Protein kinase C-dependent phosphorylation of a ciliary membrane protein and inhibition of ciliary beating

Matthias A Salathe, M. M. Pratt, Adam Wanner

Research output: Contribution to journalArticle

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Abstract

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 [γ-32P]ATP or [γ-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.

Original languageEnglish
Pages (from-to)1211-1220
Number of pages10
JournalJournal of Cell Science
Volume106
Issue number4
StatePublished - Dec 1 1993

Fingerprint

Protein Kinase C
Membrane Proteins
Phosphorylation
Sheep
Cilia
Protein Kinase Inhibitors
Membranes
Calcium-Calmodulin-Dependent Protein Kinase Kinase
Adenosine Triphosphate
Annexin A2
Annexin A1
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
Proteins
Protein C Inhibitor
Octoxynol
Protein-Tyrosine Kinases
Serine
Tyrosine
Electrophoresis
Polyacrylamide Gel Electrophoresis

Keywords

  • Ciliary beat frequency
  • Ciliary phosphorylation
  • Protein kinase C
  • Tyrosine kinase

ASJC Scopus subject areas

  • Cell Biology

Cite this

Protein kinase C-dependent phosphorylation of a ciliary membrane protein and inhibition of ciliary beating. / Salathe, Matthias A; Pratt, M. M.; Wanner, Adam.

In: Journal of Cell Science, Vol. 106, No. 4, 01.12.1993, p. 1211-1220.

Research output: Contribution to journalArticle

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