacrylamide Gel Phosphorylation Myristoylated Alanine-Rich C Kinase Substrate Protein Kinase C |
| Zdroj: | The Journal of biological chemistry. 267(26) |
| ISSN: | 0021-9258 |
| Popis: | In order to further evaluate the role of protein kinase C activation in glucose-induced insulin secretion, the extent of phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) was examined in freshly isolated rat pancreatic islets prelabeled with [32P]orthophosphate. The islets were incubated with either 2.75 mM glucose alone, 2.75 mM glucose + 1 microM phorbol myristate acetate, 20 mM glucose, or 20 mM glucose + 50 nM staurosporine. After stimulation, the homogenized islets were processed by immunoprecipitation with a specific polyclonal anti-MARCKS antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densitometric analysis of autoradiograms revealed that phorbol myristate acetate caused a 3.78 +/- 0.97-fold increase in MARCKS phosphorylation over control. In the islets exposed to 20 mM glucose, an increase of 3.43 +/- 0.46-fold over control was observed. In islets exposed to G20 + 50 nM staurosporine, MARCKS phosphorylation was inhibited by 90 +/- 4% compared with control islets exposed to 20 mM glucose alone. Islets similarly treated (but incubated without 32P) were examined by immunocytochemistry using an alpha-PKC-specific monoclonal antibody and visualized by confocal immunofluorescence microscopy. The alpha-PKC redistributed from the cytosol to the plasma membrane in the beta-cells of islets exposed to 20 mM glucose. In separate experiments, unlabeled but similarly treated islets were shown to respond with a 5-7-fold increase in insulin secretion in static incubation. Thus, when freshly isolated rat pancreatic islets are exposed to stimulatory glucose concentrations, they exhibit both a translocation of alpha-PKC and a significant increase in the extent of phosphorylation of MARCKS protein. These data suggest that alpha-PKC is activated during glucose-induced insulin secretion. |
| Databáze: | OpenAIRE |
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