Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers

Jerome E. Stasek, Carolyn E. Patterson, Joe GN Garcia

Research output: Contribution to journalArticle

147 Citations (Scopus)

Abstract

Cytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non-muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca2+-phospholipid-dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and α-thrombin, a receptor-mediated agonist known to increase endothelial cell permeability, both induced rapid, dose-dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by γ-[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist-mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist-induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (∼200% increase with 10-6 MPMA, ∼400% increase with 10-8 M α-thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC-mediated phosphorylation of two specific CSPs, the actin- and calmodulin-binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]-labeled BPAEC monolayers stimulated with either PMA or α-thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented α-thrombin- and PMA-induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist-induced barrier dysfunction. These results demonstrate that agonist-stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayers, an event which occurs in concert with agonist-mediated endothelial cell contraction and resultant barrier dysfunction.

Original languageEnglish (US)
Pages (from-to)62-75
Number of pages14
JournalJournal of Cellular Physiology
Volume153
Issue number1
StatePublished - Oct 1992
Externally publishedYes

Fingerprint

Endothelial cells
Vimentin
Protein Kinase C
Pulmonary Artery
Albumins
Monolayers
Permeability
Endothelial Cells
Phosphorylation
Thrombin
Chemical activation
Cytoskeletal Proteins
Tetradecanoylphorbol Acetate
Calmodulin-Binding Proteins
Muscle
Thrombin Receptors
Intermediate Filament Proteins
Microfilament Proteins
Contractile Proteins
Staurosporine

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. / Stasek, Jerome E.; Patterson, Carolyn E.; Garcia, Joe GN.

In: Journal of Cellular Physiology, Vol. 153, No. 1, 10.1992, p. 62-75.

Research output: Contribution to journalArticle

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abstract = "Cytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non-muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca2+-phospholipid-dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and α-thrombin, a receptor-mediated agonist known to increase endothelial cell permeability, both induced rapid, dose-dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by γ-[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist-mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist-induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (∼200{\%} increase with 10-6 MPMA, ∼400{\%} increase with 10-8 M α-thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC-mediated phosphorylation of two specific CSPs, the actin- and calmodulin-binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77 and vimentin phosphorylation were observed in intact [32P]-labeled BPAEC monolayers stimulated with either PMA or α-thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented α-thrombin- and PMA-induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist-induced barrier dysfunction. These results demonstrate that agonist-stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayers, an event which occurs in concert with agonist-mediated endothelial cell contraction and resultant barrier dysfunction.",
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