Specificity of Interactions between mDia Isoforms and Rho Proteins
2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês
10.1074/jbc.m805634200
ISSN1083-351X
AutoresMichael Lammers, Simon F. De Meyer, Dorothee Kühlmann, Alfred Wittinghofer,
Tópico(s)Muscle Physiology and Disorders
ResumoFormins are key regulators of actin nucleation and polymerization. They contain formin homology 1 (FH1) and 2 (FH2) domains as the catalytic machinery for the formation of linear actin cables. A subclass of formins constitutes the Diaphanous-related formins, members of which are regulated by the binding of a small GTP-binding protein of the Rho subfamily. Binding of these molecular switch proteins to the regulatory N-terminal mDiaN, including the GTPase-binding domain, leads to the release of auto-inhibition. From the three mDia isoforms, mDia1 is activated only by Rho (RhoA, -B, and -C), in contrast to mDia2 and -3, which is also activated by Rac and Cdc42. Little is known about the determinants of specificity. Here we report on the interactions of RhoA, Rac1, and Cdc42 with mDia1 and an mDia1 mutant (mDiaN-Thr-Ser-His (TSH)), which based on structural information should mimic mDia2 and -3. Specificity is analyzed by biochemical studies and a structural analysis of a complex between Cdc42·Gpp(NH)p and mDiaN-TSH. A triple NNN motif in mDia1 (amino acids 164-166), corresponding to the TSH motif in mDia2/3 (amino acids 183-185 and 190-192), and the epitope interacting with the Rho insert helix are essential for high affinity binding. The triple N motif of mDia1 allows tight interaction with Rho because of the presence of Phe-106, whereas the corresponding His-104 in Rac and Cdc42 forms a complementary interface with the TSH motif in mDia2/3. We also show that the F106H and H104F mutations drastically alter the affinities and thermodynamics of mDia interactions. Formins are key regulators of actin nucleation and polymerization. They contain formin homology 1 (FH1) and 2 (FH2) domains as the catalytic machinery for the formation of linear actin cables. A subclass of formins constitutes the Diaphanous-related formins, members of which are regulated by the binding of a small GTP-binding protein of the Rho subfamily. Binding of these molecular switch proteins to the regulatory N-terminal mDiaN, including the GTPase-binding domain, leads to the release of auto-inhibition. From the three mDia isoforms, mDia1 is activated only by Rho (RhoA, -B, and -C), in contrast to mDia2 and -3, which is also activated by Rac and Cdc42. Little is known about the determinants of specificity. Here we report on the interactions of RhoA, Rac1, and Cdc42 with mDia1 and an mDia1 mutant (mDiaN-Thr-Ser-His (TSH)), which based on structural information should mimic mDia2 and -3. Specificity is analyzed by biochemical studies and a structural analysis of a complex between Cdc42·Gpp(NH)p and mDiaN-TSH. A triple NNN motif in mDia1 (amino acids 164-166), corresponding to the TSH motif in mDia2/3 (amino acids 183-185 and 190-192), and the epitope interacting with the Rho insert helix are essential for high affinity binding. The triple N motif of mDia1 allows tight interaction with Rho because of the presence of Phe-106, whereas the corresponding His-104 in Rac and Cdc42 forms a complementary interface with the TSH motif in mDia2/3. We also show that the F106H and H104F mutations drastically alter the affinities and thermodynamics of mDia interactions. The dynamics of the actin cytoskeleton play an important role in many physiological processes, e.g. cytokinesis, cell migration, cell morphology, and phagocytosis (1.Castrillon D.H. Wasserman S.A. Development (Camb.). 1994; 120: 3367-3377Crossref PubMed Google Scholar, 2.Pantaloni D. Le Clainche C. Carlier M.F. Science. 2001; 292: 1502-1506Crossref PubMed Scopus (565) Google Scholar, 3.Pollard T.D. Borisy G.G. 