All proteins were expressed in BL21(DE3) cells, and protein expression was induced by the addition of 0

All proteins were expressed in BL21(DE3) cells, and protein expression was induced by the addition of 0.5 mM IPTG at 20C overnight. degradation of cargoes is determined. Here, we show that multiple extracellular stimuli, including starvation, LPS, IL-6, and EGF treatment, can strongly inhibit endocytic recycling of multiple cargoes through the activation of MAPK11/14. The stress-induced kinases in turn directly phosphorylate SNX27, a key regulator of endocytic recycling, at serine 51 (Ser51). Phosphorylation of SNX27 at Ser51 alters the conformation of its cargo-binding pocket and decreases the conversation between SNX27 and cargo proteins, thereby inhibiting endocytic recycling. Our study indicates that endocytic recycling is usually highly dynamic and can crosstalk with cellular stressCsignaling pathways. Suppression of endocytic recycling and enhancement of receptor lysosomal degradation serve as new mechanisms for cells to cope with stress and save energy. Introduction Integral cell surface proteins, together with associated ligands, proteins, and lipids, are internalized in a clathrin-dependent or impartial manner. Endocytosed proteins can be delivered to lysosomes for degradation or the TGN or plasma membrane (PM) for recycling (Burd and Cullen, 2014; Cullen and Steinberg, 2018; Wang et al., 2018). As one of the most fundamental cellular processes, endocytic recycling determines the fate of many receptors, thus fine-tuning a large number of signal pathways. Accordingly, it is also tightly regulated by many intracellular and extracellular events, such Bis-NH2-C1-PEG3 as gene expression, protein posttranslational modifications, nutritional availability, and bacterial and viral contamination (Cullen and Steinberg, 2018). Genetic variations in endocytic recycling components have been linked to a wide range of human diseases, including Alzheimers disease, Parkinsons disease, cancer, and diabetes (Cullen and Steinberg, 2018; McMillan et al., 2017; Tu et al., 2020). Rabbit Polyclonal to NXF1 Since the discovery of retromer, a central regulator of endocytic recycling, over 20 yr ago, we have gained significant insights into the process of endocytic recycling. Several Bis-NH2-C1-PEG3 highly conserved proteins or protein complexes, including retromer (VPS35/VPS26/VPS29 in higher eukaryotes; Hierro et al., 2007; Kovtun et al., 2018; Seaman et al., 1998), retriever (McNally et al., 2017; VPS35L/VPS26C/VPS29), TBC1D5 (Jia et al., 2016; Seaman et al., 2009), and Bis-NH2-C1-PEG3 multiple members of the sorting nexin (SNX) family (Lucas et al., 2016; Steinberg et al., 2013; Temkin et al., 2011), have been shown to mediate the biogenesis of cargo-enriched transport carriers or promote endocytic recycling via other mechanisms. SNX27 is usually a key regulator of endocytic recycling and often mediates recycling through cooperation with retromer and SNX-Bin/Amphiphysin/Rvs (BAR) proteins (Steinberg et al., 2013; Temkin et al., 2011). The PDZ domain name of SNX27 directly binds to the PDZ-binding motifs (PDZbms) located at the C terminus of many proteins, and conversation with the VPS26 subunit of retromer further increases its binding with PDZbms (Clairfeuille et al., 2016; Gallon et al., 2014). Established cargoes of SNX27 include the glucose transporter GLUT1, SEMA4C, G-proteinCcoupled receptors such as the 2 adrenergic receptor, and parathyroid hormone receptor (PTHR; Chan et al., 2016; McGarvey et al., 2016; Steinberg et al., 2013; Temkin et al., 2011). While the major protein machineries regulating both the recycling and degradation pathways are known, it remains obscure what determines the recycling versus degradation pathways for cargo proteins. Cells are subjected to different types of stresses, including (but not limited to) starvation, inflammation, DNA damage, and oxidative stress (Hotamisligil Bis-NH2-C1-PEG3 and Davis, 2016). One of the best-described pathways directing cellular responses to stresses is the conserved MAPK pathway. Multiple stimuli activate the MAPK through a three-kinase module, and the activation involves the MAP2K-mediated phosphorylation of MAPKs on a threonine and a tyrosine residue of the activation loop. In mammals, the MAPK pathway is usually organized into several different signaling modules, including p38, JNK, ERK1/2, and ERK5 (Johnson and Lapadat, 2002). Among them, members of the p38 MAPK family, including MAPK14 (p38) and Bis-NH2-C1-PEG3 MAPK11 (p38), phosphorylate a wide range of intracellular targets that include membrane transporters, cytoskeletal elements, transcription factors, and other protein kinases (Ono and Han, 2000). As a result, p38 MAPK activation contributes to inflammation, apoptosis, cell differentiation, and cell cycle regulation (Zarubin and Han, 2005). However, it remains unclear whether p38 MAPKs also regulate endocytic recycling. To explore how cellular stress influences endosomal sorting, we investigated endocytic trafficking of multiple cargoes under various treatments. We found that multiple modes of cellular stress inhibit the endocytic recycling of GLUT1 and other cargoes. The inhibition involves MAPK11/14-mediated phosphorylation of SNX27 at serine 51 (Ser51) within the PDZ domain name but is usually impartial of TBC1D5 (Roy et al., 2017). This phosphorylation event inhibits the binding of SNX27 to PDZbm-containing proteins and specifically suppresses endocytic recycling of PDZbm-containing cargoes. Thus, inhibition of endocytic recycling could help cells and organisms adapt to changing environmental conditions..