A structure-function analysis of a Rho GTPase exchange factor (GEF)-GTPase activating protein (GAP) interaction

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2011

Authors

Fraval, Hamilton

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Abstract

Cytokinesis is the final stage of cell division where major rearrangements of the cytoskeleton occur that result in the formation and constriction of a contractile ring at the equator of the cell. The GTPase Rho is essential for cytokinesis and it is the role of the Rho GTPase Exchange Factor (GEF) Pbl to stimulate the accumulation of an equatorial band of active Rho. Accumulation of Pbl at the midzone requires an interaction with the GTPase Activating Protein (GAP) Tum, which forms a heterotetrameric complex with the microtubule motor protein Pavarotti (Pav). It is the motor activity of Pav that localizes Pbl and Tum to the equator where they activate downstream cytokinetic effectors and thereby induce contractile ring formation and constriction. However, the mechanism/s regulate the Pbl-Tum interaction during cytokinesis are unknown. This thesis describes two strategies that probe the Pbl-Tum interaction using a structure-function analysis. The first strategy details attempts to over-express and purify Pbl and Tum in an effort to obtain enough pure protein with which to perform structural studies. The second strategy compares the effect of wild-type and mutant transgenes to identify phosphorylation events that mediate the Pbl-Tum interaction. Through this work I identified a previously uncharacterized phosphorylation event in Tum that is required to bind and localize Pbl to the equator in vivo in Drosophila.

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Thesis (PhD)

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Open Access

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