title: Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells.
abstract: Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.
authors.affiliation: Department of Pharmacology, Nagoya University School of Medicine, Aichi.
authors.forename: M
authors.initials: M
authors.lastname: Terasawa
authors.forename: H
authors.initials: H
authors.lastname: Tokumitsu
authors.forename: R
authors.initials: R
authors.lastname: Kobayashi
authors.forename: H
authors.initials: H
authors.lastname: Hidaka
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D008970:Molecular Weight; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
chemical_list: D003598:Cytoskeletal Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases