The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKC∊ isoform, these are Thr566 in the activation loop, Thr710 in the turn motif and Ser729 in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKC∊ kinase activity in controlling the phosphorylation of Thr566 and Ser729. In NIH 3T3 fibroblasts, PKC∊ migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr566 and Ser729. Cells transfected with an active PDK-1 allele also resulted in increased PKC∊ Thr566 and Ser729 phosphorylation, whereas an active myristoylated PKC∊ mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKC∊ were not phosphorylated at Ser729 in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr566, an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKC∊ in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P3, concomitant with an increase in Ser729 phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.
- phosphoinositide-dependent kinase 1
- phosphoinositide 3-kinase
- second messengers
- signal transduction
Abbreviations used: A.T.C.C., American Type Culture Collection; BIM I, bisindolylmaleimide I; DAG, diacylglycerol; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FLAG, MDYKDDDDK [methionine (M) was added by PCR as it represents the initiator residue)]; GST, glutathione S-transferase; HEK293, human embryonic kidney 293 cells; MBP, myelin basic protein; mTOR, mammalian target of rapamycin; OA, okadaic acid; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PDK-1, phosphoinositide-dependent kinase 1; PI 3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B; PKC, protein kinase C; PtdCho, phosphatidylcholine; PtdSer, phosphatidylserine; T566A (etc.), Thr566→Ala mutant; TBS, Tris-buffered saline; pThr (etc.), phosphothreonine (etc.).
- The Biochemical Society, London ©2002