PROTEIN-KINASE C-DEPENDENT PHOSPHORYLATION OF A CILIARY MEMBRANE-PROTEIN AND INHIBITION OF CILIARY BEATING

The present study examined whether protein kinase C phosphorylated a c iliary protein and whether this phosphorylation event was temporally c orrelated with a decrease in ciliary beat frequency. Activation of pro tein kinase C decreased ciliary beat frequency of sheep tracheal epith elium, an effect fully blockable by pretreatment of the tissue pieces with H-7, a protein kinase inhibitor. Using cilia removed from these e pithelial surfaces and incubated in solutions containing stimulators o f protein kinase C along with [gamma-P-32]ATP or [gamma(35)S]ATP, a si ngle protein target of ciliary protein kinase C activity was identifie d. The protein is a polypeptide of molecular mass 37 kDa (p37) as esti mated by SDS-polyacrylamide gel electrophoresis. Protein kinase C depe ndency of p37 phosphorylation was proven by showing that Calphostin C, a specific protein kinase C inhibitor, blocked label incorporation in to p37 completely, and by demonstrating that purified protein kinase C phosphorylated p37. Inhibitors of cAMP-dependent kinase and calcium/c almodulin-dependent kinase did not change the phosphorylation of p37 i n the presence of protein kinase C activators. p37 was recovered in a Triton X-100-extractable fraction of this ciliary preparation, suggest ing that p37 is membrane associated. This hypothesis was further suppo rted by the fact that p37 was present in a pellet representing reconst ituted membranes. Thin-layer electrophoresis revealed that p37 was pho sphorylated on serine and tyrosine residues, suggesting that the activ ation of protein kinase C also stimulated tyrosine kinase activity. p3 7 did not precipitate with annexin I or II antibodies. These results s how that sheep tracheal cilia contain protein kinase C activity and th at activated protein kinase C phosphorylates a membrane-associated ovi ne ciliary target, an effect temporally related to a protein kinase C- mediated decrease in ciliary beat frequency.