Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes

Abstract

SummaryMyotubularin-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.

Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling

“…4b, c), had marked elevation in phosphorylated MTMR2 (Fig. 7f), a form of the protein that has been previously shown to be unable to bind to endosomes 58 . To investigate if defective MTMR2 recruitment to endosomes is responsible for the phenotype resulting from COMMD3 deficiency, we expressed HA-tagged MTMR2 wild type (WT) or phosphorylation-resistant mutant (S58A).…”

“…Given the physical interaction of MTMR2 with CCDC22, we investigated whether the endosomal recruitment of MTMR2 was affected by CCC deficiency. Phosphorylation of MTMR2 on serine 58 has a profound effect on its cellular localization, preventing its recruitment to endosomes 58 . This residue is preceded by phenylalanine and upon phosphorylation may be recognized by a phosphoserine antibody raised against this motif (phosphoserine preceded by tyrosine, phenylalanine, or tryptophan).…”

Starvation‐induced MTMR 13 and RAB 21 activity regulates VAMP 8 to promote autophagosome–lysosome fusion

“…MTMR13 post‐translational modifications are one possible mechanism for this specific regulated response. Phosphorylation of either Sbf/MTMR13 and/or an interacting protein, such as MTMR2 , might regulate MTMR13 endosomal interactions, GEF activity or stability . In this regard, Drosophila Sbf and human MTMR13 both have conserved demonstrated phosphorylation sites .…”

Membrane Heterogeneity Controls Cellular Endocytic Trafficking

“…The transition from the endocytic pit to APPL + endosomes is driven by the conversion of PI(4,5)P 2 to PI by OCRL and INPP5B. Under specific signaling conditions, the 3-phosphatase MTMR2 specifically associates with APPL and, in conjunction with OCRL, can also generate further PI from PI(3,4)P 2 (Erdmann et al, 2007;Franklin et al, 2013). The APPL + endosome is initially completely devoid of PI(3)P. The substitution of APPL for Rab5 is promoted by acquisition of PI(3)P (Zoncu et al, 2009;Kalaidzidis et al, 2015).…”