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dc.contributor.advisorRosen, Michael K.en
dc.creatorIsmail, Ayman Mohameden
dc.date.accessioned2010-07-12T17:47:44Zen
dc.date.accessioned2014-02-19T22:01:43Z
dc.date.available2010-07-12T17:47:44Zen
dc.date.available2014-02-19T22:01:43Z
dc.date.issued2009-09-04en
dc.identifier.other760312934en
dc.identifier.urihttp://hdl.handle.net/2152.5/370en
dc.description.abstractMembers of the Wiskott-Aldrich syndrome protein (WASP) family (WASP, N-WASP, WAVE 1-3) have a central role in the transmission of the extracellular signals to the actin cytoskeleton. These proteins use their C-terminal VCA domain to stimulate the actin-nucleating activity of Arp2/3 complex in response to upstream signals from the Rho family GTPases Cdc42 and Rac1. While WASP regulation by GTPases and kinases is well characterized both biochemically and structurally, little is known about WAVE regulation. WAVE exists as part of a five protein complex termed the WAVE Regulatory Complex (WRC). It consists of WAVE, Sra1, Nap1, Abi2 and HSPC300. Biochemical studies of WRC have been hampered by the difficulty of expressing WRC components in bacterial, insect or yeast expression systems. Baculoviruses yielding high expression of each component of WRC were obtained using a modified pFastBac vector, where translation is driven by the lobster tropomyosin promoter. Co-infection into Sf9 cells allowed efficient expression and purification of WRC and two sub-complexes, Sra-Nap and Abi2-WAVE1-HSPC300. We show that WRC is inactive toward Arp2/3 complex in pyrene based actin assembly assays. However, Abi2-WAVE1-HSPC300 heterotrimer is active and Sra1-Nap1 heterodimer inhibits it suggesting that WRC is autoinhibited. A modified WRC complex, where WAVE1 has a PreScission protease site between its VCA domain (the active domain) and its N-terminus and is lacking the proline rich domain, is also inactive toward Arp2/3 complex. However, upon digestion with PreScission protease, this modified complex becomes active. This suggests that the affinity between VCA and the Sra-Nap heterodimer is inherently weak and the heterodimer requires the linkage provided by the Abi2-WAVE1-HSPC300 heterotrimer to VCA to efficiently inhibit it from activating the Arp2/3 complex. The same results are obtained using all Drosophila components. Finally, Rac1-GTP is able to activate WRC towards Arp2/3. However, no dissociation of the complex is detected upon activation by Rac1. In addition to WRC regulation, we have established the mechanism for hyperactivation of VCA through dimerization. We found that a dimeric VCA construct binds Arp2/3 complex with a two VCAs to one Arp2/3 ratio. The affinity of dimeric VCA for Arp2/3 is at least 100 fold higher than monomeric VCA. That explains the potentiation of VCA toward Arp2/3 observed upon VCA dimerization, and provides a mechanistic framework for a new model of WASP regulation superimposed upon allostery. We have also demonstrated that N-WASP and WRC may be able to form a hetero-VCA dimer through the interaction of Abi2 SH3 domain and N-WASP PRD. Such interaction increases the complexity and the signal integration potential of WASp family proteins.en
dc.format.mediumElectronicen
dc.format.mimetypeapplication/pdfen
dc.language.isoenen
dc.subjectWiskott-Aldrich Syndrome Protein Familyen
dc.subjectModels, Molecularen
dc.subjectActinsen
dc.titleBiochemical Reconstitution and Functional Characterization of the WAVE Regulatory Complexen
dc.type.genredissertationen
dc.type.materialTexten
thesis.degree.nameDoctor of Philosophyen
thesis.degree.levelPh.D.en
thesis.degree.disciplineMolecular Biophysicsen
thesis.degree.grantorGraduate School of Biomedical Sciencesen
thesis.degree.departmenten
dc.format.digitalOriginborn digitalen
thesis.date.available2011-09-04en


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