Biochemical characterization of the Rho GTPase-regulated actin assembly by diaphanous-related formins, mDia1 and Daam1, in platelets.
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.
Higashi
Tomohito
T
Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Ikeda
Tomoyuki
T
Shirakawa
Ryutaro
R
Kondo
Hirokazu
H
Kawato
Mitsunori
M
Horiguchi
Masahito
M
Okuda
Tomohiko
T
Okawa
Katsuya
K
Fukai
Shuya
S
Nureki
Osamu
O
Kita
Toru
T
Horiuchi
Hisanori
H
D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001669:Biochemical Phenomena; D001671:Biochemistry; D001792:Blood Platelets / Q000378:metabolism*; D000080865:Formins; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C499094:DAAM1 protein, human; C108962:DIAPH1 protein, human; D000080865:Formins; D008840:Microfilament Proteins; D020741:rho GTP-Binding Proteins