Dissecting Requirements for Auto-inhibition of Actin Nucleation by the Formin, mDia1

The mammalian formin, mDia1, is an actin nucleation factor. Experiments in cells and in vitro show that the N-terminal region potently inhibits nucleation by the formin homology 2 (FH2) domain-containing C terminus and that RhoA binding to the N terminus partially relieves this inhibition. Cellular experiments suggest that potent inhibition depends upon the presence of the diaphanous auto-regulatory domain (DAD) C-terminal to FH2. In this study, we examine in detail the N-terminal and C-terminal regions required for this inhibition and for RhoA relief. Limited proteolysis of an N-terminal construct from residues 1–548 identifies two stable truncations: 129–548 and 129–369. Analytical ultracentrifugation suggests that 1–548 and 129–548 are dimers, whereas 129–369 is monomeric. All three N-terminal constructs inhibit nucleation by the full C terminus. Although inhibition by 1–548 is partially relieved by RhoA, inhibition by 129–548 or 129–369 is RhoA-resistant. At the C terminus, DAD deletion does not affect nucleation but decreases inhibitory potency of 1–548 by 20,000-fold. Synthetic DAD peptide binds both 1–548 and 129–548 with similar affinity and partially relieves nucleation inhibition. C-terminal constructs are stable dimers. Our conclusions are as follows: 1) DAD is an affinity-enhancing motif for auto-inhibition; 2) an N-terminal domain spanning residues 129–369 (called DID for diaphanous inhibitory domain) is sufficient for auto-inhibition; 3) a dimerization region C-terminal to DID increases the inhibitory ability of DID; and 4) DID alone is not sufficient for RhoA relief of auto-inhibition, suggesting that sequences N-terminal to DID are important to RhoA binding. An additional finding is that FH2 domain-containing constructs of mDia1 and mDia2 lose >75% nucleation activity upon freeze-thaw. The mammalian formin, mDia1, is an actin nucleation factor. Experiments in cells and in vitro show that the N-terminal region potently inhibits nucleation by the formin homology 2 (FH2) domain-containing C terminus and that RhoA binding to the N terminus partially relieves this inhibition. Cellular experiments suggest that potent inhibition depends upon the presence of the diaphanous auto-regulatory domain (DAD) C-terminal to FH2. In this study, we examine in detail the N-terminal and C-terminal regions required for this inhibition and for RhoA relief. Limited proteolysis of an N-terminal construct from residues 1–548 identifies two stable truncations: 129–548 and 129–369. Analytical ultracentrifugation suggests that 1–548 and 129–548 are dimers, whereas 129–369 is monomeric. All three N-terminal constructs inhibit nucleation by the full C terminus. Although inhibition by 1–548 is partially relieved by RhoA, inhibition by 129–548 or 129–369 is RhoA-resistant. At the C terminus, DAD deletion does not affect nucleation but decreases inhibitory potency of 1–548 by 20,000-fold. Synthetic DAD peptide binds both 1–548 and 129–548 with similar affinity and partially relieves nucleation inhibition. C-terminal constructs are stable dimers. Our conclusions are as follows: 1) DAD is an affinity-enhancing motif for auto-inhibition; 2) an N-terminal domain spanning residues 129–369 (called DID for diaphanous inhibitory domain) is sufficient for auto-inhibition; 3) a dimerization region C-terminal to DID increases the inhibitory ability of DID; and 4) DID alone is not sufficient for RhoA relief of auto-inhibition, suggesting that sequences N-terminal to DID are important to RhoA binding. An additional finding is that FH2 domain-containing constructs of mDia1 and mDia2 lose >75% nucleation activity upon freeze-thaw. Formin proteins are emerging as regulators of many cellular actin-based structures (1Wallar B.J. Alberts A.S. Trends Cell Biol. 2003; 13: 435-446Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 2Zigmond S.H. Curr. Opin. Cell Biol. 2004; 16: 99-105Crossref PubMed Scopus (204) Google Scholar). Biochemically, formins exert several effects on actin polymerization dynamics, including acceleration of filament nucleation from monomers, inhibition of barbed end elongation rate, inhibition of complete barbed end capping by heterodimeric capping protein, and filament severing (3Pruyne D. Evangelista M. Yang C. Bi E. Zigmond S. Bretscher A. Boone C. Science. 2002; 297: 612-615Crossref PubMed Scopus (570) Google Scholar, 4Sagot I. Rodal A.A. Moseley J. Goode B.L. Pellman D. Nat. Cell Biol. 2002; 8: 626-631Crossref Scopus (392) Google Scholar, 5Pring M. Evangelista M. Boone C. Yang C. Zigmond S.H. Biochemistry. 