Abdusamad Mai, Guo Xiao, Ramirez Ivan, Velasquez Erick F, Cohn Whitaker, Gholkar Ankur A, Whitelegge Julian P, Torres Jorge Z
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
bioRxiv. 2025 Jan 14:2025.01.13.632830. doi: 10.1101/2025.01.13.632830.
Protein phosphatases are critical for regulating cell signaling, cell cycle, and cell fate decisions, and their dysregulation leads to an array of human diseases like cancer. The dual specificity phosphatases (DUSPs) have emerged as important factors driving tumorigenesis and cancer therapy resistance. DUSP12 is a poorly characterized atypical DUSP widely conserved throughout evolution. Although no direct substrate has been firmly established, DUSP12 that has been implicated in protecting cells from stress, regulating ribosomal biogenesis, and modulating cellular DNA content. In this study, we used affinity- and proximity-based biochemical purification approaches coupled to mass spectrometry to identify the zinc finger protein ZPR9 as a novel DUSP12 interactor, which was validated by in-cell and IP assays. Interestingly, ZPR9 binds to the unique zinc-binding domain of DUSP12, which previous reports indicated was important for many of DUSP12's functions within the cell. Prior studies had implicated ZPR9 as a modulator of apoptosis, but it remained unclear if and how ZPR9 participated in the cell cycle and, more so, how it promoted cell death. Using mass spectrometry analyses, we found that overexpression of DUSP12 promoted de-phosphorylation of ZPR9 at Ser. Overexpression of ZPR9, but not Ser phosphomimetic and phosphorylation-deficient mutants, led to an increase in pre-metaphase mitotic defects while knockdown of DUSP12 also showed mitotic defects in metaphase. Furthermore, knockdown of DUSP12 promoted, while knockdown of ZPR9 suppressed, stress-induced apoptosis. Our results support a model where DUSP12 protects cells from stress-induced apoptosis by promoting de-phosphorylation of ZPR9.
蛋白磷酸酶对于调节细胞信号传导、细胞周期和细胞命运决定至关重要，其失调会导致一系列人类疾病，如癌症。双特异性磷酸酶（DUSPs）已成为驱动肿瘤发生和癌症治疗耐药性的重要因素。DUSP12是一种在进化过程中广泛保守但特征不明的非典型DUSP。尽管尚未明确确定其直接底物，但DUSP12已被认为与保护细胞免受应激、调节核糖体生物合成以及调节细胞DNA含量有关。在本研究中，我们使用基于亲和和邻近的生化纯化方法结合质谱来鉴定锌指蛋白ZPR9作为一种新型的DUSP12相互作用蛋白，这通过细胞内和免疫沉淀试验得到了验证。有趣的是，ZPR9与DUSP12独特的锌结合结构域结合，先前的报道表明该结构域对于DUSP12在细胞内的许多功能很重要。先前的研究表明ZPR9是细胞凋亡的调节剂，但ZPR9是否以及如何参与细胞周期，以及它如何促进细胞死亡仍不清楚。通过质谱分析，我们发现DUSP12的过表达促进了ZPR9丝氨酸位点的去磷酸化。ZPR9的过表达，但不是丝氨酸磷酸模拟和磷酸化缺陷突变体，导致前期有丝分裂缺陷增加，而DUSP12的敲低也显示中期有丝分裂缺陷。此外，DUSP12的敲低促进了应激诱导的细胞凋亡，而ZPR9的敲低则抑制了这种凋亡。我们的结果支持一个模型，即DUSP12通过促进ZPR9的去磷酸化来保护细胞免受应激诱导的细胞凋亡。
