Adducin in platelets: activation-induced phosphorylation by PKC and proteolysis by calpain
| Autor: | Rami Sarid, Diana M. Gilligan, Joleen Weese |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Adult
Blood Platelets Platelet Aggregation Immunology Biology In Vitro Techniques Biochemistry chemistry.chemical_compound Thrombin medicine Staurosporine Humans Platelet activation Phosphorylation Protein kinase C Protein Kinase C Calpain Cell Biology Hematology Dipeptides Platelet Activation Molecular biology Recombinant Proteins Cell biology Cytosol Cytoskeletal Proteins Kinetics chemistry biology.protein Phorbol Regression Analysis Calmodulin-Binding Proteins medicine.drug |
| Zdroj: | Blood. 99(7) |
| ISSN: | 0006-4971 |
| Popis: | Adducins are a family of cytoskeletal proteins encoded by 3 genes (alpha, beta, and gamma). Platelets express alpha and gamma adducins, in contrast to red blood cells that express alpha and beta adducins. During platelet activation with thrombin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate, alpha and gamma adducins were phosphorylated by protein kinase C (PKC) as detected by an antibody specific for a phosphopeptide sequence in the highly conserved carboxy terminus. Platelet activation also led to adducin proteolysis; inhibition by calpeptin suggests that the protease was calpain. The kinase inhibitor staurosporine inhibited PKC phosphorylation of adducin and also inhibited proteolysis of adducin. Experiments with recombinant alpha adducin demonstrated that the PKC-phosphorylated form was proteolyzed at a significantly faster rate than the unphosphorylated form. The concentration of adducin in platelets was estimated at 6 μM, similar to the concentration of capping protein. Fractionation of platelets into high-speed supernatant (cytosol) and pellet (membrane and cytoskeleton) revealed a shift of PKC-phosphorylated adducin to the cytosol during platelet activation. Platelet aggregation detected turbidometrically was decreased in the presence of staurosporine and was completely inhibited by calpeptin. Thrombin-induced changes in morphology were assessed by confocal microscopy with fluorescein phalloidin and were not prevented by staurosporine or calpeptin. Our results suggest that regulation of adducin function by PKC and calpain may play a role in platelet aggregation. |
| Databáze: | OpenAIRE |
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