The DUSP domain of pseudophosphatase MK-STYX interacts with G3BP1 to decrease stress granules

Abstract

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 (HCX5R). 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.