Related Experiment Videos
Cyclin-dependent kinase 5 promotes insulin exocytosis
1Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Abstract:
Cyclin-dependent kinase 5 (Cdk5) is widely expressed although kinase activity has been described preferentially in neuronal systems. Cdk5 has an impact on actin polymerization during neuronal migration and neurite outgrowth and deregulation of the kinase has been implicated in the promotion of neurodegeneration. Recently it was shown that Cdk5 modulates dopamine signaling in neurons by regulating DARPP-32 function. In addition, Cdk5 phosphorylates munc-18 and synapsin I, two essential components of the exocytotic machinery. We have shown by reverse transcriptase-polymerase chain reaction, immunocytochemistry, and Western blotting that Cdk5 is present in the insulin-secreting pancreatic beta-cell. Subcellular fractionation of isolated beta-cells revealed a glucose-induced translocation of membrane-bound Cdk5 protein to lower density fractions. Inhibition of Cdk5 with roscovitine reduced insulin secretion with approximately 35% compared with control after glucose stimulation and with approximately 65% after depolarization with glucose and KCl. Capacitance measurements performed on single beta-cells that expressed a dominant-negative Cdk5 mutant showed impaired exocytosis. The effect on exocytosis by Cdk5 appeared to be independent of changes in free cytoplasmic Ca(2+) concentration. Taken together these results show that Cdk5 is present in beta-cells and acts as a positive regulator of insulin exocytosis.
Insights
Cyclin-dependent kinase 5 (Cdk5) regulates insulin secretion in pancreatic beta-cells. This kinase is crucial for insulin exocytosis, impacting glucose-stimulated insulin release and depolarization.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Cyclin-dependent kinase 5 (Cdk5) is primarily known for its roles in neuronal development and function.
- Deregulation of Cdk5 is linked to neurodegenerative diseases.
- Cdk5 influences cellular processes through protein phosphorylation, including components of the exocytotic machinery.
Purpose of the Study:
- To investigate the presence and function of Cdk5 in pancreatic beta-cells.
- To determine if Cdk5 plays a role in regulating insulin secretion.
Main Methods:
- Reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry, and Western blotting to detect Cdk5 expression in beta-cells.
- Subcellular fractionation to analyze Cdk5 localization.
- Pharmacological inhibition of Cdk5 using roscovitine.
- Electrophysiological measurements (capacitance) using dominant-negative Cdk5 mutants.
Main Results:
- Cdk5 protein was detected in insulin-secreting pancreatic beta-cells.
- Glucose stimulation induced translocation of membrane-bound Cdk5 to lower density fractions.
- Inhibition of Cdk5 significantly reduced glucose- and depolarization-stimulated insulin secretion.
- Expression of a dominant-negative Cdk5 mutant impaired exocytosis, independent of cytoplasmic calcium levels.
Conclusions:
- Cdk5 is present and functional in pancreatic beta-cells.
- Cdk5 acts as a positive regulator of insulin exocytosis.
- Cdk5 plays a significant role in the glucose-stimulated insulin secretion pathway.