Biochemical Characterization of the Diaphanous Autoregulatory Interaction in the Formin Homology Protein FHOD1

Abstract

Diaphanous related formins (DRFs) are cytoskeleton remodeling proteins that mediate specific upstream GTPase signals to regulate cellular processes such as cytokinesis, cell polarity, and organelle motility. Previous work on the Rho-interacting DRF mDia has established that the biological activity of DRFs is regulated by an autoinhibitory interaction of a C-terminal diaphanous autoregulatory domain (DAD) with the DRF N terminus. This autoinhibition is released upon competitive binding of an activated GTPase to the N terminus of the DRF. Analyzing autoregulation of the Rac1-interacting DRF FHOD1, we utilized in vitro binding studies to identify a 60-amino acid DAD at the protein C terminus that recognizes an N-terminal formin homology (FH) 3 domain. Importantly, the FH3 domain of FHOD1 does not overlap with the proposed Rac1-binding domain. The FHOD1 DAD was found to contain one functional hydrophobic autoregulatory motif, while a previously uncharacterized basic cluster that is conserved in all DRF family DADs also contributed to the FH3-DAD interaction. Simultaneous mutation of both motifs efficiently released autoinhibition of FHOD1 in NIH3T3 cells resulting in the formation of actin stress fibers and increased serum response element transcription. A second putative hydrophobic autoregulatory motif N-terminal of the DAD belongs to a unique FHOD subdomain of yet undefined function. NMR structural analysis and size exclusion chromatography experiments revealed that the FHOD1 DAD is intrinsically unstructured with a tendency for a helical conformation in the hydrophobic autoregulation motif. Together, these data suggest that in FHOD1, DAD acts as signal sequence for binding to the well folded and monomeric FH3 domain and imply an activation mechanism that differs from competitive binding of Rac1 and DAD to one interaction site.Formin proteins are involved in the regulation of many cytoskeletal processes including cytokinesis, actin cable and stress fiber formation, polarity establishment, neurite outgrowth, and intracellular trafficking (reviewed in Refs. 1 and 2). These functions are achieved by their ability to promote F-actin assembly at the filament barbed end and to move processively with the barbed end as it elongates (3-7). Formins are large proteins of typically more than 1000 amino acids that are defined by the presence of two conserved regions, namely the formin homology 1 and 2 (FH1 and FH2) 2 domains (1). Additional conserved domains such as a N-terminal GTPase-binding domain (GBD) and a C-terminal diaphanous autoregulation domain (DAD) were found to constitute a formin subfamily, the diaphanous related formins (DRFs) (8). Over the last years, DRFs have emerged as a group of proteins with the potential to bridge between G-protein signals and the cytoskeleton via their ability to bind activated small GTPases and to subsequently remodel the cytoskeleton (9 -13).At present, phylogenetic analyses of FH2 domains suggest that metazoan formins fall into seven groups, termed Dia (diaphanous), DAAM (dishevelle...

Regulation of the Formin for3p by cdc42p and bud6p

“…This mild phenotype is in contrast to studies in other cell types in which more dramatic effects are seen upon overexpression of truncated formin fragments (Watanabe et al, 1999;Tominaga et al, 2000;Evangelista et al, 2002;Sagot et al, 2002a;Dong et al, 2003;Koka et al, 2003;Schonichen et al, 2006). With these other studies, the more extreme effects may have been caused in part by formin overexpression and in part by the fact that formin fragments were likely delocalized from their intracellular location.…”

“…Initially discovered in diaphanous-related formins, this mechanism relies on the interaction of a conserved C-terminal motif, named Diaphanous Autoregulatory Domain (DAD), with the Diaphanous Inhibitory Domain (DID) in the N-terminal region (Watanabe et al, 1999;Alberts, 2001;Higgs, 2003, 2004;Wallar et al, 2006). Support for this model largely came from the observation that exogenous expression of truncated formin constructs, lacking either N or C terminus, or harboring a mutated DAD region, led to an overabundance of actin structures, such as filopodia or stress fibers (Watanabe et al, 1999;Tominaga et al, 2000;Koka et al, 2003;Schonichen et al, 2006;Wallar et al, 2006). It is not clear whether all formins use an autoinhibitory mechanism for regulation, because many formins do not contain an obvious DAD-like sequence at the primary sequence level (Higgs and Peterson, 2004).…”

Interaction of the N- and C-terminal Autoregulatory Domains of FRL2 Does Not Inhibit FRL2 Activity

“…FHOD1, like FRL2 and FRL3, also possesses two C-terminal DAD-like motifs, with the more distal DAD mediating FHOD1 autoregulation (32). For FHOD1 and for mDia2, efficient DID/DAD dimerization and autoinhibition is dependent upon a cluster of basic residues C-terminal to DAD (32,33).…”

Structures of the Dual Bromodomains of the P-TEFb-activating Protein Brd4 at Atomic Resolution

“…Size Exclusion Chromatography-Analytical gel filtration experiments were performed using a Superdex S200 10/300 column at a flow rate of 1.0 ml/min similarly as described (32). Prior to injection of the protein samples, the column was equilibrated in 50 mM Hepes (pH 7.5), 50 mM NaCl, 1 mM TCEP buffer.…”