Sorting nexins (SNXs) containing the Phox (PX) domain are implicated in the regulation of membrane trafficking and sorting processes of epithelial growth factor receptor (EGFR). In this study, we investigated whether SNX16 regulates EGF-induced cell signaling by regulating EGFR trafficking. SNX16 is localized in early and recycling endosomes via its PX domain. Mutation of the PX domain disrupted the association between SNX16 and phosphatidylinositol 3-phosphate [PtdIns(3)P]. Treatment with wortmannin, a PtdIns 3-kinase inhibitor, abolished the endosomal localization of SNX16, suggesting that the intracellular localization of SNX16 is regulated by PtdIns 3-kinase activity. SNX16 was found to associate with EGFR after stimulation with EGF in COS-7 cells. Moreover, overexpression of SNX16 increased the rate of EGF-induced EGFR degradation and inhibited the EGF-induced up-regulation of ERK and serum response element (SRE). In addition, mutation in the PX domain significantly blocked the inhibitory effect of SNX16 on EGF-induced activation of ERK and SRE. From these results, we suggest that SNX16 directs the sorting of EGFR to the endosomal compartment and thus regulates EGF-induced cell signaling.
Dual-specificity phosphatases (DUSPs) constitute a subfamily of protein tyrosine phosphatases, and are intimately involved in the regulation of diverse parameters of cellular signaling and essential biological processes. DUSP28 is one of the DUSP subfamily members that is known to be implicated in the progression of hepatocellular and pancreatic cancers, and its biological functions and enzymatic characteristics are mostly unknown. Herein, we present the crystal structure of human DUSP28 determined to 2.1 Å resolution. DUSP28 adopts a typical DUSP fold, which is composed of a central β-sheet covered by α-helices on both sides and contains a well-ordered activation loop, as do other enzymatically active DUSP proteins. The catalytic pocket of DUSP28, however, appears hardly accessible to a substrate because of the presence of nonconserved bulky residues in the protein tyrosine phosphatase signature motif. Accordingly, DUSP28 showed an atypically low phosphatase activity in the biochemical assay, which was remarkably improved by mutations of two nonconserved residues in the activation loop. Overall, this work reports the structural and biochemical basis for understanding a putative oncological therapeutic target, DUSP28, and also provides a unique mechanism for the regulation of enzymatic activity in the DUSP subfamily proteins.
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