The Ca2+-independent, but diacylglycerol-regulated, novel protein kinase C (PKC) theta (θ) is highly expressed in hematopoietic cells where it participates in immune signaling and platelet function. Mounting evidence suggests that PKCθ may be involved in cancer, particularly blood cancers, breast cancer, and gastrointestinal stromal tumors, yet how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Here, we examine the effect of four cancer-associated mutations, R145H/C in the autoinhibitory pseudosubstrate, E161K in the regulatory C1A domain, and R635W in the regulatory C-terminal tail, on the cellular activity and stability of PKCθ. Live-cell imaging studies using the genetically-encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter 2 (CKAR2), revealed that the pseudosubstrate and C1A domain mutations impaired autoinhibition to increase basal signaling. This impaired autoinhibition resulted in decreased stability of the protein, consistent with the well-characterized behavior of Ca2+-regulated PKC isozymes wherein mutations that impair autoinhibition are paradoxically loss-of-function because the mutant protein is degraded. In marked contrast, the C-terminal tail mutation resulted in enhanced autoinhibition and enhanced stability. Thus, the examined mutations were loss-of-function by different mechanisms: mutations that impaired autoinhibition promoted the degradation of PKC, and those that enhanced autoinhibition stabilized an inactive PKC. Supporting a general loss-of-function of PKCθ in cancer, bioinformatics analysis revealed that protein levels of PKCθ are reduced in diverse cancers, including lung, renal, head and neck, and pancreatic. Our results reveal that PKCθ function is lost in cancer.

中文翻译：

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蛋白激酶 C θ 的癌症相关突变是功能丧失


Ca2+ 非依赖性但受甘油二酯调节的新型蛋白激酶 C （PKC） θ （θ） 在造血细胞中高度表达，参与免疫信号转导和血小板功能。越来越多的证据表明，PKCθ 可能与癌症有关，尤其是血癌、乳腺癌和胃肠道间质瘤，但如何靶向这种激酶（作为癌基因或肿瘤抑制因子）尚未确定。在这里，我们检查了四种癌症相关突变，即自身抑制假底物中的 R145H/C、调节性 C1A 结构域中的 E161K 和调节性 C 末端尾部的 R635W 对 PKCθ 细胞活性和稳定性的影响。使用基于基因编码的基于荧光共振能量转移的 PKC 活性报告基因 C 激酶活性报告基因 2 （CKAR2） 的活细胞成像研究表明，假底物和 C1A 结构域突变损害了自身抑制以增加基础信号传导。这种受损的自抑制导致蛋白质的稳定性降低，这与 Ca2+ 调节的 PKC 同工酶的明确表征行为一致，其中损害自身抑制的突变自相矛盾地丧失了功能，因为突变蛋白被降解。与此形成鲜明对比的是，C 端尾部突变导致增强的自身抑制和增强的稳定性。因此，检查的突变是不同机制的功能丧失：损害自身抑制的突变促进了 PKC 的降解，而增强自身抑制的突变稳定了失活的 PKC。生物信息学分析支持 PKCθ 在癌症中的普遍功能丧失，生物信息学分析显示 PKCθ 的蛋白质水平在多种癌症中降低，包括肺癌、肾癌、头颈癌和胰腺癌。 我们的结果显示 PKCθ 功能在癌症中丢失。