Department of Pharmacology, University of California at San Diego, La Jolla, California, USA; Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, California, USA.
Department of Pharmacology, University of California at San Diego, La Jolla, California, USA.
J Biol Chem. 2021 Jan-Jun;296:100445. doi: 10.1016/j.jbc.2021.100445. Epub 2021 Feb 20.
Within the AGC kinase superfamily, gene fusions resulting from chromosomal rearrangements have been most frequently described for protein kinase C (PKC), with gene fragments encoding either the C-terminal catalytic domain or the N-terminal regulatory moiety fused to other genes. Kinase fusions that eliminate regulatory domains are typically gain of function and often oncogenic. However, several quality control pathways prevent accumulation of aberrant PKC, suggesting that PKC fusions may paradoxically be loss of function. To explore this topic, we used biochemical, cellular, and genome editing approaches to investigate the function of fusions that retain the portion of the gene encoding either the catalytic domain or regulatory domain of PKC. Overexpression studies revealed that PKC catalytic domain fusions were constitutively active but vulnerable to degradation. Genome editing of endogenous genes to generate a cancer-associated PKC fusion resulted in cells with detectable levels of fusion transcript but no detectable protein. Hence, PKC catalytic domain fusions are paradoxically loss of function as a result of their instability, preventing appreciable accumulation of protein in cells. Overexpression of a PKC regulatory domain fusion suppressed both basal and agonist-induced endogenous PKC activity, acting in a dominant-negative manner by competing for diacylglycerol. For both catalytic and regulatory domain fusions, the PKC component of the fusion proteins mediated the effects of the full-length fusions on the parameters examined, suggesting that the partner protein is dispensable in these contexts. Taken together, our findings reveal that PKC gene fusions are distinct from oncogenic fusions and present a mechanism by which loss of PKC function occurs in cancer.
在 AGC 激酶超家族中，染色体重排导致的基因融合最常发生在蛋白激酶 C（PKC）中，基因片段编码 C 末端催化结构域或 N 末端调节部分融合到其他基因上。消除调节结构域的激酶融合通常是功能获得性的，并且常常是致癌的。然而，有几个质量控制途径可以防止异常 PKC 的积累，这表明 PKC 融合可能是功能丧失。为了探讨这个课题，我们使用生化、细胞和基因组编辑方法来研究保留编码 PKC 催化结构域或调节结构域部分的基因融合的功能。过表达研究表明，PKC 催化结构域融合是组成性激活的，但易降解。通过基因组编辑内源基因产生与癌症相关的 PKC 融合，导致融合转录本可检测到，但无融合蛋白可检测。因此，PKC 催化结构域融合由于其不稳定性而表现出功能丧失，阻止了细胞内大量蛋白质的积累。PKC 调节结构域融合的过表达抑制了基础和激动剂诱导的内源性 PKC 活性，通过与二酰基甘油竞争以显性负性方式发挥作用。对于催化和调节结构域融合，融合蛋白中的 PKC 成分介导了全长融合对所检查参数的影响，这表明在这些情况下，伴侣蛋白是可有可无的。总之，我们的发现表明，PKC 基因融合与致癌融合不同，并且揭示了 PKC 功能丧失在癌症中发生的机制。
