title: Fibronectin phosphorylation by ecto-protein kinase.
abstract: The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with [gamma-32]ATP for 10 min at 37 degrees C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with [gamma-32P]ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.
authors.affiliation: Meiji Institute of Health Science, Meiji Milk Products Co., Inc., Odawara, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Imada
authors.forename: Y
authors.initials: Y
authors.lastname: Sugiyama
authors.forename: M
authors.initials: M
authors.lastname: Imada
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D005353:Fibronectins / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D005353:Fibronectins; D010748:Phosphopeptides; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C034454:ectoprotein kinase