2003; 42: 486-496Crossref PubMed Scopus (187) Google Scholar, 6Zigmond 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 (214) Google Scholar, 7Moseley J.B. Sagot I. Manning A.L. Xu Y. Eck M.J. Pellman D. Goode B.L. Mol. Biol. Cell. 2004; 15: 896-907Crossref PubMed Scopus (222) Google Scholar, 8Kovar D.R. Kuhn J.R. Tichy A.L. Pollard T.D. J. Cell Biol. 2003; 161: 875-887Crossref PubMed Scopus (272) Google Scholar, 9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 10Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). These in vitro activities are generally considered to result from the ability of formins to bind at or near the filament barbed end and to move processively with the barbed end as it elongates (11Higashida C. Miyoshi T. Fujita A. Oceguera-Yanez F. Monypenny J. Andou Y. Narumiya S. Watanabe N. Science. 2004; 303: 2007-2010Crossref PubMed Scopus (249) Google Scholar, 12Kovar D.R. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14725-14730Crossref PubMed Scopus (354) Google Scholar). Essential to these properties is the formin homology 2 (FH2) 1The abbreviations used are: FH, formin homology; DAD, diaphanous auto-regulatory domain; DID, diaphanous inhibitory domain; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; GMP-PNP, guanosine 5′-β,γ-imidotriphosphate; GDP, guanosine 5′-diphosphate. 1The abbreviations used are: FH, formin homology; DAD, diaphanous auto-regulatory domain; DID, diaphanous inhibitory domain; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; GMP-PNP, guanosine 5′-β,γ-imidotriphosphate; GDP, guanosine 5′-diphosphate. domain, a 400-residue region generally found in the C-terminal half of the protein. Biochemical and structural studies show that the FH2 domain is dimeric for several formins (10Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 13Xu Y. Moseley J. Sagot I. Poy F. Pellman D. Goode B.L. Eck M.J. Cell. 2004; 116: 711-723Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 14Shimada A. Nyitrai M. Vetter I.R. Kuhlmann D. Bugyi B. Narumiya S. Geeves M.A. Wittinghofer A. Mol. Cell. 2004; 13: 511-522Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), although longer constructs of the budding yeast formin, Bni1p, can tetramerize (6Zigmond 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 (214) Google Scholar). Mammals possess 15 formin genes, in seven distinct phylogenetic groups (15Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2004; 16: 1-13Crossref PubMed Scopus (201) Google Scholar). For one mammalian formin, mDia1, the mechanisms regulating effects on actin have begun to be elucidated. The in vitro nucleation activity of the FH2-containing C terminus of mDia1 is inhibited potently by inclusion of a separate polypeptide containing the mDia1 N terminus (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), suggesting an auto-inhibitory regulatory mechanism. Cellular experiments and two-hybrid interactions implicate a short sequence C-terminal to FH2, known as the diaphanous auto-regulatory domain (DAD), as a critical binding site for the N terminus in both mDia1 and the related protein, mDia2 (16Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Direct effects of DAD on auto-inhibition of actin nucleation in vitro have not been examined. Cellular studies suggest that binding of the Rho family GTPase, RhoA, to the N terminus of mDia1 can relieve auto-inhibition (17Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (724) Google Scholar). Biochemical studies on actin nucleation support these findings (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), with the caveat that RhoA does not relieve completely the auto-inhibitory effect of the N terminus of mDia1. This incomplete relief by RhoA might imply that a second, non-RhoA dependent, auto-inhibitory interaction between the N and C terminus might exist. In this study, we examine mDia1 auto-inhibition in more detail. Deletion experiments show that auto-inhibition can be uncoupled from RhoA relief, suggesting that mDia1's N-terminal binding sites for RhoA and for the mDia1 C terminus are not identical. In addition, deletion of DAD from the C terminus does not affect nucleation but decreases the inhibitory potency of the N terminus 20,000-fold, suggesting that DAD mediates a high affinity interaction important for potent auto-inhibition. A synthetic DAD peptide binds the N terminus and partially disrupts auto-inhibition, supporting the role of DAD. DNA Constructs—Constructs of mouse mDia1 (accession number U96963) were generated by reverse transcription-PCR and cloned into pGEX-KT, as described previously (9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Deletion constructs were generated by PCR from longer constructs, using Pfu DNA polymerase (Stratagene). The mouse mDia2 521–1171 construct in pGEX-KT was a kind gift from Dr. Arthur Alberts (Van Andel Research Institute). Protein Preparation and Purification—All proteins were expressed and purified through the thrombin cleavage