Thesis Title: Modulation of the actin cytoskeleton through phosphorylation of actin regulators
PhD student: Pavithra Singaravelu1, 2
Supervisors: Robert Robinson1, 3, Kunchithapadam Swaminathan2
1 Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore
2 Department of Biological Sciences, National University of Singapore, Singapore.
3 Department of Biochemistry, National University of Singapore, Singapore.
Yersinia species cause a range of human diseases, including yersiniosis (Yersinia pseudotuberculosis and Yersinia enterocolitica) and the plague (Yersinia pestis). Yersinia modulate and evade host immune systems through injection of Yersinia outer proteins (Yops) into phagocytic cells. YopO/YpkA impairs phagocytosis through actin filament disruption. YopO/YpkA uses monomeric actin as bait to recruit and phosphorylate host actin polymerization regulating proteins. The functions of these phosphorylation events are largely unknown. Here, we report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals the active site architecture of the kinase domain. We identify the YopO phosphorylation sites on gelsolin, mDia1, VASP, EVL and WASP by mass spectrometry. YopO phosphorylation of the actin filament elongating proteins VASP, EVL and mDia1 attenuates their abilities to accelerate in vitro actin polymerization. Phosphorylation sites on VASP and mDia1 were validated with mutational studies. In contrast, phosphorylation of WASP by YopO has no observable effect on ARP2/3 activation. Finally, we determine that YopO phosphorylates gelsolin in the linker region between G3-G4 to give rise to calcium-independent activity. Together, these results indicate that phosphorylation of actin polymerization regulators by YopO leads to altered activities, which collectively contribute to reduction in filamentous actin.
Figure legend: The effects of YopO on actin dynamics.
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