Cancer‐Associated Fusions of the Protein Kinase C Kinase Domain are Loss‐of‐Function

Abstract

Gain‐of‐function fusion proteins have emerged as novel cancer drivers and potential therapeutic targets, exemplified by the successful targeting of the oncogenic Bcr‐Abl gene fusion that drives chronic myelogenous leukemia. Over 60 fusions of protein kinase C (PKC) family members with other genes have been identified in diverse cancers, accounting for the most abundant fusions in the AGC family of kinases. Given our recent findings that PKC isozymes generally function as tumor suppressors, we explored whether such fusions may provide another mechanism for loss of PKC function in cancer. Specifically, we addressed the cellular activity of fusions in which the C‐terminal catalytic domain of PKC was retained, but the N‐terminal regulatory domain was truncated by fusion to the N‐terminus of unrelated genes: these fusions are TANC2‐PRKCA, which encodes a protein containing the first 46 residues of the adaptor protein TANC2 fused to the N‐terminus of the C2 domain of PKCα, and GGA2‐PRKCB, which encodes a protein containing the first 158 residues of the Golgi trafficking protein GGA2 fused to the C1A domain of PKCβ. Overexpression in COS7 cells revealed that both fusion proteins are constitutively active as assessed by the FRET‐based C Kinase Activity Reporter (CKAR). This constitutive activity is not a result of the various proteins to which PKC is fused, but rather is a consequence of loss of the autoinhibitory pseudosubstrate from within the PKC regulatory moiety; constructs of PKC deleted in the pseudosubstrate are also constitutively active in cells. However, the fusion proteins are unstable as assessed by their accelerated turnover compared to wild‐type PKC following inhibition of protein synthesis. Because active PKC is in a degradation‐sensitive conformation, we reasoned that PKC fusion proteins would be too unstable for significant levels to accumulate in cancer cells. To test this, we used CRISPR/Cas9 to edit one allele of PKCα to express the TANC2‐PRKCA fusion observed in cancer. Western blot analysis of these clones revealed readily detectable PKCα from the intact allele but no detectable fusion protein, verifying that the PKC fusion is indeed too unstable to accumulate in cells. Thus, while the fusion proteins are rendered constitutively active due to a loss of autoinhibitory constraints, their inherent instability results in a dramatic reduction of their steady‐state levels, effectively making them loss‐of‐function. PKC fusion proteins therefore present another mechanism by which loss of PKC activity can occur in the context of 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 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.