Role of protein kinase C in chick embryo skeletal myoblast fusion

Abstract

The involvement of Ca 2+ and PGE 1 in myoblast fusion has been well documented. Extracellular Ca 2+ is essential for myoblast adhesion, alignment, and fusion. There is an obligatory increase in Ca 2+ influx immediately preceding fusion and the Ca 2+ ionophore A23187 promotes precocious fusion. PGE 1 receptors appear just prior to fusion, and an antagonist of PGE 1 binding to cell surface receptors blocks fusion when added prior to the Ca 2+ influx. Finally, exogenous PGE 1 induces precocious fusion. The present study was an initial test of the hypothesis that membrane protein phosphorylation by protein kinase C (PKC) links PGE 1 receptor occupancy and the increase in Ca 2+ influx. Our conclusion that PKC is an essential component in the regulation of myoblast fusion is based in part on the following evidence: (1) an activator of PKC, the tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA), at low concentration and for a brief exposure period, induces precocious fusion and stimulates Ca 2+ influx; (2) 4α-phorbol-12,13-didecanoate, an inactive analog of TPA, has no discernible effect on fusion or Ca 2+ influx; (3) 1-oleoyl-2-acetyl glycerol, an analog of endogenous diacylglycerol (DAG) which activates PKC, promotes precocious fusion, as does the DAG kinase inhibitor R59022 (6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl]ethyl]-7-methyl-5 H-thiazole-[3,2α]-pyrimidin-5-one) which raises the level of endogenous DAG by inhibiting its catabolism; (4) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a highly potent PKC inhibitor, reversibly blocks myogenesis at a point between alignment and fusion; and (5) H-7 also blocks the normal increase in Ca 2+ influx preceding fusion.