Specific in situ phosphorylation of plectin in detergent-resistant cytoskeletons from cultured Chinese hamster ovary cells.
Plectin (Mr = 300,000) was found to be an abundant polypeptide component of Chinese hamster ovary cells accounting for up to 1% of cellular protein. Seventy-five per cent of the plectin were present in cytoskeletons prepared by extraction of attached cells with 0.15% Triton X-100. As shown by immunofluorescence microscopy, plectin's spatial arrangement within these cytoskeletal preparations appeared well preserved, though slightly more filamentous compared to nonextracted cells. Upon in situ incubation of cytoskeletons with [gamma-32P] ATP followed by solubilization and immunoprecipitation, plectin was identified as one of the major phosphoacceptors. A basic phosphorylation of the protein was accomplished by a type I cAMP-independent protein kinase, while a cAMP-dependent protein kinase enhanced its phosphorylation up to 2-fold. Peptide mapping revealed that the two kinases phosphorylated different molecular sites. Peptide maps generated from cytoskeletal plectin phosphorylated in vitro using [gamma-32P]ATP and plectin phosphorylated in vivo using 32Pi were virtually identical demonstrating that the in situ phosphorylation of plectin in preparations of cytoskeletons was specific. Moreover, the specific radioactivity of cytoskeletal plectin was three times higher than that of detergent-extracted plectin, suggesting that phosphorylation is important for the protein's association with the cytoskeleton.
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003599:Cytoskeleton / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D007381:Intermediate Filament Proteins; D017830:Octoxynol; D010053:Ovary / Q000648:ultrastructure*; D010766:Phosphorylation; D051190:Plectin; D011092:Polyethylene Glycols; D011506:Proteins / Q000378:metabolism*
D007381:Intermediate Filament Proteins; D051190:Plectin; D011506:Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol