The diaphanous-related formins are actin nucleating and elongating factors. They are kept in an inactive state by an intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous-autoregulatory domain (DAD). It is considered that the dissociation of this autoinhibitory interaction upon binding of GTP-bound Rho to the GTPase binding domain next to DID induces exposure of the FH1-FH2 domains, which assemble actin filaments. Here, we isolated two diaphanous-related formins, mDia1 and Daam1, in platelet extracts by GTP-RhoA affinity column chromatography. We characterized them by a novel assay, where beads coated with the FH1-FH2-DAD domains of either mDia1 or Daam1 were incubated with platelet cytosol, and the assembled actin filaments were observed after staining with rhodamine-phalloidin. Both formins generated fluorescent filamentous structures on the beads. Quantification of the fluorescence intensity of the beads revealed that the initial velocity in the presence of mDia1 was more than 10 times faster than in the presence of Daam1. The actin assembly activities of both FH1-FH2-DADs were inhibited by adding cognate DID domains. GTP-RhoA, -RhoB, and -RhoC, but not GTP-Rac1 or -Cdc42, bound to both mDia1 and Daam1 and efficiently neutralized the inhibition by the DID domains. The association between RhoA and Daam1 was induced by thrombin stimulation in platelets, and RhoA-bound endogenous formins induced actin assembly, which was inhibited by the DID domains of Daam1 and mDia1. Thus, mDia1 and Daam1 are platelet actin assembly factors having distinct efficiencies, and they are directly regulated by Rho GTPases.

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Posttranslational modification by ubiquitin regulates a broad range of cellular activities such as protein degradation, transcriptional regulation, endocytosis, and DNA repair. p62 is one of the proteins which is known to recognize poly-linked ubiquitin chains through the ubiquitin associated (UBA) domain. Physiological function of p62 is implicated in the formation of protein inclusions which can be observed in neurodegenerative diseases such as Huntington's disease. p62 is known to accumulate in ubiquitin-positive inclusions of polyubiquitinated proteins, and p62 protein lacking the UBA domain failed to form inclusions in HEK cells, suggesting the important role of the p62 UBA domain in the formation of those inclusions. We solved the crystal structure of the p62 UBA domain at 1.4 angstrom resolution. Phases were obtained by MAD technique using the selenomethionine derivative of the p62 UBA domain. The structure of the p62 UBA domain was a homodimer in a crystallographic asymmetric unit. Two molecules form a dimer with a large hydrophobic interface. Results of analytical ultracentrifugation and NMR spectroscopy strongly supported that the p62 UBA domain also adopts a dimer configuration under an aqueous condition. We further examining how this dimeric structure changes upon ubiquitin binding and whether the dimerization affects the binding to the specific polyubiquitin chains. 1 The University of Tokyo, Medical Genome Sciences, 1-1-1 Yayoi, B u n k y o -k u , To k y o , 11 3 -0 0 3 2 , J a p a n , 2 K y o t o U n i v e r s i t y, 5 4 Shogoinkawara-machi, Sakyo-ku, Kyoto 606-8507, Japan, 3 Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan, 4 Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe-shi, Hyogo 650-0017, Japan, E-mail kk087351@mgs. k.u-tokyo.ac.jp Reorganization of actin filaments is an essential process for cell motility, cell-cell attachment, and intracellular transport. Formin protein family promotes nucleation and elongation of the actin filament, which is catalyzed by the conserved Formin-homology 2 (FH2) domain. FH2 forms a dimer and directly binds to the barbed end of the actin filament. The active dimeric FH2 structure has been reported in yeast formin, Bni1p, but not in any mammalian formin. Dishevelled-associated activator of morphogenesis (DAAM) is a Rho-regulated formin implicated in neuronal development. To elucidate the mechanism of the actin filament assembly by mammalian FH2, we crystallized human DAAM1 FH2. The native crystal belongs to the triclinic space group P1, with unit-cell parameters a = 69.2 Å, b = 91.9 Å, c = 97.7 Å, α = 98.1 , β = 90.3 , γ = 104.8 , and diffracts to 2.8 Å resolution. The structure was solved by multiple-wavelength anomalous dispersion method using the SeMet-labeled crystal, and refined to an Rfree value of 28.9% at 2.8 Å resolution. The present DAAM1 FH2 structure consists of five subdomains (termed as "lasso", "linker", "knob", "coiled-coil", and "post"), and forms a dimeric ring in a head-to-tail manner si...

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