Date of Award

2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Vacratsis, Panayiotis

Keywords

Endocytosis; Myotubilarin; Parkinson; Receptor Mediated Endocytosis 8; Retrograde; Signalling

Rights

CC BY-NC-ND 4.0

Abstract

Myotubularin related protein 2 (MTMR2) is a member of the myotubularin family of phosphoinositide lipid phosphatases whose subcellular localization is regulated by a phosphorylation event on Ser 58. Our laboratory has shown that the phosphorylationmimetic mutant (S58E) targets MTMR2 to the cytoplasm, whereas the phosphorylationdeficient variant MTMR2 (S58A) targets it to Rab5-positive endosomes resulting in PI(3)P depletion. Although MTMR2 dephosphorylates PI(3)P and PI(3,5)P2, the phosphoinositide binding proteins that are regulated by MTMR2 are poorly characterized. In this study, we identified RME-8 as a novel PI(3)P binding protein implicated in the translocation of Hsc70 to early endosomes for clathrin removal during retrograde transport. Remarkably, the depletion of PI(3)P by MTMR2 S58A attenuated RME-8 endosomal localization. Moreover, we have identified the amino acid determinants required for PI(3)P binding within a region predicted to adopt a pleckstrin homology-like fold in the N terminus of RME-8. The ability of RME-8 to associate with PI(3)P and early endosomes is largely abolished when residues Lys17, Trp20, Tyr24, or Arg26 are mutated resulting in diffuse cytoplasmic localization of RME-8 while maintaining the ability to interact with Hsc70. We also provide evidence that RME-8 PI(3)P binding regulates the early endosomal clathrin dynamics and alters the steady state localization of the cation independent mannose 6-phosphate receptor.

In addition, once a phosphorylation-deficient variant (S58A) targets MTMR2 to Rab5-positive endosomes and depleting PI(3)P, it results in increased endosomal signaling, such as a significant increase in ERK1/2 activation. Using in vitro kinase assays, cellular MAPK inhibitors, siRNA knockdown and a phosphospecific-Ser58 antibody, we 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.

Taken together, our results suggest a model in which the localization of RME-8 to endosomal compartments is spatially mediated by PI(3)P binding and temporally regulated by MTMR2 activity and compartmentalization of MTMR2 and potential subsequent effects on endosome maturation and endosome signaling are dynamically regulated through MAPK-mediated differential phosphorylation events.

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