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From the structure of the FH2 alone and in complex with tetramethylrhodamine-labeled actin and from biochemical experiments, the mechanism of actin polymerization by the formin was described as a stair-stepping mechanism, where the FH2 dimer binds to the barbed ends of actin filaments as a leaky capper that processively catalyzes barbed-end actin assembly (11.Romero S. Le Clainche C. Didry D. Egile C. Pantaloni D. Carlier M.F. Cell. 2004; 119: 419-429Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 13.Zigmond S.H. Evangelista M. Boone C. Yang C. Dar A.C. Sicheri F. Forkey J. Pring M. Curr. Biol. 2003; 13: 1820-1823Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 17.Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 22.Higashida C. Miyoshi T. Fujita A. Oceguera-Yanez F. Monypenny J. Andou Y. Narumiya S. Watanabe N. 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Cell. 2004; 119: 419-429Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 22.Higashida C. Miyoshi T. Fujita A. Oceguera-Yanez F. Monypenny J. Andou Y. Narumiya S. Watanabe N. Science. 2004; 303: 2007-2010Crossref PubMed Scopus (251) Google Scholar, 23.Kovar D.R. Pollard T.D. Nat. Cell Biol. 2004; 6: 1158-1159Crossref PubMed Scopus (42) Google Scholar). For most formins, the tandem arrangement of FH1-FH2 domains is necessary for function. FH1 contains several stretches of poly-l-prolines that are targets for Src homology 3 and WW domain-containing proteins and for profilin (19.Pring M. Evangelista M. Boone C. Yang C. Zigmond S.H. Biochemistry. 2003; 42: 486-496Crossref PubMed Scopus (190) Google Scholar, 29.Bedford M.T. Chan D.C. Leder P. EMBO J. 1997; 16: 2376-2383Crossref PubMed Scopus (190) Google Scholar, 30.Macias M.J. Wiesner S. Sudol M. FEBS Lett. 2002; 513: 30-37Crossref PubMed Scopus (393) Google Scholar). By binding to G-actin as well as to FH1, profilin delivers actin monomers to the barbed end of the filament and increases the concentration of actin near the FH2 domain (13.Zigmond S.H. Evangelista M. Boone C. Yang C. Dar A.C. Sicheri F. Forkey J. Pring M. Curr. Biol. 2003; 13: 1820-1823Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 28.Otomo T. Tomchick D.R. Otomo C. Panchal S.C. Machius M. Rosen M.K. Nature. 2005; 433: 488-494Crossref PubMed Scopus (290) Google Scholar). Therefore, FH1-FH2 accelerates the association rate for ATP-G-actin monomers at the barbed end, but it also accelerates the hydrolysis rate of actin-bound ATP (11.Romero S. Le Clainche C. Didry D. Egile C. Pantaloni D. Carlier M.F. Cell. 2004; 119: 419-429Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 22.Higashida C. Miyoshi T. Fujita A. Oceguera-Yanez F. Monypenny J. Andou Y. Narumiya S. Watanabe N. Science. 2004; 303: 2007-2010Crossref PubMed Scopus (251) Google Scholar). This leads to the release of profilin from the barbed-end making this site accessible for the next round of G-actin incorporation (11.Romero S. Le Clainche C. Didry D. Egile C. Pantaloni D. Carlier M.F. Cell. 2004; 119: 419-429Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). N-terminal to the FH1/FH2 domains, the subclass of Diaphanous-related formins contains a regulatory unit consisting of the GBD and FH3 domains (mDiaN in short), which interact with the C-terminal Diaphanous-autoregulatory domain (DAD) (31.Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 32.Lammers M. Rose R. Scrima A. Wittinghofer A. EMBO J. 2005; 24: 4176-4187Crossref PubMed Scopus (136) Google Scholar, 33.Otomo T. Otomo C. Tomchick D.R. Machius M. Rosen M.K. Mol. Cell. 2005; 18: 273-281Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 34.Rose R. Wittinghofer A. Weyand M. Acta Crystallogr. F Struct. Biol. 