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Review
. 2018 Apr;53(2):208-230.
doi: 10.1080/10409238.2018.1442408.

Protein Kinase C: Perfectly Balanced

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Free PMC article
Review

Protein Kinase C: Perfectly Balanced

Alexandra C Newton. Crit Rev Biochem Mol Biol. .
Free PMC article

Abstract

Protein kinase C (PKC) isozymes belong to a family of Ser/Thr kinases whose activity is governed by reversible release of an autoinhibitory pseudosubstrate. For conventional and novel isozymes, this is effected by binding the lipid second messenger, diacylglycerol, but for atypical PKC isozymes, this is effected by binding protein scaffolds. PKC shot into the limelight following the discovery in the 1980s that the diacylglycerol-sensitive isozymes are "receptors" for the potent tumor-promoting phorbol esters. This set in place a concept that PKC isozymes are oncoproteins. Yet three decades of cancer clinical trials targeting PKC with inhibitors failed and, in some cases, worsened patient outcome. Emerging evidence from cancer-associated mutations and protein expression levels provide a reason: PKC isozymes generally function as tumor suppressors and their activity should be restored, not inhibited, in cancer therapies. And whereas not enough activity is associated with cancer, variants with enhanced activity are associated with degenerative diseases such as Alzheimer's disease. This review describes the tightly controlled mechanisms that ensure PKC activity is perfectly balanced and what happens when these controls are deregulated. PKC isozymes serve as a paradigm for the wisdom of Confucius: "to go beyond is as wrong as to fall short."

Keywords: Alzheimer’s disease; Protein kinase C; cancer; diacylglycerol; phorbol esters; phosphorylation; pseudosubstrate; tumor suppressor.

Conflict of interest statement

Declaration of interest:

The author has no competing interests. This work was supported by NIH R35 GM122523 and the Cure Alzheimer’s Fund.

