Protein kinase C (PKC) isoenzymes are a prototypical class of serine/threonine kinases that are conserved throughout evolution and widely expressed in almost all cell types. They consist of domain permutations in their N-terminal regulatory regions linked by variable sequences to a highly conserved C-terminal kinase domain. The biological role of PKC isoenzymes has been intensely studied in both various physiological and pathological conditions. In addition, PKC isoenzymes are implicated in multiple signal transduction pathways that turn environment cues into cellular actions. The PKC signaling pathway has critical roles in regulating gene expression, as well as cell proliferation, differentiation, migration, survival and death. Inactivation or dysregulation of PKC isoenzymes may cause different pathologies, such as allergy, inflammatory diseases, diabetes and autoimmune diseases. Recent scientific breakthroughs and technological advancements have improved our understanding of the functional roles of PKCs in cancer development and progression. Moreover, PKC enzymes can serve as promising targets for cancer treatment. In this review, we focus on the regulatory activities of PKC isoforms and discuss their involvement in cancer progression. Furthermore, we provide a summary of clinical trials with PKC inhibitors in cancers and highlight the latest development of PKCs as targeted anti-cancer therapies.
PKC isoforms were first identified and found to be activated by proteolysis in 1977. These isoforms are now known to be critical mediators in signal transduction pathways, which regulate a set of biological functions such as cell proliferation, differentiation, death and tumorigenesis.
The PKC family members are classified into three subfamilies according to their cofactor dependence: (i) conventional or classical PKC isozymes (cPKCs), including PKCα, PKCβ1, PKCβ2 and PKCγ, which are calcium-dependent and activated by both diacylglycerol (DAG) and phosphatidylserine (PS); (ii) novel PKC isozymes (nPKCs), including PKCδ, PKCε, PKCη and PKCθ, which are calcium-independent and regulated by DAG and PS; and (iii) atypical PKC isozymes (aPKCs), including PKCζ and PKCλ/ι, which are calcium-independent and do not require DAG for activation, although PS can regulate their activity.
Members of the PKC family share a common architecture of an N-terminal regulatory moiety (approximately 20–40 kDa) and a C-terminal kinase domain (approximately 45 kDa), composed of conserved regions (C1–C4) interspersed with variable regions (V0–V5), which are of lower homology. The C1 domain, a cysteine-rich motif, is duplicated in most PKC isoenzymes and forms the DAG/phorbol ester binding site, which is preceded by an autoinhibitory pseudosubstrate sequence. The C2 domain contains a calcium-binding site or recognition site for acidic lipids. The C3 and C4 domains constitute the ATP- and substrate-binding lobes of the kinase core. There is a hinge region between the regulatory and catalytic domains that becomes proteolytically labile when the enzyme is membrane-bound.
PKC is initially considered an unphosphorylated form (immature) associated with the membrane and under acute structural and spatial regulation including its phosphorylation state, conformation and subcellular location. Activation of PKC isoforms strictly relies on a posttranslational maturation process. Thus, when processed by three ordered phosphorylation (activation loop phosphorylation, turn motif phosphorylation, and hydrophobic motif phosphorylation), PKCs will expose their pseudosubstrate and position at the specific intracellular location, transducing signaling. The first step of this maturation process is regulated by 3-phosphoinositide-dependent protein kinase-1 (PDK1), which phosphorylates threonine residues at the activation loop in the C4 region of PKC isoforms. Such phosphorylation then leads to the autophosphorylation of the “turn” and “hydrophobic” motifs within the C4 region and stabilizes these isoenzymes (maturation). At this point, the mature form of PKC is prepared for activation. Its inactive form (“folded conformation”) resides predominantly in the cytosol, with an interaction between the C-terminal domain and the N-terminal pseudosubstrate motif. Under physiological conditions, PKC can be activated by interacting with extracellular agonists such as hormones, cytokines and growth factors, which sequentially translocates to different subcellular compartments, thus conferring specific substrate phosphorylation and distinct cellular functions. The translocation of PKC is regulated by anchoring/scaffolding proteins to ensure proper spatio-temporal distribution of PKC isoforms. Receptors for activated C kinase (RACKs) are intracellular scaffolding proteins that bind to individual PKC isoforms following their activation and provide anchorage of each isoform next to its physiological substrates. Subsequently, the translocated PKC phosphorylates specific substrates such as membrane-associated phospholipase C (PLC), which hydrolyzes the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to generate DAG and inositol 1,4,5-trisphosphate (IP3). IP3 then interacts with the inositol trisphosphate receptor (InsP3R) and triggers the rapid release of Ca 2+ from the intracellular store to the endoplasmic reticulum (ER). DAG is phosphorylated by diacylglycerol kinases (DGK) and converts to phosphatidic acid (PA) in a reaction that terminates the PKC-regulated signals. The reversal of PKC activity is regulated by dephosphorylation, ubiquitination, and caveolin-dependent endocytosis. In summary, PKC signaling is initiated by phosphorylation, pseudosubstrate exposure and subcellular localization, which ultimately leads to a specific cellular response.
The PKC isoforms show variable expression profiles during cancer progression depending on cell types. For example, in the transitional cell carcinoma of the bladder, PKCα and β2 immunostains were intense in both normal urothelium and all grade tumor tissues. At the same time, PKCβ1 and δ immunostains were intense in normal urothelium and low-grade tumors, but weak in high-grade tumors. In human liver cancer, the level of membrane-bound PKCα was significantly lower in cancer tissues than in adjacent normal tissues. The levels of PKCδ and λ/ι in both cytosolic and membrane fractions were significantly higher in cancer tissues than in adjacent normal tissues. Moreover, Western blot results showed that the expression levels of PKC isoforms (α and λ/ι) were remarkably higher in cancerous tissues than in normal breast tissues. The expression of PKCε was correlated with high histologic grade and poor disease-free and overall survival in breast cancer patients. PKC isoforms (α, β, ε and η) were found to be highly expressed in prostate cancer compared with benign prostatic hyperplasia.
The PKC family contains nine genes and is frequently mutated in human cancer. Notably, there are 20–25% PKC mutations verified in melanoma, colorectal cancer and lung squamous cell carcinoma, while less than 5% in ovarian cancer, glioblastoma and breast cancer. PKCα mutation (D294G in the hinge region) was the first reported cancer-associated mutation in PKC, which separated the regulatory and catalytic moieties and abolished the F-actin dependent PKCα accumulation at the cell-cell contacts.