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The structure of the mDiaN in complex with activated RhoC shows that Rho interacts via switch I and II with the GBD but also with the ARR via the Rho insert helix. The DAD-binding site on mDiaN (DID) has also been described biochemically and structurally (32.Lammers M. Rose R. Scrima A. Wittinghofer A. EMBO J. 2005; 24: 4176-4187Crossref PubMed Scopus (136) Google Scholar). The structure shows that the 16 highly conserved residues of the DAD core region (DCR) form an α-helix, which binds to the ARR in a site not directly overlapping with the Rho-binding site (32.Lammers M. Rose R. Scrima A. Wittinghofer A. EMBO J. 2005; 24: 4176-4187Crossref PubMed Scopus (136) Google Scholar, 41.Nezami A. Poy F. Eck M. Structure (Lond.). 2006; 14: 257-263Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Nevertheless, Rho actively dissociates DAD from its binding site via long range electrostatic repulsion and short range steric clashes (32.Lammers M. Rose R. Scrima A. 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Very often, different signal transduction pathways via Rho proteins lead to similar or overlapping cellular responses (58.Van Aelst L. D'Souza-Schorey C. Gen. Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2101) Google Scholar). Effectors of Rho proteins are nevertheless not promiscuous in responding to either Rho and its homologues, i.e. RhoA, -B, -C, or to Cdc42 and Rac and its homologues such as TC10. Because a particular Rho protein interacts with a number of structurally different effectors via the highly conserved switch I and II regions, a high plasticity in the interface is required (59.Dvorsky R. Ahmadian M.R. EMBO Rep. 2004; 12: 1130-1136Crossref Scopus (125) Google Scholar). Numerous Rho family effectors have been described so far, defined as proteins that only bind to the GTP-bound state. ROCK I and II and Rhotekin specifically interact with and are activated by binding of Rho but not Rac or Cdc42 (39.Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. 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Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (727) Google Scholar), whereas mDia2 and -3 seem to be highly promiscuous and activated by RhoA-C, Cdc42, and RhoD (36.Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (688) Google Scholar, 39.Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (727) Google Scholar, 62.Gasman S. Kalaidzidis Y. Zerial M. Nat. Cell Biol. 2003; 5: 195-204Crossref PubMed Scopus (178) Google Scholar, 63.Yasuda S. Oceguera-Yanez F. Kato T. Okamoto M. Yonemura S. Terada Y. Ishizaki T. Narumiya S. Nature. 2004; 428: 767-771Crossref PubMed Scopus (162) Google Scholar). The question to ask is whether, like in protein kinase C-related kinase, the effector binds to multiple Rho family members at different binding sites or whether different G proteins use the same binding site. Here we analyzed the specificity of interaction between mDia isoforms and Rho proteins. We show that Rho, Rac, and Cdc42 use a similar binding site and that most of the specificity is determined by a Asn-Asn-Asn (triple N) motif of mDia1 that is replaced by Thr-Ser-His (TSH) in mDia2 and -3. On the Rho site, specificity is determined by a Phe-His substitution of Phe-106. We also observe for the first time that the insert helix, a specific hallmark of Rho proteins, is involved in effector binding and specificity. Recombinant Proteins—Proteins were expressed as glutathione S-transferase fusions using the vector pGEX-4T1 (Amersham Biosciences). All proteins were expressed in Escherichia coli BL21(DE3) cells. Cells were grown to an A600 of 0.7 (37 °C, 160 rpm) and induced with 100 μm isopropyl β-d-thiogalactopyranoside overnight at 20 °C for mDiaN and mDiaN-TSH and at 18 °C for RhoA, Rac1, and Cdc42. Cells were harvested and resuspended in buffer A (20 mm Tris/HCl, pH 7.5, 100 mm NaCl, 2 mm β-mercaptoethanol, and 5 mm MgCl2 in cases of RhoA, Rac1, and Cdc42) containing 0.1 mm phenylmethylsulfonyl fluoride and 2 mm EDTA. Cells were lysed by microfluidizing; the extract was applied to a GSH-Sepharose column (Amersham Biosciences), and the column was washed with buffer B (20 mm Tris/HCl, pH 