title: Vimentin is a target of PKCβ phosphorylation in MCP-1-activated primary human monocytes.
abstract: OBJECTIVE
  We designed a study to detect downstream phosphorylation targets of PKCβ in MCP-1-induced human monocytes.
  
  
  METHODS
  Two-dimensional gel electrophoresis was performed for monocytes treated with MCP-1 in the presence or absence of PKCβ antisense oligodeoxyribonucleotides (AS-ODN) or a PKCβ inhibitor peptide, followed by phospho- and total protein staining. Proteins that stained less intensely with the phospho-stain, when normalized to the total protein stain, in the presence of PKCβ AS-ODN or the PKCβ inhibitor peptide, were sequenced.
  
  
  RESULTS
  Of the proteins identified, vimentin was consistently identified using both experimental approaches. Upon (32)P-labeling and vimentin immunoprecipitation, increased phosphorylation of vimentin was observed in MCP-1 treated monocytes as compared to the untreated monocytes. Both PKCβ AS-ODN and the PKCβ inhibitor reduced MCP-1-induced vimentin phosphorylation. The IP of monocytes with anti-vimentin antibody and immunoblotting with a PKCβ antibody revealed that increased PKCβ becomes associated with vimentin upon MCP-1 activation. Upon MCP-1 treatment, monocytes were shown to secrete vimentin and secretion depended on PKCβ expression and activity.
  
  
  CONCLUSIONS
  We conclude that vimentin, a major intermediate filament protein, is a phosphorylation target of PKCβ in MCP-1-treated monocytes and that PKCβ phosphorylation is essential for vimentin secretion. Our recently published studies have implicated vimentin as a potent stimulator of the innate immune receptor Dectin-1 as reported by Thiagarajan et al. (Cardiovasc Res 99:494-504, 2013). Taken together our findings suggest that inhibition of PKCβ regulates vimentin secretion and, thereby, its interaction with Dectin-1 and downstream stimulation of superoxide anion production. Thus, PKCβ phosphorylation of vimentin likely plays an important role in propagating inflammatory responses.
authors: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH, 44195, USA.
authors: Praveena S
authors: PS
authors: Thiagarajan
authors: Ayse C
authors: AC
authors: Akbasli
authors: Michael T
authors: MT
authors: Kinter
authors: Belinda
authors: B
authors: Willard
authors: Martha K
authors: MK
authors: Cathcart
mesh_terms: D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000276:immunology*; D006801:Humans; D009000:Monocytes / Q000276:immunology*; D010766:Phosphorylation; D064546:Protein Kinase C beta / Q000276:immunology*; D014746:Vimentin / Q000276:immunology*
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D014746:Vimentin; D064546:Protein Kinase C beta
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