Accumulation of unphosphorylated calponin in the submembranous cytoskeletons of arachidonic acid-stimulated human platelets.
Calponin, a basic smooth-muscle protein capable of binding to F-actin, tropomyosin and calmodulin in vitro, was tested for its expression and subcellular localization in resting and stimulated human platelets. Using immunoblotting techniques calponin was revealed as a single protein band with a molecular weight of 34 kDa. Although calponin has been shown to be proteolytically degraded by calpain, in the presence of the calpain inhibitor E-64 and EGTA a significant hydrolysis of calponin could not be detected. Upon stimulation with 10 microM arachidonic acid calponin became increasingly incorporated into Triton X-100 insoluble cytoskeletal fractions reaching a plateau after 15 s. The accumulation of calponin in the cytoskeletons of stimulated platelets paralleled the polymerization of actin into newly formed microfilaments. Immunofluorescence microscopy revealed a submembranous co-localization of calponin and actin in aggregated platelets. Since isolated calponin is phosphorylated by protein kinase C and Ca2+/calmodulin-dependent protein kinase II thereby losing its inhibitory effect on the actomyosin MgATPase activity, we examined whether changes in cell shape due to platelet stimulation are accompanied by a phosphorylation of calponin. By performing immunoblotting analysis on either resting or stimulated platelets phosphorylation of calponin on tyrosine, serine or threonine residues could not be demonstrated. In line, [32P]radiolabeling experiments were unable to detect phosphate incorporation into calponin. These observations support the hypothesis that calponin plays a physiological role in regulating contraction and secretion of human platelets even in the absence of its phosphorylation.
Meyer
T
T
Department of Cardiology, University of Göttingen, Germany.
Unterberg
C
C
Kreuzer
H
H
Buchwald
A B
AB
D000199:Actins / Q000097:blood / Q000648:ultrastructure; D016718:Arachidonic Acid / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000097:blood*; D003598:Cytoskeletal Proteins / Q000097:blood*; D006801:Humans; D008840:Microfilament Proteins; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins / Q000097:blood*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000097:blood; D015539:Platelet Activation / Q000187:drug effects*; D011485:Protein Binding; D012694:Serine / Q000737:chemistry; D013268:Stimulation, Chemical; D013912:Threonine / Q000737:chemistry; D000096985:Calponins
D000199:Actins; D016718:Arachidonic Acid; D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D019000:Phosphotyrosine; D012694:Serine; D013912:Threonine; D000096985:Calponins