Prostaglandin F2α-activated protein kinase Cα phosphorylates myristoylated alanine-rich C kinase substrate protein in bovine luteal cells

Prostaglandin F2α (PGF2α)-induced secretion of oxytocin by the bovine corpus luteum involves the phosphorylation of a unique protein kinase C (PKC) substrate, myristoylated alanine-rich C kinase substrate (MARCKS) protein. This study was conducted to determine the specific PKC isoform engaged in phosphorylation of MARCKS protein in bovine luteal cells. In experiment 1, dispersed luteal cells recovered from the corpus luteum on d 8 of the estrous cycle were preincubated with [32P] orthophosphate and then exposed to PGF2α alone or in combination with PKC inhibitors. Autoradiography and densitometry of Western blots revealed that MARCKS protein was phosphorylated by a conventional PKC (cPKC) isoform. Experiment 2 was conducted to identify the specific cPKC isoform that phosphorylates MARCKS protein in luteal cells. Corpora lutea were removed from control and PGF2α-treated heifers on d 8 of the cycle, and PKC isoforms associated with membrane and cytosolic fractions were determined. Treatment with PGF2α increased membrane concentrations of PKCα within 5 min after treatment (p<0.005). Collectively, these data suggest that phosphorylation of MARCKS protein coinciding with oxytocin secretion is mediated by PKCα.