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

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.

Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis

“…Surprisingly, Dia1 (Diap1 -Mouse Genome Informatics) knockout mice were developmentally/morphologically normal, albeit with an agedependant myeloproliferative defect (Peng et al, 2007). Despite that, Dia1 displays significantly stronger activity in promoting actin nucleation than do other formin family members (Higashi et al, 2008). These findings strongly suggest that each formin plays a unique and specific physiological function, largely depending on its individual expression profile, upstream activators, localization and inherent activity.…”

“…capacity of directly mediating actin polymerization in vitro (Higashi et al, 2008;Moseley et al, 2006), we closely examined potential alterations of cytoskeletal organization in Daam1 gt/gt mice. Our work demonstrates that there is a significant reduction of F-actin in Daam1 gt/gt myocardia and aberrant sarcomeric organization, cell adhesion and alignment in Daam1 gt/gt cardiomyocytes.…”

“…Previous in vitro studies have demonstrated that Daam1 regulates filamentous actin (F-actin) assembly (Higashi et al, 2008;Moseley et al, 2006). However, the impact of Daam1 on the actin cytoskeleton has not been examined in an in vivo mammalian system.…”

Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms

“…Characteristic features of formins are the forming homology (FH) domains that are essential for mediating linear actin polymerization, functions distinct from proteins such as Arp2/3, which initiate and elongate branched actin filaments. DAAM1 was initially identified as a disheveled interacting protein mediating the Wnt/planar cell polarity pathway, 37 and recent data have established both the functional capacity of platelet DAAM1 in actin assembly, 29 and the relevance of the canonical Wnt signaling pathway as a negative modulator of platelet responsiveness. 30 The latter is especially relevant given known platelet functional abnormalities and thrombohemorrhagic consequences in ET.…”

“…28 Limited information is available for platelet DAAM1, although recent data suggest that it functions as an activation-dependent intermediary linking Rho GTPase to actin assembly 29 ; furthermore, its inhibitory effects on dishevelled may modulate noncanonical Wnt signaling pathways in platelets. 28,30 We confirmed DAAM1 target identification using a reporter construct encompassing the 3Ј-UTR and synthetic doublestranded RNAs specific for either miR 490 5p or miR 490 3p, thereby providing further dissection of the relative roles of either of the strands in modulating DAAM1 protein expression.…”