When cells are exposed to stress such as high temperature or ultraviolet rays from the sun, they respond quickly to ensure survival. One of the most rapid responses is stopping protein production, which allows cells to adapt to stress. Cells maintain this control by sequestering messenger RNAs, which provide the blueprint for protein synthesis, to special compartments called stress granules. A messenger RNA binding protein named G3BP-1 is widely accepted as an important component of stress granules. Researchers recently reported that another protein called MK-STYX interacts with G3BP-1 and inhibits stress granule formation. Although stress granules have been studied extensively for the past eight years, most studies have focused exclusively on G3BP-1 and other components involved in regulating stress granule assembly. This research explores the novel role MK-STYX has in disassembly of stress granules and in blocking their formation. This study will provide insight into the role MK-STYX plays in the stress response, as a regulator of messenger RNA stability and in the dynamics of stress granule formation. The following specific aims will be addressed: (1) Determine the effect of MK-STYX on the cellular pathway responsible for protein production. Does MK-STYX indirectly activate or bypass this pathway to block stress granule assembly? Does MK-STYX stabilize protein production, which would prevent stress granule formation, or is it required for their disassembly? (2) Determine the effect of MK-STYX on the G3BP-1 complex required for stress granule assembly. Does MK-STYX disrupt interactions between G3BP-1 and other scaffold proteins, which are required for stress granule assembly? (3) Determine the effect of MK-STYX on the cell cytoskeleton and chaperones required for stress granule dynamics. Does MK-STYX interact with heat shock proteins required for disassembly, such as Hsp70?

Broader Impacts. This project provides the opportunity to integrate seamlessly research and education. It will allow the investigator to enhance the biology curriculum at the College of William & Mary and to serve as a positive role model for underrepresented students. One of the investigator's goals is to design a lecture and laboratory course in biological chemistry, which will expose students to the study of protein-protein interactions and their function. Aims and results of her research will be incorporated into her teaching. The investigator has always been committed to programs that enhance participation of underrepresented groups in science. However, she has realized that just being a faculty member is not enough to retain African-Americans and other minorities in the sciences. It is imperative to encourage individuals from underrepresented groups to obtain PhDs become independent investigators themselves. The investigator will also participate in an HHMI-funded program that provides underrepresented groups with a unique undergraduate research experience and that and promotes collaborations with local historically black-serving institutions such as Hampton University, Norfolk State University, and Virginia State University to foster professional development and mentoring relationships between faculty and students. The investigator will continue to provide real research opportunities for her students so that they may engage in the exciting process of scientific discovery.

Project Report

ver the past 2 years, we have made significant progress in our research addressing how MK-STYX (MAP kinase serine/threonine/tyrosine binding protein) inhibits stress granule formation and its role in neuronal differentiation. This work has allowed formulation of the proposed work. To date, a manuscript has been published with 3 undergraduate co-authors, another manuscript is under revision for resubmission,and students have presented posters at the American Society for Cell Biology (1 in 2011, 3 in 2012, including the REU student, and 1 in 2013) and the Virginia Academy of Sciences (2012). In addition, two high school students received training in cellular and molecular techniques and worked on independent projects. One of these high school students won first place for her poster presentation at the Beta Kappa Chi/National Institute of Sciences Annual Meeting (2013) and at the National Association for the Advancement of Colored People -Afro-Academic, Cultural, Technological, and Scientific Olympics (NAACP ACT-SO) regional competition. Furthermore, she won third place at the national NAACP ACT-SO conference that resulted in a $1500 college scholarship [featured in The Virginia Gazette (July 31, 2013) and W & M webpage (www.wm.edu/news/stories/2013/whos-that-in-the-lab-it-just-might-be-a-high-school-student123.php)]. The following report highlights exciting outcomes of this NSF funded project (1113167). INTELLECTUAL MERIT: Pseudophosphatase MK-STYX inhibits stress granule formation independently of G3BP-1 phosphorylation (Barr et al., 2013): The pseudophosphatase MK-STYX is a member of the protein tyrosine phosphatase (PTP) family, but lacks the essential cysteine in the PTP signature motif that is critical for catalysis. We previously showed that MK-STYX inhibits the assembly of stress granules, which are cytoplasmic storage sites for mRNA that form as a protective mechanism against stressors such as heat shock, UV irradiation, and hypoxia (Hinton et al., 2010). Furthermore, MK-STYX interacts with a key component of stress granules, G3BP-1 (Ras-GTPase activating protein SH3 domain binding protein-1). Since G3BP-1 dephosphorylation at serine 149 induces stress granule assembly, we initially hypothesized that MK-STYX inhibition of stress granules was G3BP-1 phosphorylation-dependent. In our published report, arising from NSF #MCB 113167, we showed that MK-STYX inhibits stress granule formation independently of G3BP-1’s phosphorylation status at serine 149 (Barr et al., 2013). We also showed that an active mutant of MK-STYX, in which catalytic activity is restored, has the opposite effects to wild-type MK-STYX. Our results provided compelling evidence that MK-STYX plays a key role in the cellular response to stress. Publication Resulting from NSF Award #MCB113167 (*W&M undergraduates; † Hampton University undergraduate): *Barr, J.E., * Munyikwa, M.R.,†Frazier, E.A., and Hinton, S.D. The Pseudophosphatase MK-STYX Inhibits Stress Granule Assembly Independently of Ser149 Phosphorylation of G3BP-1. Federation of European Biochemical Societies (FEBS) Journal, 280 (2013), 273-284. BROADER IMPACTS: Contribution of the Completed Work to the Development of Human Resources in Sciences: Within 2 years, the PI has established a very productive lab that is actively engaged in recruiting and training students. To date she has developed 1 Master’s, 11 undergraduates (1 REU-Hampton University); 2 high school students (1 RAHSS).These students have contributed to the significant findings highlighted above, as well as the exciting findings discussed in the Preliminary Data. Since students helped set up the P.I.’s new lab, they have gained experience in lab management. Also, veteran lab members develop teaching skills by helping train the newest lab members. Lab members participate in weekly lab meetings, where everyone takes turn presenting their latest data. In addition, undergraduates are encouraged to complete an Honor’s Thesis (4 to date). Notably, Justin Barr won the 2012 Mangum Prize for Outstanding Research in Biology. Lab members are also encouraged to participate in scientific conferences (7 including REU and RAHSS awardess). All these experiences have led to careers in research or other professionally related disciplines (7 to date). Furthermore,to engage younger students, the P.I. developed a scientific program called "Open Lab," where her lab hosts children from Preschool/Elementary and Afterschool programs along with their parents for an entire day to help expose them to life as a scientist. "Open Lab" starts with an interactive PowerPoint, to introduce my research, rules of the lab, and the experiments to be performed by the children. Students in the PI’s lab create and write the script for the presentation. Once the children completed their experiments they were assessed on what they learned by painting art canvases for display in the Biology Dept. Parents were assessed with Pre and Post-Surveys. These surveys showed that 14.3% of parents had never visited W&M, although most (71.4%) had visited Colonial Williamsburg, in the College’s backyard. Before Open Lab all parents preferred that their child become a medical doctor; most were not aware of what scientists do. Open Lab changed their minds (71.4%). Photos of the event are on the PI’s website, and the Biology Dept website(www.wm.edu/as/biology/news/open_lab.php).

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Gregory W. Warr
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College of William and Mary
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