The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function. Here, we identified by mass spectrometry a phosphorylated serine at the GLS N-terminal domain (at position 95) and investigated its role on regulating GAC activity. The ectopic expression of the phosphomimetic mutant (GAC.S95D) in breast cancer cells, compared to wild-type GAC (GAC.WT), led to decreased glutaminase activity, glutamine uptake, glutamate release and intracellular glutamate levels, without changing GAC sub-cellular localization. Interestingly, cells expressing the GAC.S95D mutant, compared to GAC.WT, presented decreased migration and vimentin level, an epithelial-to-mesenchymal transition marker. These results reveal that GAC is post-translationally regulated by phosphorylation, which affects cellular glutamine metabolism and glutaminase-related cell phenotype.

线粒体磷酸激活的谷氨酰胺酶C（GAC）是通过GLS基因的选择性剪接产生的。与其他GLS相比异构体，即肾型谷氨酰胺酶（KGA），GAC在酶促方面更为有效，对于癌细胞的生长尤为重要。尽管其催化机理已广为人知，但对翻译后修饰如何影响GAC功能的了解甚少。在这里，我们通过质谱法鉴定了位于GLS N末端结构域（位于95位）的磷酸化丝氨酸，并研究了其在调节GAC活性中的作用。与野生型GAC（GAC.WT）相比，乳腺癌细胞中模拟磷酸化突变体（GAC.S95D）的异位表达导致谷氨酰胺酶活性，谷氨酰胺吸收，谷氨酸释放和细胞内谷氨酸水平降低，而不会改变GAC亚细胞定位。有趣的是，与GAC.WT相比，表达GAC.S95D突变体的细胞呈现出降低的迁移和波形蛋白水平，上皮到间质的过渡标记。这些结果表明，GAC在翻译后受磷酸化调节，从而影响细胞的谷氨酰胺代谢和谷氨酰胺酶相关的细胞表型。