Gravin binds to endogenous PKA and PKC in vascular endothelial cells

Research output: Contribution to journalArticle

Abstract

Gravin, a 300 kDa intracellular protein, is upregulated in cells at the wound edge in wounded endothelial cell monolayers. Inhibition of gravin expression using antisense oligonucleotides inhibits endothelial wound healing. We postulate that gravin may play a role in endothelial wound healing, but the mechanism is unclear. A recent study showing that PKA and PKCα bind to gravin in HEL cells, however, suggests that gravin may play a role in endothelial wound healing through interactions with PKA and PKC. To determine if endogenous PKA and PKC bind to gravin in vascular endothelial cells, lysates prepared from cultured umbilical vein endothelial cells were immunoprecipitated with an anti-gravin antibody on protein A agarose beads and assayed for PKA and PKC. PKA was assayed by eluting the beads with 1 mM cAMP and analyzing the eluate for kinase activity and for the presence of the PKA catalytic subunit. The presence of PKC in the immunoprecipitates was assessed by Western blotting. Cyclic AMP eluates from anti-gravin immunoprecipitates showed kinase activity that was substantially higher than that in control eluates and which was inhibitable with a PKA specific inhibitor. Immunoblots probed with an anti-PKA catalytic subunit antibody confirmed the presence of the 40 kDa subunit in the gravin specific eluates. Immunoblots of the immunoprecipitates probed with an anti-PKCα antibody revealed an 80 kDa immunoreactive band indicating the presence of PKCα. These data support the hypothesis that PKA and PKCα interact with gravin. We postulate that gravin is a protein kinase binding protein that regulates PKA and PKC dependent pathways associated with endothelial wound healing.

Original languageEnglish (US)
JournalFASEB Journal
Volume12
Issue number5
StatePublished - Mar 20 1998
Externally publishedYes

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Endothelial cells
tissue repair
blood vessels
Wound Healing
endothelial cells
Endothelial Cells
Antibodies
Phosphotransferases
protein subunits
antibodies
Anti-Idiotypic Antibodies
Catalytic Domain
phosphotransferases (kinases)
Catalytic Antibodies
Antisense Oligonucleotides
Staphylococcal Protein A
Cyclic AMP
Sepharose
Protein Kinases
Umbilical Veins

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Gravin binds to endogenous PKA and PKC in vascular endothelial cells. / Grove, Bryon.

In: FASEB Journal, Vol. 12, No. 5, 20.03.1998.

Research output: Contribution to journalArticle

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AB - Gravin, a 300 kDa intracellular protein, is upregulated in cells at the wound edge in wounded endothelial cell monolayers. Inhibition of gravin expression using antisense oligonucleotides inhibits endothelial wound healing. We postulate that gravin may play a role in endothelial wound healing, but the mechanism is unclear. A recent study showing that PKA and PKCα bind to gravin in HEL cells, however, suggests that gravin may play a role in endothelial wound healing through interactions with PKA and PKC. To determine if endogenous PKA and PKC bind to gravin in vascular endothelial cells, lysates prepared from cultured umbilical vein endothelial cells were immunoprecipitated with an anti-gravin antibody on protein A agarose beads and assayed for PKA and PKC. PKA was assayed by eluting the beads with 1 mM cAMP and analyzing the eluate for kinase activity and for the presence of the PKA catalytic subunit. The presence of PKC in the immunoprecipitates was assessed by Western blotting. Cyclic AMP eluates from anti-gravin immunoprecipitates showed kinase activity that was substantially higher than that in control eluates and which was inhibitable with a PKA specific inhibitor. Immunoblots probed with an anti-PKA catalytic subunit antibody confirmed the presence of the 40 kDa subunit in the gravin specific eluates. Immunoblots of the immunoprecipitates probed with an anti-PKCα antibody revealed an 80 kDa immunoreactive band indicating the presence of PKCα. These data support the hypothesis that PKA and PKCα interact with gravin. We postulate that gravin is a protein kinase binding protein that regulates PKA and PKC dependent pathways associated with endothelial wound healing.

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