Research article

Changes in protein kinase C ∊ phosphorylation status and intracellular localization as 3T3 and 3T6 fibroblasts grow to confluency and quiescence: a role for phosphorylation at Ser-729?

Karen ENGLAND, Martin G. RUMSBY

Abstract

Protein kinase C (PKC) ε in 3T3 and 3T6 fibroblasts and in C6 glioma cells migrated on SDS/PAGE predominantly as a doublet with molecular masses of 87 and 95kDa (PKC ε87 and PKC ε95 respectively). PKC ε95 predominates when cells reach confluency but PKC ε87 was the main form detected within 15min when confluent cells were passaged at low cell density into fresh medium containing serum and allowed to adhere. Matrix-assisted laser-desorption ionization–time-of-flight MS analysis and experiments with phosphospecific antibodies revealed that PKC ε87 is phosphorylated at Thr-566 and Ser-703, and PKC ε95 is additionally phosphorylated at Ser-729. Cell fractionation studies revealed that PKC ε95 is associated with the nuclear fraction, whereas PKC ε87 was found in the 100000g cytosol fraction. Immunofluorescence studies confirmed these findings and showed that PKC ε95 had a perinuclear, probably Golgi, localization and PKC ε87 was distributed in the cytosol. It is proposed that phosphorylation at Ser-729 may be important for determining the intracellular localization of PKC ε, and that a specific Ser-729 phosphatase may be activated on cell passage to convert PKC ε95 to PKC ε87.

  • fibroblasts
  • Golgi
  • MALDITOF-MS
  • phosphorylation
  • protein kinase C