Phorbol ester stimulates the synthesis and phosphorylation of a 48 kDa-intracellular protein in plasminogen activator secreting melanoma cells.
Phorbol ester (12-O-tetradecanoyl-phorbol 13-acetate) stimulates the secretion of tissue-type plasminogen activator by the melanoma cell line, Bowes. This effect is associated with increased levels of mRNAs for both tissue-type plasminogen activator and a 48 kDa-protein. Labelling of melanoma cells with L-[35S]methionine allowed to identify an intracellular protein which, by 3 criteria, was identical with the in vitro translation product of the 48kDa-protein mRNA: a Mr of 48,000 on electrophoresis in the presence of sodium dodecyl sulphate; inducibility by phorbol ester and failure of reducing agents to affect electrophoretic mobility. As detectable by L-[35S]methionine labelling, the protein was mainly localized in the cytosol. In vitro phosphorylation reactions, carried out on subcellular fractions revealed a membrane-associated protein which also had the three characteristics of the aforementioned 48 kDa-protein. Phosphorylation did not require Ca2+-ions. Addition of phorbol ester to the reaction mixtures increased the phosphorylation. Reconstitution experiments between membrane and cytosol fractions of phorbol ester-treated and untreated cells showed that the 48kDa protein occurs in a cytosolic, unphosphorylated and a membrane-bound, phosphorylated form and that the former is converted to the latter by a phorbol ester activated, membrane-associated protein kinase.
D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000378:metabolism*; D010704:Phorbols / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D009363:Neoplasm Proteins; D010704:Phorbols; D010750:Phosphoproteins; D010959:Tissue Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate