Association between Ras GTPase-activatingprotein and the neuronal guidance receptorDSCAM in Drosophila melanogaster

Abstract

AbstractRas GTPase-activating protein (RasGAP) is an important regulator of Ras proteins,converting active GTP-bound Ras to inactive GDP-bound Ras. Through its regulationof Ras, RasGAP plays an important role in regulating the strength and duration ofsignalling by receptor tyrosine kinases (RTKs). Although Ras is known to functiondownstream of receptors other than RTKs, whether RasGAP also functions in othersignalling pathways is not known. A recent study in this laboratory identifiedDrosophila Down syndrome cell adhesion molecule (Dscam), a cell surface receptorthat plays an important role in axon guidance in the nervous system, as a potentialRasGAP interacting partner. In this study I have investigated the potential interactionbetween RasGAP and Dscam using biochemical and genetic approaches. RasGAPco-immunoprecipitated with Dscam when both proteins were overexpressed in S2 cells.Further studies showed that the N-terminal SH2 (Src homology 2) domain of RasGAPwas sufficient to mediate association with the cytoplasmic domain of Dscam. There wasa single tyrosine residue in the cytoplasmic domain of Dscam matching the preferredbinding motif (phosphoTyr-X-X-Pro-X-Asp) for the RasGAP SH2 domain. However, amutant Dscam protein with the putative tyrosine mutated to phenylalanine stillefficiently associated both with full length RasGAP and with the isolated N-terminalSH2 domain. These biochemical studies show that RasGAP and Dscam can associate invitro, but it is not known whether they interact directly or are part of the same proteincomplex. As Dscam is involved in formation of the embryonic ventral nerve cord (VNC)in Drosophila, I investigated whether RasGAP also had a role in VNC formation.RasGAP mutant embryos affected the structure of the VNC with defects in thecontinuity of the outer longitudinal fascicules. However, there was no evidence for astrong genetic interaction between RasGAP and Dscam mutants with respect to VNCformation. In conclusion, although RasGAP and Dscam proteins associate in vitro thereis no evidence that they function together during VNC formation in Drosophilaembryos.

Date of Award	31 Dec 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Hughes (Supervisor) & Andreas Prokop (Supervisor)