Article

Regulation of novel protein kinase C ε by phosphorylation

Boston Biomedical Research Institute, Watertown, MA 02472, USA.
Biochemical Journal (Impact Factor: 4.78). 06/2002; 363(Pt 3):537-45.
Source: PubMed

ABSTRACT 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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    • "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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    ABSTRACT: Protein kinase C (PKC) is a key enzyme involved in agonist-induced smooth muscle contraction. In some cases, regulatory phosphorylation of PKC is required for full activation of the enzyme. However, this issue has largely been ignored with respect to PKC-dependent regulation of contractile vascular smooth muscle (VSM) contractility. The first event in PKC regulation is a transphosphorylation by PDK at a conserved threonine in the activation loop of PKC, followed by the subsequent autophosphorylation at the turn motif and hydrophobic motif sites. In the present study, we determined whether phosphorylation of PKC is a regulated process in VSM and also investigated a potential role of calponin in the regulation of PKC. We found that calponin increases the level of in vitro PKCα phosphorylation at the PDK and hydrophobic sites, but not the turn motif site. In vascular tissues, phosphorylation of the PKC hydrophobic site, but not turn motif site, as well as phosphorylation of PDK at S241 increased in response to phenylephrine. Calponin knockdown inhibits autophosphorylation of cellular PKC in response to phenylephrine, confirming results with recombinant PKC. Thus these results show that autophosphorylation of PKC is regulated in dVSM and calponin is necessary for autophosphorylation of PKC in VSM.
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    • "This provides evidence that TORC2 also regulates the level of phosphorylation of mature PKC, consistent with a role for TORC2 in the regulated phosphorylation of the hydrophobic site by signaling. There are many examples of this regulation (Cenni et al. 2002; Newton and Messing 2010), but there is little previous evidence for a role of TORC2 in this regulation. "
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    • "PKC isoenzymes are processed by a series of ordered phosphorylations that are required to gain full catalytic activity of the enzyme and correct intracellular localization. The phosphorylation of PKCs on the hydrophobic motif is enhanced upon growth factor stimulation and correlates with activation [29] [30]. Using an antibody directed against phospho-Ser675 of PKCη we show timedependent increased phosphorylation on the hydrophobic motif in response to IGF-I stimulation (Fig. 4B). "
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