The guanine nucleotide exchange factor RasGRF1 directly binds microtubules via DHPH2-mediated interaction.
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 approximately = 2 microM) 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 RasGRF1-microtubule interaction. In vivo colocalization of RasGRF1 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 RasGRF1 may help to elucidate the signaling network in which RasGRF1 is involved.
Forlani
Greta
G
Department of Biomolecular Sciences and Biotechnology, University of Milan, Italy.
Baldassa
Simona
S
Lavagni
Paola
P
Sturani
Emmapaola
E
Zippel
Renata
R
D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics; D020663:ras Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D020666:ras-GRF1 / Q000235:genetics / Q000378:metabolism*
C504491:Rasgrf2 protein, mouse; D011994:Recombinant Proteins; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1