Phosphorylation Of SNX27 Ser51 By MAPK11/14 Links Cellular Stress Signaling Pathways With Endocytic Recycling

Integral cell surface proteins,together with associated ligands,proteins and lipids,are internalized,in a clathrin-dependent or-independent manner.Endocytosed proteins can be delivered to lysosomes for degradation,or delivered to the trans-Golgi network(TGN)or the plasma membrane(PM)for recycling.As one of the most fundamental cellular processes,endocytic recycling maintains cellular homeostasis.Accordingly,it is also tightly regulated by many intracellular and extracellular events,such as gene expression,protein post-translational modifications,nutritional availability,and bacterial and viral infection.Genetic variations in endocytic recycling components have been linked to a wide range of human diseases which include Alzheimer’s disease,Parkinson’s disease,cancer,and diabetes.Since the discovery of Retromer,a central regulator of endocytic recycling,over 20 years ago,we have gained significant insights into the process of endocytic recycling.Several highly-conserved proteins or protein complexes,including Retromer(VPS35/VPS26/VPS29 in higher eukaryotes),Retriever(VPS35L/VPS26C/VPS29),WASH complex,TBC1D5,and multiple members of the sorting nexin(SNX)family,have been shown to mediate the biogenesis of cargo-enriched transport carriers or promote endocytic recycling via other mechanisms.At present,34 kinds of SNXs have been found in mammals,which can be divided into three categories according to the composition of domains,including SNX-PX,SNX-PX-BAR and SNX-PX-other.SNX27 is a key regulator of endocytic recycling,and often mediates recycling through cooperation with Retromer and SNX-BARs.The PDZ domain of SNX27 directly binds to the PDZ-binding motifs(PDZbms)located at the C-terminus of many proteins,and interaction with the VPS26 subunit of Retromer further increases its binding with PDZbms.Established cargoes of SNX27 include glucose transporter GLUT1,SEMA4 C,G-protein-coupled receptors(GPCRs)such as the β2 adrenergic receptor(β2AR),and parathyroid hormone receptor(PTHR).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.To investigate endocytic trafficking under starvation,we did some experiments,such as immunofluorescence,surface protein biotinylation and endocytic recycling of CD8 A tagged cargoes.The results showed that starvation could perturb the normal recycling of GLUT1,SEMA4 C and PTHR.But starvation treatment had no effect on TRAILR1 distribution.Since nutrient starvation induces autophagy,we next tested whether autophagy is required for starvation-mediated inhibition of endocytic recycling.In the present study,we showed that starvation decreases GLUT1 accumulation in the plasma membrane,which is distinct from a recent study which concluded that autophagy promotes cell surface expression of GLUT1.After a series of experiments,we found that wild-type 293 T cells,but not ATG7-KO cells,showed a reduction of co-localization between GLUT1 and Phalloidin upon starvation.Taken together,our data indicate that starvation-induced autophagy may impair endocytic recycling either directly or indirectly by compromising signaling pathways that can regulate endocytic recycling.TBC1D5 is proposed to shuttle between Retromer-positive structures and autophagosome,depending on cellular metabolic states.A prerequisite of this model is that TBC1D5 binds to Retromer and LC3 competitively.Thus,we next assessed the interactions of TBC1D5 and VPS35 under normal and starvation conditions.Our results confirmed that TBC1D5 associates with LC3 B through LIR4,and IP experiments proved that starvation does not dissociate TBC1D5 from Retromer.Thus,TBC1D5 is unlikely to mediate starvation-induced inhibition of GLUT1 trafficking.We have proved that TBC1D5 had no significant effects on GLUT1 trafficking.So which protein of endosomal sorting is affected by starvation? A previous study indicated that treatment of cells with cholera toxin increases phosphorylation of rat SNX27 at S49.We next asked whether SNX27,another key