Figures

Figure 1
Figure 1. Cartoon illustrating activation of conventional protein kinase C
Receptor-mediated hydrolysis of phosphatidyl-inositol-4,5,-bisphosphate (PIP2) generates inositol trisphosphate (IP3), which mobilizes intracellular Ca2+ and diacylglycerol, the allosteric activator of conventional and novel PKC. Binding of these second messengers localizes PKC in an active conformation at the plasma membrane, where it phosphorylates diverse substrates, including membrane proteins such as transporters (e.g. drug transporters) and receptors (e.g. receptor tyrosine kinases).
Figure 2
Figure 2. PKC isozymes are AGC kinases with N-terminal modules that control activity
A. The AGC branch of the human kinome (reproduced from www.cellsignal.com/reference/kinase and courtesy of Gerard Manning) showing the position of the PKC isozymes. This branch includes Akt, p70S6 kinase and PDK-1. Most closely related to the PKC isozymes are the PKN family members that diverge first from the branch, followed by the atypical PKC isozymes (purple), the novel PKC isozymes (orange), and finally, the conventional PKC isozymes (pink), which are at the tip of the branch. B. Domain composition of PKC family members showing pseudosubstrate (red rectangle), C1 domain (orange rectangle; Y/W switch that dictates affinity for diacylglycerol-containing membranes indicated by circle in C1B domain), C2 domain (yellow rectangle; basic patch that drives binding to PIP2 indicated by ++ on domain), connecting hinge segment, kinase domain (cyan), and carboxyl-terminal tail (CT, grey rectangle). Also shown are the three priming phosphorylations: the activation loop in the kinase domain (magenta circle) and the turn motif (orange circle) and hydrophobic motif (green circle) in the carboxyl-terminal tail (note atypical PKC isozymes have Glu at phospho-acceptor position of hydrophobic motif). Table shows dependence of protein kinase C family members on second messengers (diacyglycerol (DG) and Ca2+) and pharmacological tools to activate (phorbol esters) or inhibit (Gö 6983, Gö 6976, and PZ09) PKC; +, ++, and +++ indicate relative affinity for C1 domain ligands.
Figure 3
Figure 3. Cartoon showing the life cycle of a conventional PKC
Following its biosynthesis, PKC is in an open and degradation-sensitive conformation in which all its regulatory modules are unmasked (species i). It is then processed by three ordered phosphorylations that depend on the binding of Hsp90 and Cdc37 (to a conserved PXXP motif in the kinase domain), the kinase complex mTORC2, and the activation loop kinase, PDK-1. Phosphorylation at these priming sites, the activation loop (magenta circle), the turn motif (orange circle) and the hydrophobic motif (green circle), promote PKC to adopt an autoinhibited conformation. Specifically, the Ca2+-sensing C2 domain (yellow) clamps the autoinhibitory pseudosubstrate segment (red) in the substrate-binding cavity of the kinase domain (cyan), and the diacyglycerol-sensing C1 domains (orange) become masked (species ii). The masking of the C1 domains, in particular, effectively prevents basal signaling in the absence of agonists. Agonists that bind Gq-coupled receptors (R) cause phospholipase C (PLC)-catalyzed hydrolysis of PIP2 generating diacylglycerol and Ca2+. This promotes Ca2+-dependent recruitment of PKC to the plasma membrane via engagement of the Ca2+-bound C2 domain (species iii), where PKC binds its membrane-embedded ligand, diacylglycerol, via primarily the C1B domain (species iv). This active PKC phosphorylates downstream substrates, with one function being to suppress oncogenic signaling via its inactivating phosphorylation of proteins such as Ras and the EGF receptor. PKC returns to the autoinhibited conformation following the decay of its second messengers. The kinetics of activation mirror those for the rise in intracellular Ca2+ and the kinetics of inactivation mirror those for the decay in diacylglycerol. Note the membrane-bound conformation of PKC is sensitive to dephosphorylation, with the first dephosphorylation event on the hydrophobic motif catalyzed by PHLPP; subsequent dephosphorylation by PP2A produces a fully dephosphorylated PKC that is degraded via a proteasomal pathway (species v). However, binding of Hsp70 to the dephosphorylated turn motif allows PKC to become rephosphorylated to sustain the signaling lifetime of the enzyme. Phorbol esters bind the C1B domain with two-orders of magnitude higher affinity than diacylglycerol and are not readily metabolized, trapping PKC in the open, phosphatase-sensitive conformation and resulting in chronic loss, or down-regulation, of PKC. Novel PKC isozymes are regulated by similar mechanisms except their C2 domain does not function as a Ca2+ or plasma membrane sensor, resulting in the localization of novel PKC isozymes primarily to the more abundant and diacylglycerol-rich Golgi membranes. Atypical PKC isozymes are activated upon binding to specific protein scaffolds that tether the pseudosubstrate out of the substrate-binding cavity. Proteins indicated in grey are key regulators of the steady-state levels of PKC: Hsp70, Hsp90, mTORC2, and PDK-1 function to increase the steady-state levels of PKC by permitting/catalyzing processing phosphorylations; Pin1 and the phosphatases PHLPP and PP2A function to decrease the steady-state levels of PKC by permitting/catalyzing the dephosphorylation of PKC. Targeting any of these proteins will disrupt the balance of PKC signaling. Inset shows the structure of the autoinhibited kinase domain of PKCβII, showing autoinhibitory pseudosubstrate locked in the substrate-binding cavity of the kinase domain (cyan) by the C2 domain (yellow); C-terminal tail is indicated in grey.
Figure 4
Figure 4. Phorbol esters: potent tumor promoters
A. Drawing of the plant Croton tiglium, whose milky white sap contains phorbol esters (from Franz Eugen Köhler, Köhler’s Medizinal-Pflanzen (1887)). Also shown is the structure of phorbol ester, whose potency depends on the esterified group (R) at positions 12 and 13: phorbol-12-myristate-13-acetate (PMA) and phorbol-12, 13-dibutyrate (PDBu) are relatively water soluble phorbol esters used in cell biology (Courtesy of P. Blumberg, NIH.). B. Skin model of tumor promotion: painting a subthreshold amount of either a carcinogen (such as DMBA) or phorbol esters (such as PMA) alone on the skin of nude mouse does not result in papilloma formation. However, painting a subthreshold amount of the carcinogen followed by repetitive treatment with phorbol esters causes papillomas to form, which eventually develop into carcinomas. C. Treatment of NIH-3T3 fibroblasts with either PMA or bryostatin 1 causes endogenous conventional and novel PKC redistribution from the cytosolic fraction (soluble; sol.) to the detergent-solubilized membrane fraction (membrane; mem) and detergent-insoluble fraction (insoluble; insol.) within minutes, followed by down-regulation of the protein (apparent as loss of total (tot) PKC). PKCα, δ, and ε have different kinetics of down-regulation following PMA vs bryostatin 1 treatment, but all are effectively depleted following 48 hours of treatment. The atypical PKCζ is not down-regulated by these C1 ligands. Reproduced courtesy of P. Blumberg from (Szallasi et al. 1994).

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