Phosphorylation at Ser729 specifies a Golgi localisation for protein kinase C epsilon (PKCepsilon) in 3T3 fibroblasts.
We demonstrate that GFP-PKCepsilon concentrates at a perinuclear site in living fibroblasts and that cell passage induces rapid translocation of PKCepsilon to the periphery where it appears to colocalise with F-actin. When newly passaged cells have adhered and are proliferating again, GFP-PKCepsilon returns to its perinuclear site. GFP-PKCepsilon co-localises with wheat germ agglutinin suggesting that it is associated with the Golgi at the perinuclear site. In support, PKCepsilon is detected in a Golgi-enriched fraction in pre-passage cells but is lost from the fraction after passage. PKCepsilon at the perinuclear Golgi site is phosphorylated at Ser729 but cell passage induces the loss of the phosphate at this site as reported previously [England et al. (2001) J. Biol. Chem. 276, 10437-10442]. PKCepsilon S729A, S729E and S729T mutants, which are not recognised by a specific antiphosphoPKCepsilon (Ser729) antibody, do not concentrate at a perinuclear/Golgi site in proliferating fibroblasts. This suggests that both phosphorylation and serine rather than threonine are needed at position 729 to locate PKCepsilon at its perinuclear/Golgi site. Phorbol ester induced translocation of PKCepsilon to the nucleus also requires dephosphorylation at Ser729; after translocation nuclear PKCepsilon lacks a phosphate at Ser729. Sulphation and secretion of glycosaminoglycan (GAG) chains from fibroblasts increases on passage and returns to basal as cells proliferate showing that cell passage influences secretory events at the Golgi. The results indicate that Ser729 phosphorylation plays a role in determining PKCepsilon localisation in fibroblasts.
Xu
Tian-Rui
TR
SHWFGF-Proteomics Section, Joseph Black Building, University of Glasgow, University Avenue, Glasgow, G12 8QQ, UK.
He
Guiyuan
G
Dobson
Kath
K
England
Karen
K
Rumsby
Martin
M
D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D049109:Cell Proliferation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006025:Glycosaminoglycans / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism; D041681:NIH 3T3 Cells; D010703:Phorbol Esters / Q000494:pharmacology; D010768:Phosphoserine / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D013455:Sulfur / Q000378:metabolism
D045163:Antibodies, Phospho-Specific; D006025:Glycosaminoglycans; D050505:Mutant Proteins; D010703:Phorbol Esters; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010768:Phosphoserine; D013455:Sulfur; D051744:Protein Kinase C-epsilon