Date Thesis Awarded
Bachelors of Science (BS)
Shantá D. Hinton
Mark H. Forsyth
Regulation of cell signaling is critical for determining fundamental processes such as whether a cell will grow, change shape, differentiate or die. Phosphorylation is a post-translational modification of proteins that is key for transducing signaling messages. The major players in phosphorylation cascades are proteins called kinases and phosphatases. Pseudokinases and pseudophosphatases are catalytically inactive proteins that add complexity to the regulation of cell signaling. In this thesis, the pseudophosphatase MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] is implicated in the stress response pathway. MK-STYX interacts with G3BP-1 (Ras-GTPase activating protein SH3 domain binding protein-1), and inhibits stress granule assembly. Stress granules, cytoplasmic storage sites for mRNA, form as a protective mechanism against stressors such as UV irradiation, hypoxia, and heat shock. In addition, the overexpression of G3BP-1 induces stress granule assembly. The initial hypothesis for how MK-STYX attenuates stress granule assembly was through a mechanism dependent on the phosphorylation status of G3BP-1 at serine 149. However, data with a G3BP-1 phosphomimetic mutant suggests differently. The eukaryotic initiation factor 2 alpha (eIF2α) is investigated for playing a role in the reduction of stress granules by MK-STYX. eIF2α initiates translation by forming a ternary complex with methionine bound to tRNA. Phosphorylation of eIF2α arrests translation and results in stress granule formation, whereas dephosphorylation promotes polysomes. To determine if MK-STYX inhibits stress granule assembly via upstream interactions, we investigated its effects on eIF2α phosphorylation. HeLa cells were transfected with pMT2-FLAG-MK-STYX-FLAG or pMT2 expression vectors, and heat shocked for an hour to induce stress. Cells were lysed and immunoblots for phosphorylation of eIF2α were performed. We show that the presence of MK- STYX reduced the phosphorylation of eIF2α. Also, MK-STYX was immunoprecipitated with eIF2α, suggesting that MK-STYX and eIF2α interact. These data are significant because they suggest MK-STYX may play a role in stress granule assembly. MK-STYX involvement in disassembly was also investigated by immunoblotting for Hsp70. Hsp70 is thought to be necessary for stress granule disassembly and reduces protein aggregation. The overexpression of MK-STYX correlates with increased expression of Hsp70, suggesting MK-STYX may also play a role in stress granule disassembly. This thesis implicates MK-STYX, a pseudophosphatase, as a regulator of cellular processes.
Barr, Justin, "The Role of MK-STYX in Stress Granule Assembly/Disassembly" (2012). Undergraduate Honors Theses. Paper 863.
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