TAILIEUCHUNG - Báo cáo khoa học: The guanine nucleotide exchange factor RasGRF1 directly binds microtubules via DHPH2-mediated interaction

RasGRF is a family of guanine nucleotide exchange factors with dual spe-cificity for both Ras and Rac GTPases. In this study, using mouse brain extracts, we show that both RasGRF1 and RasGRF2 interact with micro-tubules in an in vitro microtubule assembly system and this binding is very tight. To characterize this association, recombinant purified proteins con-taining different regions of RasGRF1 were tested for their ability to bind microtubules preassembled from pure tubulin. | ềFEBS Journal The guanine nucleotide exchange factor RasGRFI directly binds microtubules via DHPH2-mediated interaction Greta Forlani Simona Baldassa Paola Lavagni Emmapaola Sturani and Renata Zippel Department of Biomolecular Sciences and Biotechnology University of Milan Italy Keywords DHPH2 module microtubule Ras RasGRF sodium arsenate Correspondence R. Zippel Department of Biomolecular Sciences and Biotechnology University of Milan via Celoria 26 20133 Milan Italy Fax 39 025031 4912 Tel 39 025031 4914 E-mail Received 16 January 2006 revised 7 March 2006 accepted 13 March 2006 doi RasGRF is a family of guanine nucleotide exchange factors with dual specificity for both Ras and Rac GTPases. In this study using mouse brain extracts we show that both RasGRF1 and RasGRF2 interact with microtubules in an in vitro microtubule assembly system and this binding is very tight. To characterize this association recombinant purified proteins containing different regions of RasGRF1 were tested for their ability to bind microtubules preassembled from pure tubulin. Only the DHPH2 tandem directly associates with microtubules whereas the isolated DH or PH2 domains do not indicating that the entire DHPH2 region is required for this association. The interaction occurs with high affinity Kd w 2 pM and with a stoichiometry at saturating conditions of one DHPH2 molecule for two tubulin dimers. Competition experiments support the hypothesis that the DHPH2 module is largely responsible for RasGRFI-microtubule interaction. In vivo colocalization of RasGRFI and microtubules was also observed by fluorescence confocal microscopy in nonneuronal cells after stimulation with an oxidative stress agent and in highly differentiated neuron-like cells. Identification of microtubules as new binding partners of RasGRFI may help to elucidate the signaling network in which RasGRFI is involved. Microtubules are crucial elements in the generation and

• Báo cáo khoa học
• Report medicine
• traditional medicine
• scientific reports
• molecular Biology
• tumor cells
• Antimicrobial
• medical knowledge
• medical research
• analysis of cytoplasmic
• oxidation
• Crystal structure
• bacteria
• hemoglobin
• medicine
• cell proliferation
• breast cancer
• chemical reaction
• gastric cancer
• hormone medicine
• biochemical studies
• environmental medicine