Biochemical Characterization of the Rho GTPase-regulated Actin Assembly by Diaphanous-related Formins, mDia1 and Daam1, in Platelets

Tomohito Higashi,Tomoyuki Ikeda,Ryutaro Shirakawa,Hirokazu Kondo,Mitsunori Kawato,Masahito Horiguchi,Tomohiko Okuda,Katsuya Okawa,Shuya Fukai,Osamu Nureki,Toru Kita,Hisanori Horiuchi

doi:10.1074/jbc.m707839200

Abstract

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.

Highlights

Small GTPase Rho proteins regulate various cellular functions such as stress fiber formation, cell shape change, and cytokinesis via rearrangement of the actin cytoskeleton [1]
It is considered that the autoinhibitory interaction between diaphanous inhibitory domain (DID) and diaphanous-autoregulatory domain (DAD) is disrupted when the active GTP-bound Rho binds to the GTPase binding domain (GBD), which is localized at the N-terminal end next to DID, resulting in exposure of the FH1 and FH2 domains (24 –27)
By means of a novel actin assembly assay using beads coated with FH1-FH2 domains, we demonstrated that both formins possess actin assembly activity in the cytosol and that the autoinhibition of both formins was relieved by active Rho GTPases

Summary

EXPERIMENTAL PROCEDURES

Actin Assembly Assay Using Formin-coated Beads and Platelet Cytosol—Glutathione beads coated with GST-mDia CT, GST-Daam CT, or their mutants were incubated at 37 °C with platelet cytosol at 2 mg proteins/ml for 10 min in the presence of ATP regeneration system unless otherwise specified. Association of mDia NT and Daam NT with Various Rho Family GTPases—Glutathione beads coated with GST-mDia NT and -Daam NT were incubated at 4 °C for 2 h with His6tagged various Rho family GTPases bound to GTP␥S or GDP in 200 ␮l of buffer A containing 4 mg/ml bovine serum albumin. Bead-associated proteins were subjected to SDS-PAGE followed by immunoblot analysis using an anti-His antibody (Sigma). Bead-associated proteins were analyzed by immunoblotting with anti-RhoA monoclonal and anti-Daam rat polyclonal antibodies

RESULTS

There is a possibility that actin assembly occurred in the

RhoA was also confirmed by the observation of each bead for

The pyrene assay has traditionally

RhoC and weakly with RhoD but not with other GTPases including