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Phosphorylation at Ser729 specifies a Golgi localisation for protein kinase C epsilon (PKCepsilon) in 3T3 fibroblasts

Tian-Rui Xu1, Guiyuan He, Kath Dobson

  • 1SHWFGF-Proteomics Section, Joseph Black Building, University of Glasgow, University Avenue, Glasgow, G12 8QQ, UK.

Cellular Signalling
|July 6, 2007
PubMed

Insights

Cell passage causes protein kinase C epsilon (PKCepsilon) to move from the Golgi to the cell periphery. Serine 729 phosphorylation is crucial for PKCepsilon localization at the Golgi in fibroblasts.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Protein kinase C epsilon (PKCepsilon) is involved in various cellular processes.
  • The localization of PKCepsilon within cells is dynamic and can be influenced by cellular events.
  • Previous studies have indicated a role for PKCepsilon phosphorylation in its function.

Purpose of the Study:

  • To investigate the localization dynamics of GFP-PKCepsilon in living fibroblasts.
  • To determine the role of cell passage and phosphorylation at Serine 729 in PKCepsilon localization.
  • To explore the relationship between PKCepsilon localization and fibroblast secretory events.

Main Methods:

  • Live-cell imaging of GFP-PKCepsilon in fibroblasts.
  • Analysis of PKCepsilon colocalization with F-actin and Golgi markers (wheat germ agglutinin).
  • Biochemical fractionation to isolate Golgi-enriched fractions.
  • Site-directed mutagenesis of PKCepsilon at Serine 729 (S729A, S729E, S729T).
  • Assessment of glycosaminoglycan (GAG) secretion.

Main Results:

  • GFP-PKCepsilon concentrates at a perinuclear/Golgi site in proliferating fibroblasts.
  • Cell passage induces rapid translocation of PKCepsilon to the cell periphery, colocalizing with F-actin.
  • PKCepsilon returns to the perinuclear site as cells adhere and proliferate.
  • Phosphorylation at Ser729 is necessary for PKCepsilon localization at the perinuclear/Golgi site; dephosphorylation occurs upon translocation.
  • Mutants lacking phosphorylation at Ser729 do not localize to the perinuclear/Golgi site.
  • Cell passage increases GAG secretion, indicating an influence on Golgi secretory events.

Conclusions:

  • Serine 729 phosphorylation is critical for anchoring PKCepsilon at the perinuclear/Golgi site in fibroblasts.
  • Cell passage triggers a dynamic relocalization of PKCepsilon, linked to changes in secretory function.
  • Phosphorylation status at Ser729 regulates PKCepsilon localization and potentially its function in response to cellular events.

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