Fusion Gene TANC2‐PRKCA Reveals Another Mechanism for Loss of Protein Kinase C Function in Cancer

Abstract

The protein kinase C (PKC) family of serine/threonine kinases plays a critical role in signal transduction, and dysregulation of PKC activity is implicated in a variety of diseases, including cancer. Over 60 fusion genes involving PKC isozymes have been identified in an array of cancers, making PKC the most frequently fused member of the AGC kinase superfamily. With previous studies in the laboratory revealing a tumor suppressive role for PKC, we sought to characterize these PKC fusions and determine whether they may be loss‐of‐function in cancer. For our analysis, we selected TANC2‐PRKCA, a fusion gene identified in lung squamous cell carcinoma. This fusion retains the C‐terminal catalytic domain of PKC while having a truncated N‐terminal regulatory moiety due to fusion with TANC2. Overexpression in cells reveals that the fusion protein lacks the conserved priming phosphorylations necessary for proper PKC maturation yet is constitutively active, a result of the loss of the autoinhibitory pseudosubstrate from within the PKC regulatory moiety. As an additional consequence, the fusion is also markedly unstable, unable to adopt the stable, autoinhibited conformation of wild‐type PKC. We thus reasoned that TANC2‐PKCα, in an active and degradation‐sensitive conformation, would be too unstable to significantly accumulate in cells. To address this, we utilized CRISPR/Cas9 gene‐editing to express the TANC2‐PRKCA fusion in cells. While the fusion mRNA was detected in the CRISPR‐edited clones, the fusion protein was not, consistent with the protein's inherent instability. Thus, while the loss of autoinhibition renders the fusion constitutively active, the resulting instability leads to a dramatic reduction of its steady‐state levels, making it paradoxically loss‐of‐function. While overexpression studies showed that the fusion protein is more ubiquitinated compared to wild‐type protein, inhibition of proteasomal, lysosomal, and endosomal degradative pathways in the CRISPR‐edited cells did not result in accumulation of the fusion protein. Knockdown of RINCK (TRIM41), an E3 ligase for PKC, and CHIP (STUB1), an E3 ligase for misfolded proteins, by siRNA similarly did not result in accumulation of the fusion protein. Taken together, our analyses show that the TANC2‐PKCα fusion protein is constitutively active but locked in an open and unstable conformation, resulting in its downregulation. Fusion of PKC's catalytic domain to other genes thus presents another mechanism by which loss of PKC activity can occur in cancer, 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).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.