Date Thesis Awarded
5-2023
Access Type
Honors Thesis -- Access Restricted On-Campus Only
Degree Name
Bachelors of Science (BS)
Department
Biology
Advisor
Shantá D. Hinton
Committee Members
Mark Forsyth
Dan Cristol
Larry Leemis
Abstract
MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein) is a pseudophosphatase belonging to the MAPK phosphatase (MKP) subfamily within the protein tyrosine phosphatase (PTP) superfamily. The absence of a histidine and critical cysteine in the signature active motif render MK-STYX catalytically inactive, earning it the prefix pseudo. A mass spectrometry study revealed that MK-STYX and the cytoskeletal protein vimentin are binding partners. Vimentin is expressed in the early stage of neuronal development, contributes to organelle organization, and localizes at the perinucleus, mitochondria, and golgi. It has been reported that the downregulation of vimentin decreases neuritogenesis. Previous research revealed that MK-STYX increases neurite outgrowth in rat pheochromocytoma PC-12 cells and rat hippocampal primary neurons. This interaction of MK-STYX and vimentin and the role of MK-STYX in neurite formation point towards a possible relationship between the two proteins that may contribute to neuritogenesis. Our goal is to further confirm and characterize the interaction between MK-STYX and vimentin. HEK-293 cells were cotransfected with the following mammalian expression vectors: GFP and mCherry, GFP-MK-STYX and mCherry, GFP-MK-STYX(active mutant) and mCherry, mCherry-vimentin and GFP, GFP-MK-STYX and mCherry-vimentin, or GFP-MK-STYX(active mutant) and mCherry-vimentin followed by western blot analysis. Vimentin expression was increased in the presence of MK-STYX. To further characterize the dynamics between MK-STYX and vimentin, we performed colocalization experiments and used fluorescence microscopy to analyze them. The conditions were GFP, mCherry, GFP and mCherry, GFP-MK-STYX, mCherry-vimentin, GFP and mCherry-vimentin, GFP-MK-STYX(active mutant) and mCherry-vimentin, or GFP-MK-STYX and mCherry-vimentin. Higher rates of partial colocalization were observed in GFP-MK-STYX and mCherry-vimentin conditions (≥75%) than in the mCherry-vimentin and GFP condition, confirming the pattern observed in our pilot colocalization experiments. All instances of colocalization were observed in the cytoplasm and future studies will investigate vimentin localization at the perinucleus, mitochondria, or golgi in the presence of MK-STYX and GFP-MK-STYX(active mutant) which has phosphatase activity. Taken together the colocalization pattern of MK-STYX and vimentin and the increase of vimentin in the presence of MK-STYX support the hypothesis that MK-STYX and vimentin are interacting and together may serve as important players in neuritogenesis.
Recommended Citation
Wagner, Gwen, "Investigating the Interaction between Pseudophosphatase MK-STYX and Vimentin" (2023). Undergraduate Honors Theses. William & Mary. Paper 1983.
https://scholarworks.wm.edu/honorstheses/1983