modulator of endosomal trafficking,could regulate GLUT1 trafficking in response to starvation.Next assess the protein expression and degradation of SNX27 under autophagy.IP experiments showed that the protein levels of SNX27 were not affected by starvation.Next,to identify the phosphorylation site(s)in SNX27,we utilized mass spectroscopy(MS)-based approach and custom-made phospho-SNX27 S51-specific antibody.Results indicate that starvation increases phosphorylation of S51 in the PDZ domain of SNX27.Then,we sought to determine which kinase is responsible for phosphorylation of SNX27-S51.The GST-pulldown experiment demonstrated that both the PX and FERM domains,but not the PDZ domain of SNX27,mediated the interaction with MAPK11/14.In vitro and in vivo kinase assay showed that MAPK11/14 phosphorylates SNX27 on Ser51.As phosphorylation of Ser51 occurs within the PDZ domain,we speculated that phosphorylation may reduce SNX27 binding to its cargoes.IP and ITC experiments further confirmed starvation significantly decreased the affinity between SNX27 and its substrates,such as DGKζ,PTHR and SEMA4 C.Finally,to explore how phosphorylation at S51(p S51)affects SNX27 binding to PDZbm,we performed molecular dynamics(MD)simulations starting from the crystal structure of the SNX27 PDZ domain in complex with the DGKζ peptide.Our data indicates that p S51 decreases the binding between SNX27 PDZ and PDZbm,most likely by altering the conformation of the in-situ loop and adjacent helix 2.We subsequently find 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.GST-pulldown experiments revealed that treatment with LPS,IL-6,EGF,excessive ATP and erastin significantly increased phosphorylation of SNX27.On the other hand,treatment with CCCP or TG did not alter the level of SNX27 phosphorylation.Notably,besides MAPK11/14,other kinases in the MAPK pathway,including ERK1/2 and JNK,are also involved in coping with cell stress.To determine whether these kinases can also phosphorylate SNX27 S51,cells expressing SNX27 were pre-treated with different inhibitors.GST-pulldown and immunofluorescence experiments showed that SNX27 is specifically mediated by MAPK11/14,but not by other related kinases in the MAPK pathway.To determine whether MAPK11/14 can regulate endocytic trafficking under physiological conditions,we used zebrafish as a model system.Similar to previous studies,immunofluorescence experiments showed that activated p38 promotes SNX27 phosphorylation and inhibits GLUT1 trafficking in zebrafish.To assess how phosphorylation of S51 affects endocytic trafficking of GLUT1 in zebrafish,we co-injected SNX27 MO together with m RNA encoding human SNX27 WT,S51 A or S51 D.Similar to what we have observed in cultured cells,re-expression of SNX27 WT or S51 A,but not S51 D,increased the co-localization of GLUT1 andβ-catenin,a plasma membrane marker.Hence,phosphorylation of S51 can serve as a switch to regulate endocytic trafficking in both cultured cells and in zebrafish.Using both mammalian cells as well as a zebrafish model,we show that phosphorylation of SNX27 at S51 alters the conformation of its cargo-binding pocket,decreases the interaction between SNX27 and cargo proteins,thereby inhibiting endocytic recycling.Our study indicates that endocytic recycling is highly dynamic and can crosstalk with cellular stress signaling pathways.Several conclusions come from this study.(1)For the first time,we demonstrate that multiple stimulis such as starvation suppress endocytic recycling of PDZbm-containing cargoes.(2)We refined that the inhibition involves MAPK11/14-mediated phosphorylation of SNX27 at S51 within the PDZ domain,but is independent of TBC1D5.(3)We also determined that MAPK11/14 regulate endocytic recycling via phosphorylating SNX27.(4)The molecular dynamics(MD)simulations offer one explanation from a structural point of view for the affinity of SNX27-p S51 with PDZbm-containing cargo.Suppressing of endocytic recycling and enhancing of receptors lysosomal degradation serve as a new mechanism for cells to cope with stress and to save energy.