Date of Award
5-16-2024
Publication Type
Thesis
Degree Name
M.Sc.
Department
Chemistry and Biochemistry
Supervisor
Panayiotis Vacratsis
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Abstract
MTMR2 is a phosphoinositide lipid phosphatase that plays a crucial role for endosomal membrane trafficking. Mutations in MTMR2 are associated with CMT4B1, an inheritable neuromuscular disorder affecting proper Schwann cell myelination. Reversible phosphorylation of Ser58 is ERK-mediated in a negative feedback loop and attenuates localization of MTMR2 to PI3P positive vesicles. Unphosphorylated MTMR2 sequesters to early endosomes where it depletes PI3P, and phosphorylated MTMR2 disperses cytoplasmically. In these studies, we developed high resolution mass spectrometry (HR-MS) targeted approaches that incorporated high definition multiple reaction monitoring (HD-MRM) to advance our understanding of Myotubularin (MTM) phosphorylation dynamics. We used these analytical tools to identify novel phosphorylation sites on MTMR2 (Ser49) and MTMR5 (Ser118, Ser1229, Thr1820, and either Ser1749/Thr1750). We were able to detect changes in phosphorylation on MTMR2 and MTMR5 in response to various conditions, i.e.: Caly A treatments, MAPK inhibitor treatments, and co?transfection experiments. Our results provide evidence that MTMR2 phosphorylation at Ser58 elicits a conformational change making this region more solvent exposed. Inhibitor studies revealed that MTMR5 Ser1229 may be a novel substrate of JNK. We also used a BioID approach to identify the MTMR2 proximity proteome in response to changes in Ser58 phosphorylation. We found evidence that MTMR2 distributes to regions of the cells occupied by proteins essential for synaptic dynamics and for regulating Schwann cell polarity. We also have evidence that MTMR10 is a potential MTMR2 pseudophosphatase binding partner and competes with MTMR5 for association with MTMR2.
Recommended Citation
Hand, Kaitlyn Susan Maureen, "Investigating Phosphorylation of the Myotubularin Family Members MTMR2 and MTMR5 Using High Resolution Mass Spectrometry" (2024). Electronic Theses and Dissertations. 9409.
https://scholar.uwindsor.ca/etd/9409