RESUMEN
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.
Asunto(s)
Endosomas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Transporte de Proteínas , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anticuerpos Fosfo-Específicos/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Células HEK293 , Células HeLa , Humanos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Mutación/genética , Fosforilación/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Proteínas Tirosina Fosfatasas no Receptoras/genética , Piridinas/farmacología , ARN Interferente Pequeño/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser(58), or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.
