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 ciliary protein and whether this phosphorylation event was temporally correlated with a decrease in ciliary beat frequency. Activation of protein kinase C decreased ciliary beat frequency of sheep tracheal epithelium, an effect fully blockable by pretreatment of the tissue pieces with H-7, a protein kinase inhibitor. Using cilia removed from these epithelial surfaces and incubated in solutions containing stimulators of protein kinase C along with [gamma-32P]ATP or [gamma-35S]ATP, a single protein target of ciliary protein kinase C activity was identified. The protein is a polypeptide of molecular mass 37 kDa (p37) as estimated by SDS-polyacrylamide gel electrophoresis. Protein kinase C dependency of p37 phosphorylation was proven by showing that Calphostin C, a specific protein kinase C inhibitor, blocked label incorporation into p37 completely, and by demonstrating that purified protein kinase C phosphorylated p37. Inhibitors of cAMP-dependent kinase and calcium/calmodulin-dependent kinase did not change the phosphorylation of p37 in the presence of protein kinase C activators. p37 was recovered in a Triton X-100-extractable fraction of this ciliary preparation, suggesting that p37 is membrane associated. This hypothesis was further supported by the fact that p37 was present in a pellet representing reconstituted membranes. Thin-layer electrophoresis revealed that p37 was phosphorylated on serine and tyrosine residues, suggesting that the activation of protein kinase C also stimulated tyrosine kinase activity. p37 did not precipitate with annexin I or II antibodies. These results show that sheep tracheal cilia contain protein kinase C activity and that activated protein kinase C phosphorylates a membrane-associated ovine ciliary target, an effect temporally related to a protein kinase C-mediated decrease in ciliary beat frequency.
Salathe
M
M
Pulmonary Division (D-60), University of Miami School of Medicine, Florida 33136.
Pratt
M M
MM
Wanner
A
A
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D017302:Annexins / Q000302:isolation & purification; D002227:Carbazoles; D002458:Cell Fractionation; D002923:Cilia / Q000187:drug effects / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation; D004912:Erythrocytes / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D009068:Movement; D010766:Phosphorylation; D010768:Phosphoserine / Q000302:isolation & purification; D019000:Phosphotyrosine; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology; D012756:Sheep; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014132:Trachea / Q000166:cytology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000302:isolation & purification
D017302:Annexins; D002227:Carbazoles; D007211:Indoles; D007546:Isoquinolines; D008565:Membrane Proteins; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; C057416:KT 5720; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate