Regulation of novel protein kinase C ε by phosphorylation

Boston Biomedical Research Institute, Watertown, MA 02472, USA.
Biochemical Journal (Impact Factor: 4.4). 06/2002; 363(Pt 3):537-45. DOI: 10.1042/bj3630537
Source: PubMed


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 PKCepsilon isoform, these are Thr(566) in the activation loop, Thr(710) in the turn motif and Ser(729) in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKCepsilon kinase activity in controlling the phosphorylation of Thr(566) and Ser(729). In NIH 3T3 fibroblasts, PKCepsilon 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 Thr(566) and Ser(729). Cells transfected with an active PDK-1 allele also resulted in increased PKCepsilon Thr(566) and Ser(729) phosphorylation, whereas an active myristoylated PKCepsilon mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKCepsilon were not phosphorylated at Ser(729) in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr(566), 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 PKCepsilon in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P(3), concomitant with an increase in Ser(729) 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.

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    • "This provides a link with the phosphoinositide-3 kinase (PI3-kinase) pathway as the PI3-kinase products, PtdIns(3,4)P2 and PtdIns(3,4,5)P3, bind and recruit PDK-1 to membranes thus leading to phosphorylation of downstream substrates such as PKCε. Phosphorylation of Ser729 in the C-terminal hydrophobic motif depends on the internal catalytic activity of the kinase indicating that it occurs by autophosphorylation rather than an upstream kinase.14 Recent evidence indicates that Ser729 phosphorylation controls intracellular PKCε location and is required for the kinase to achieve mature conformation and to be primed for activation by co-factors.15,16 "
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    • "Studies of PKCí µí»¼ in a variety of cell types in culture have been consistent with the completion of such phosphorylation events only during enzyme maturation; however, for novel PKCs at least 2 groups have reported that the phosphorylations are dynamically regulated [12] [16] [19]. A study using NIH 3T3 cells reported that PKC phosphorylation increases in response to PDGF [16]. Also, PKCí µí¼€ and PKCí µí»¿ in cardiomyocytes in primary culture appear to undergo regulated and regulatory phosphorylation of the activation loop and the hydrophobic motif even in the absence of allosteric regulators [12]. "
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