Protein Kinase C Fusions Reveal Another Mechanism for Loss of Protein Kinase C Function in Cancer

Abstract

Over 60 protein kinase C (PKC) fusion genes have been identified in a wide variety of cancers, making PKC the most frequently fused AGC kinase. Our laboratory previously revealed that cancer‐associated point mutations annotated for PKC family members are generally loss‐of‐function, with functional studies revealing a tumor suppressive role for PKC. Here, we sought to determine whether PKC fusions may also be loss‐of‐function. In our analysis, we characterized TANC2‐PRKCA, a fusion identified in lung squamous cell carcinoma that retains the catalytic domain of PKCα, and PRKCA‐CDH8, a fusion identified in breast cancer that retains the regulatory domain of PKCα. Overexpression of TANC2‐PKCα in cells showed that the fusion protein is constitutively active; however, it lacks phosphorylation at key sites required for the stability of the enzyme. Most importantly, without its amino‐terminal pseudosubstrate, the fusion protein is unable to adopt the autoinhibited, stable conformation of wild‐type PKC, rendering it markedly unstable. To determine whether the fusion protein could accumulate in cells given this instability, we utilized CRISPR/Cas9‐mediated gene editing to express TANC2‐PRKCA. Indeed, while the fusion mRNA was detected in the CRISPR‐edited clones, we were unable to detect the fusion protein. Thus, although the fusion is rendered constitutively active through loss of autoinhibition, its subsequent instability prevents its accumulation in cells, making it paradoxically loss‐of‐function. In contrast to fusions that retain the PKC catalytic domain, fusions that retain the PKC regulatory domain are intrinsically loss‐of‐function with respect to PKC activity. However, we were interested in exploring whether regulatory domain fusions could also act in a dominant‐negative manner. Overexpression of PKCα‐CDH8 in cells suppressed endogenous PKC activity, both basally and following agonist stimulation. This suggests that PKC regulatory fusions are not only loss‐of‐function by loss of the catalytic domain, but also dominant negative by competition of the ligand‐sensing modules for binding the allosteric activator diacylglycerol. Taken together, our data demonstrate that both catalytic and regulatory domain PKC fusions are loss‐of‐function, revealing another mechanism by which PKC activity is lost in cancer and supporting a tumor suppressive role for PKC.Support or Funding InformationThis work was supported by NIH R35 GM122523 (ACN). ANV, TRB, and CEA were supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), and CEA was supported by an NSF Graduate Research Fellowship (DGE1144086).