Protein kinase C: poised to signal

Abstract

Nestled at the tip of a branch of the kinome, protein kinase C (PKC) family members are poised to transduce signals emanating from the cell surface. Cell membranes provide the platform for PKC function, supporting the maturation of PKC through phosphorylation, its allosteric activation by binding specific lipids, and, ultimately, promoting the downregulation of the enzyme. These regulatory mechanisms precisely control the level of signaling-competent PKC in the cell. Disruption of this regulation results in pathophysiological states, most notably cancer, where PKC levels are often grossly altered. This review introduces the PKC family and then focuses on recent advances in understanding the cellular regulation of its diacylglycerol-regulated members.

Fig. 1.

Protein kinase C (PKC) family members, showing position on branch of AGC kinome, domain composition, and cofactor dependence. A: human kinome (reproduced from www.cellsignal.com/reference/kinase), showing the position of the AGC kinases (bottom right). PKC isozymes (enlarged on right) are poised on a branch that includes Akt, p70 S6 kinase, and PDK-1 (phosphoinositide-dependent kinase-1). The PKN family diverges, then the atypical PKC isozymes, the novel PKC isozymes, and finally, most divergent, the conventional PKC isozymes. B: domain composition of PKC family members, showing pseudosubstrate (green rectangle), C1 domain [orange rectangle; Y/W switch that dictates affinity for diacylglycerol (DG)-containing membranes indicated by circle in C1B domain], C2 domain [yellow rectangle; basic patch that drives binding to PIP₂ (phosphatidylinositol-4,5-bisphosphate), indicated by oval with ++], connecting hinge segment, kinase domain (cyan), and carboxyl-terminal tail (CT; dark blue rectangle). Also shown are the 3 priming phosphorylations in the kinase domain and CT, with numbering indicated for PKCβII, PKCε, and PKCζ (note atypical PKC isozymes have Glu at phospho-acceptor position of hydrophobic motif). C: table showing dependence of PKC family members on C1 domain cofactors, DG, and phosphatidylserine (PS) and C2 domain cofactors Ca ²⁺ and PIP₂.

Fig. 2.

Structure of kinase domain of PKCβII showing priming site phosphorylations: activation loop (pink), turn motif (orange), and hydrophobic motif (green) (38). Also shown is the clamp between the PXXP motif (Pro, in green) and the conserved Tyr (yellow) of the αE helix.

Fig. 3.

Model for the life cycle of PKC, from biosynthesis to degradation. Newly synthesized PKC (leftmost species) associates with a membrane fraction, where it is processed by a series of ordered and tightly coupled phosphorylations. Heat shock protein-90 (HSP90) binds to the PXXP clamp in the kinase domain right before the CT (see text), an event required for priming phosphorylations. Two upstream kinases control priming phosphorylations: PDK-1, bound to the exposed carboxyl terminus of newly synthesized PKC, phosphorylates the activation loop (pink circle); this step appears to be first, and necessary for the processing PKC. The mTORC2 complex promotes the phosphorylation of the turn motif (orange circle), second phosphorylation event, and hydrophobic motif (green circle), the final phosphorylation event. The fully phosphorylated “mature” PKC localizes to the cytosol with the pseudosubstrate (green rectangle) occupying the substrate-binding cavity (bottom species on left). Signals that cause lipid hydrolysis recruit PKC to membranes. For conventional PKC isozymes, binding of Ca²⁺ to the C2 domain recruits them to the plasma membrane via interaction with PIP₂, an event that allows efficient binding of the membrane-embedded ligand DG (top right species). For novel PKC isozymes, the intrinsic affinity of the C1 domain is sufficiently high to allow direct recruitment to membranes by agonist-evoked levels of DG. Membrane-bound PKC adopts an open conformation, in which the pseudosubstrate is released from the kinase domain, allowing downstream signaling (top right species). This open conformation is sensitive to dephosphorylation: the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase) dephosphorylates the hydrophobic motif, an event that shunts PKC to the detergent-insoluble fraction where it is further dephosphorylated and degraded (bottom right species). Note: molecular chaperone HSP70 binds the dephosphorylated turn motif (bottom middle species), an event that promotes rephosphorylation of PKC and reentry into the pool of signaling-competent enzyme, thus sustaining the lifetime of the enzyme. Specific protein scaffolds (gray structures shown associated with matured PKC) bind specific isozymes and specific species [e.g., RACKs bind active PKC, but other scaffolds bind inactive PKC] to poise PKC in specific cellular microdomains. The amplitude of the PKC signal and similarly the responsiveness of the cell to low concentrations of agonist are ultimately controlled by how much PKC is in the cell.