title: Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes.
abstract: Myotubularin-related 2 (MTMR2) is a 3-phosphoinositide lipid phosphatase with specificity towards the D-3 position of phosphoinositol 3-phosphate [PI(3)P] and phosphoinositol 3,5-bisphosphate lipids enriched on endosomal structures. Recently, we have shown that phosphorylation of MTMR2 on Ser58 is responsible for its cytoplasmic sequestration and that a phosphorylation-deficient variant (S58A) targets MTMR2 to Rab5-positive endosomes resulting in PI(3)P depletion and an increase in endosomal signaling, including a significant increase in ERK1/2 activation. Using in vitro kinase assays, cellular MAPK inhibitors, siRNA knockdown and a phosphospecific-Ser58 antibody, we now provide evidence that ERK1/2 is the kinase responsible for phosphorylating MTMR2 at position Ser58, which suggests that the endosomal targeting of MTMR2 is regulated through an ERK1/2 negative feedback mechanism. Surprisingly, treatment with multiple MAPK inhibitors resulted in a MTMR2 localization shift from Rab5-positive endosomes to the more proximal APPL1-positive endosomes. This MTMR2 localization shift was recapitulated when a double phosphorylation-deficient mutant (MTMR2 S58A/S631A) was characterized. Moreover, expression of this double phosphorylation-deficient MTMR2 variant led to a more sustained and pronounced increase in ERK1/2 activation compared with MTMR2 S58A. Further analysis of combinatorial phospho-mimetic mutants demonstrated that it is the phosphorylation status of Ser58 that regulates general endosomal binding and that the phosphorylation status of Ser631 mediates the endosomal shuttling between Rab5 and APPL1 subtypes. Taken together, these results reveal that MTMR2 compartmentalization and potential subsequent effects on endosome maturation and endosome signaling are dynamically regulated through MAPK-mediated differential phosphorylation events.
authors: Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B3P4, Canada.
authors: Norah E
authors: NE
authors: Franklin
authors: Christopher A
authors: CA
authors: Bonham
authors: Besa
authors: B
authors: Xhabija
authors: Panayiotis O
authors: PO
authors: Vacratsis
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D054558:Protein Tyrosine Phosphatases, Non-Receptor / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
chemical_list: C464801:APPL1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D045163:Antibodies, Phospho-Specific; D007093:Imidazoles; D011725:Pyridines; D034741:RNA, Small Interfering; D048049:Extracellular Signal-Regulated MAP Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C408986:MTMR2 protein, human; D054558:Protein Tyrosine Phosphatases, Non-Receptor; D020696:rab5 GTP-Binding Proteins; C093642:SB 203580
keywords: APPL1; ERK; Endocytosis; MTMR2; PI(3)P; Phosphoinositol 3-phosphate; Phosphorylation