7.5, 100 mm NaCl, 2 mm β-mercaptoethanol, 5 mm MgCl2). The fusion protein was digested with 5 units of thrombin (Serva) per mg of glutathione S-transferase fusion protein on the column overnight at 4 °C. Final purification was done with size exclusion chromatography in buffer B using a Superdex S200 for mDiaN and mDiaN-TSH or S75 for RhoA, Rac1, and Cdc42. Point mutations were made using the QuikChange method of Stratagene. Mutant proteins were purified like the wild type proteins. Concentrations of mDiaN and mDiaN-TSH were determined using the absorption at 280 nm under denaturing conditions with the extinction coefficient deduced from the sequence, and RhoA/Cdc42/Rac1 concentrations were determined by the Bradford assay. Rho was purified and loaded with Gpp(NH)p and mant-Gpp(NH)p as described elsewhere (35.Rose R. Weyand M. Lammers M. Ishizaki T. Ahmadian M.R. Wittinghofer A. Nature. 2005; 435: 513-518Crossref PubMed Scopus (218) Google Scholar). Rac1 and Cdc42 were purified using the same strategy. Stopped-flow Kinetics—The experiments were done at 20 °C using a SX18 MW Applied Photophysics apparatus (Leather-head, UK). The mant fluorophore (Molecular Probes) was excited at 366 nm with a bandpass of 6.4 nm. Emission (at 450 nm) was recorded using a 408-nm cutoff filter. All measurements were done with a final concentration of 100 nm mant-Gpp(NH)p-loaded GNBP/mutated GNBP. Increasing concentrations of wild type or mutant mDiaN (1-15 μm) were used in the experiments using pseudo first-order conditions. The observed fluorescence transients were fitted to a single exponential function to obtain kobs. The association rate constant kon was derived from the slope of kobs versus protein concentration. The dissociation rate constants were obtained by mixing a preformed 100 nm mDiaN·RhoA·mant-Gpp(NH)p (or mutated proteins) complex with a 100-fold excess of unlabeled Rho protein. All measurements were done in buffer C (300 mm NaCl, 50 mm Tris/HCl, pH 7.5, 2 mm β-mercaptoethanol, 5 mm MgCl2), which in experiments with RhoA additionally contained 5 mm MgCl2. GraFit 3.0-5.0 was used for data analysis. ITC Measurements—The interaction of mDiaN/mDiaN-TSH and RhoA/Cdc42/Rac1 and DAD-(1145-1200) and mutants was done by ITC based on Wiseman et al. (64.Wiseman T. Williston S. Brandts J.F. Lin L.N. Anal. Biochem. 1989; 179: 131-137Crossref PubMed Scopus (2447) Google Scholar) (MicroCal™; VP-ITC MicroCalorimeter). All measurements were carried out in buffer B at 20 °C. DAD, RhoA, Cdc42, and Rac1 (or mutants) (syringe) were stepwise-injected to the mDiaN, mDiaN-TSH solution (cell). The heating power per injection was observed over the reaction time until equilibrium was reached. The data were analyzed using the software provided by the manufacturer. Crystallization, Data Collection, and Structure Solution—Crystals of the mDiaN[TSH] domain (69-451) in complex with Cdc42-(1-178)·Gpp(NH)p were grown in buffer D containing Bistris propane, pH 8.8 (pH adjusted with citric acid), 26% PEG3350, 250 mm sodium tartrate to a size of ∼100 × 200 × 100 μm using the hanging drop/vapor diffusion method. The protein was solved in buffer B containing 100 mm NaCl, 20 mm Tris/HCl, pH 7.5, 2 mm β-mercaptoethanol, and 5 mm MgCl2.1 μl of protein solution of various concentrations (2.5, 5, and 10 μg/μl) was mixed with the reservoir solution. After 1 day crystals were grown to their final size and flash-frozen in liquid nitrogen using reservoir solution with 35% PEG3350 as cryoprotectant. Data set collection has been performed at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland (Table 4). The native dataset was collected at 100 K on beam line PXII at a wavelength of 0.95 Å using a Mar225 CCD detector. Data were indexed, integrated, and scaled with the XDS package (65.Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3243) Google Scholar).TABLE 4Statistics of data co
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