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

We demonstrate that GFP-PKC epsilon concentrates at a perinuclear site in living fibroblasts and that cell passage induces rapid translocation of PKC epsilon to the periphery where it appears to colocalise with F-actin. When newly passaged cells have adhered and are proliferating again, GFP-PKC epsilon returns to its perinuclear site. GFP-PKC epsilon co-localises with wheat germ agglutinin suggesting that it is associated with the Golgi at the perinuclear site. In support, PKC epsilon is detected in a Golgi-enriched fraction in pre-passage cells but is lost from the fraction after passage. PKC epsilon 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]. PKC epsilon S729A, S729E and S729T mutants, which are not recognised by a specific antiphosphoPKC epsilon (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 PKC epsilon at its perinuclear/Golgi site. Phorbol ester induced translocation of PKC epsilon to the nucleus also requires dephosphorylation at Ser729; after translocation nuclear PKC epsilon 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 PKC epsilon localisation in fibroblasts. (c) 2007 Elsevier Inc. All rights reserved.