Similarities of cow mammary gland cytosolic 21-kDa protein, the substrate for protein kinase C, to the 20-kDa myosin light chain from smooth muscle

In the cytosol of cow mammary gland, several proteins are phosphorylated in the presence of the protein kinase C (PKC) cofactors 1-oleoyl-2-acetyl-sn-glycerol (OAG), phosphatidylserine (PS) and Ca2+. Of the substrates, the 21-kDa protein is inferred to be a 20-kDa regulatory myosin light chain (MLC20) from smooth muscle because of its molecular mass, its distribution in the cytosol, its association with melittin and sphingosine (the PKC modulators), and phosphorylation by PKC as well as by Ca2+/calmodulin-dependent myosin light chain kinase (MLCK). The present study was undertaken to examine whether the 21-kDa protein could be identified as MLC20, by adding cow uterine MLC20 to the reaction mixture containing cytosol with or without the PKC cofactors and/or calmodulin. In the absence of MLC20, the 21-kDa protein was phosphorylated when the PKC cofactors and calmodulin were added to the reaction mixture. Phosphorylation of the 21-kDa protein was inhibited by melittin or sphingosine, and the inhibition was reversed by PS, but not by calmodulin. When MLC20 was included in the reaction mixture, it was phosphorylated in the presence of the PKC cofactors, and the phosphorylated MLC20 band overlapped that of the 21-kDa protein. The indistinguishably overlapped band of the two proteins was inhibited by melittin and by sphingosine, and their inhibition was reversed by PS, not by calmodulin. It is suggested that the 21-kDa protein is the smooth muscle MLC20 and also that the 21-kDa MLC20 is phosphorylated by PKC, but not by MLCK.