Department of Biology, Integrated Science Center, William and Mary, Williamsburg, VA, 23185, USA.
Department of Biology, Integrated Science Center, William and Mary, Williamsburg, VA, 23185, USA; Department of Genetics, Washington University, St. Louis, MO, 63110, USA.
Arch Biochem Biophys. 2023 Aug;744:109702. doi: 10.1016/j.abb.2023.109702. Epub 2023 Jul 27.
Mitogen activated protein kinase phosphoserine/threonine/tyrosine-binding protein (MK-STYX) is a dual specificity (DUSP) member of the protein tyrosine phosphatase family. It is a pseudophosphatase, which lacks the essential amino acids histidine and cysteine in the catalytic active signature motif (HCXR). We previously reported that MK-STYX interacts with G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding-1] and reduces stress granules, stalled mRNA. To determine how MK-STYX reduces stress granules, truncated domains, CH2 (cell division cycle 25 phosphatase homology 2) and DUSP, of MK-STYX were used. Wild-type MK-STYX and the DUSP domain significantly decreased stressed granules that were induced by sodium arsenite, in which G3BP1 (a stress granule nucleator) was used as the marker. In addition, HEK/293 and HeLa cells co-expressing G3BP1-GFP and mCherry-MK-STYX, mCherry-MK-STYX-CH2, mCherry-MK-STYX-DUSP or mCherry showed that stress granules were significantly decreased in the presence of wild-type MK-STYX and the DUSP domain of MK-STYX. Further characterization of these dynamics in HeLa cells showed that the CH2 domain increased the number of stress granules within a cell, relative to wild-type and DUSP domain of MK-STYX. To further analyze the interaction of G3BP1 and the domains of MK-STYX, coimmunoprecipitation experiments were performed. Cells co-expressing G3BP1-GFP and mCherry, mCherry-MK-STYX, mCherry-MK-STYX-CH2, or mCherry-MK-STYX-DUSP demonstrated that the DUSP domain of MK-STYX interacts with both G3BP1-GFP and endogenous G3BP1, whereas the CH2 domain of MK-STYX did not coimmunoprecipitate with G3BP1. In addition, G3BP1 tyrosine phosphorylation, which is required for stress granule formation, was decreased in the presence of wild-type MK-STYX or the DUSP domain but increased in the presence of CH2. These data highlight a model for how MK-STYX decreases G3BP1-induced stress granules. The DUSP domain of MK-STYX interacts with G3BP1 and negatively alters its tyrosine phosphorylation- decreasing stress granule formation.
丝裂原活化蛋白激酶磷酸丝氨酸/苏氨酸/酪氨酸结合蛋白（MK-STYX）是蛋白酪氨酸磷酸酶家族的一种双特异性（DUSP）成员。它是一种伪磷酸酶，在催化活性特征基序（HCXR）中缺少必需氨基酸组氨酸和半胱氨酸。我们之前报道过，MK-STYX 与 G3BP1[Ras-GAP（GTPase-activating protein）SH3（Src homology 3）结构域结合蛋白-1]相互作用，减少应激颗粒，停滞的 mRNA。为了确定 MK-STYX 如何减少应激颗粒，我们使用了 MK-STYX 的截断结构域 CH2（细胞分裂周期 25 磷酸酶同源物 2）和 DUSP。野生型 MK-STYX 和 DUSP 结构域显著减少了由砷酸钠诱导的应激颗粒，其中 G3BP1（应激颗粒核形成因子）被用作标记物。此外，在共表达 G3BP1-GFP 和 mCherry-MK-STYX、mCherry-MK-STYX-CH2、mCherry-MK-STYX-DUSP 或 mCherry 的 HEK/293 和 HeLa 细胞中，应激颗粒明显减少，在存在野生型 MK-STYX 和 MK-STYX 的 DUSP 结构域的情况下。在 HeLa 细胞中进一步对这些动力学进行了特征描述，结果表明 CH2 结构域相对于野生型和 MK-STYX 的 DUSP 结构域增加了细胞内应激颗粒的数量。为了进一步分析 G3BP1 与 MK-STYX 结构域的相互作用，进行了免疫共沉淀实验。共表达 G3BP1-GFP 和 mCherry、mCherry-MK-STYX、mCherry-MK-STYX-CH2 或 mCherry-MK-STYX-DUSP 的细胞表明，MK-STYX 的 DUSP 结构域与 G3BP1-GFP 和内源性 G3BP1 相互作用，而 MK-STYX 的 CH2 结构域则没有与 G3BP1 共免疫沉淀。此外，应激颗粒形成所必需的 G3BP1 酪氨酸磷酸化在存在野生型 MK-STYX 或 DUSP 结构域的情况下减少，但在存在 CH2 的情况下增加。这些数据突出了 MK-STYX 减少 G3BP1 诱导的应激颗粒的模型。MK-STYX 的 DUSP 结构域与 G3BP1 相互作用并负向改变其酪氨酸磷酸化，减少应激颗粒的形成。