step following the procedure described in detail in Refs. 9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar and 10Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar. After elution of thrombin-cleaved protein from glutathione-Sepharose, further purification varied as follows. N-terminal constructs (1–548, 129–548, and 129–369) were purified by fast protein liquid chromatography on a SourceS15 5/5 or 10/10 column (Amersham Biosciences) and then concentrated on Q Sepharose Fast Flow (Amersham Biosciences) and dialyzed into the following buffer: 2 mm NaPO4, pH 7.0, 50 mm NaCl, 0.1 mm MgCl2, 0.1 mm EGTA, 0.5 mm DTT. N-terminal constructs could be frozen in aliquots of 75% of their nucleation activity when frozen. C-terminal constructs were stored at concentrations 75% nucleation potency for mDia1 and mDia2 521–1171 FH2, DAD, and C-terminal as as for mDia1 and and not were frozen in aliquots in PCR by into liquid in which the was frozen in This loss of activity of the presence of In similar of mDia1 N-terminal constructs not affect their to inhibit nucleation In this study, we the auto-inhibitory properties of mDia1 with the following cellular findings of DAD (16Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), suggesting that the role of DAD is to binding affinity to the N-terminal auto-inhibitory to be of the auto-inhibitory mechanism. we a inhibitory residues which we to as DID inhibitory we show that auto-inhibition can be uncoupled from RhoA relief of auto-inhibition inhibition by the DID or DID with a C-terminal is not by we a region between DID and the domain that mediates DID by proteolysis using The of residues 129–369 to suggests that this region a stable In addition, the ability of this region to inhibit nucleation by the mDia1 C terminus suggests that it mediates the auto-inhibitory that this region the that many including of the and as as budding yeast Bni1p, regions similar to DID (15Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2004; 16: 1-13Crossref PubMed Scopus (201) Google Scholar). All of these proteins possess auto-inhibitory through binding be to many are proteins in the and groups (15Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2004; 16: 1-13Crossref PubMed Scopus (201) Google Scholar), which we to be by In this region the 129–548 construct to with RhoA, important for RhoA binding N-terminal to two-hybrid studies found that RhoA an mDia1 construct containing residues N. T. Ishizaki T. Watanabe A. Y. Narumiya S. J. 16: PubMed Scopus Google Scholar). full affinity for RhoA residues both DID and N-terminal to of residues from to N-terminal to with affinity in the 100 nm a region is from to (15Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2004; 16: 1-13Crossref PubMed Scopus (201) Google Scholar), we that this region mediates at two does this region in a or is the of mDia1, which both this dimerization region and the dimeric FH2 to these be for a complete of mDia1 Our experiments show that DAD is required for high affinity auto-inhibition. Deletion of DAD from the C terminus the of the N-terminal 1–548 construct from 2 nm to or 20,000-fold. Synthetic DAD peptide binds constructs and partially relieves inhibition. that DAD in cells DAD in N. and A. DAD is not required for and inhibition of the C terminus can be at high concentrations of N terminus, we that the role of DAD is to a high affinity interaction that a interaction between DID and the FH2 This interaction is inhibitory to The that RhoA does not relieve auto-inhibition suggests that a is required for full This the interaction or further the The nucleation activity of mDia1 and mDia2 FH2 domain-containing constructs is by This might to mDia1 nucleation by J.B. Sagot I. Manning A.L. Xu Y. Eck M.J. Pellman D. Goode B.L. Mol. Biol. Cell. 2004; 15: 896-907Crossref PubMed Scopus (222) Google Scholar, 9Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 14Shimada A. Nyitrai M. Vetter I.R. Kuhlmann D. Bugyi B. Narumiya S. Geeves M.A. Wittinghofer A. Mol. Cell. 2004; 13: 511-522Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. D. C. D. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The to might imply that FH2 domain-containing constructs are to as that mDia1 FH2 domain-containing constructs partially in a of when stored at high concentrations at 4 °C for For these we mDia1 <10 μm at 4 in which full activity is for For longer the of glycerol to and at -20 °C full activity for several This to might not be for FH2 FH2 domain-containing constructs of (10Harris E.S. Li F. Higgs H.N. J. Biol. Chem. 2004; 279: 20076-20087Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) does not affect polymerization or severing Li and N. Dr. Arthur Alberts (Van Andel Research for the kind gift of the mDia2 construct and The of and in the Facility is