Formin proteins: a domain-based approach

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Formin proteins are potent regulators of actin dynamics. Most eukaryotes have multiple formin isoforms, suggesting diverse cellular roles. Formins are modular proteins, containing a series of domains and functional motifs. The Formin homology 2 (FH2) domain binds actin filament barbed ends and moves processively as these barbed ends elongate or depolymerize. The FH1 domain influences FH2 domain function through binding to the actin monomer-binding protein, profilin. Outside of FH1 and FH2, amino acid similarity between formins decreases, suggesting diverse mechanisms for regulation and cellular localization. Some formins are regulated by auto-inhibition through interaction between the diaphanous inhibitory domain (DID) and diaphanous auto-regulatory domain (DAD), and activated by Rho GTPase binding to GTPase-binding domains (GBD). Other formins lack DAD, DID and GBD, and their regulatory mechanisms await elucidation.

Introduction

Formin proteins have emerged as potent regulators of actin dynamics in vitro and in cells. With effects on filament nucleation rate, filament elongation rate and barbed-end capping protein function, the net activity of formins is to increase actin filament assembly. Most eukaryotes possess multiple formin genes, and the cellular roles of these proteins are just beginning to be explored.

Two well-written reviews on formins were published recently, covering cellular studies and the initial biochemical studies 1, 2. I will not duplicate these reviews but will concentrate on biochemical findings made subsequently, which have been substantial. This review takes a ‘domain-based’ approach, focusing on the structure (if known) and the biochemical function of different domains and functional motifs in formins. It should be noted that some of the regions that are commonly referred to as ‘domains’ [the Formin homology 1 domain (FH1) and the diaphanous auto-regulatory domain (DAD)] probably do not conform to the strictest definition of this term because it is doubtful that they fold into discrete tertiary structures. Nevertheless, this nomenclature has been established in the field and thus it will be maintained here.

Section snippets

Formin homology 2 domain

Formins are large proteins, generally >1000 amino acids (Figure 1). The defining feature of formin proteins is the Formin homology 2 (FH2) domain, an ∼400 amino acid sequence that is crucial for effects on actin. Database searches reveal that most eukaryotes possess multiple FH2 domain-containing sequences 3, 4, including: mammals (15 genes); Drosophila (6); Caenorhabditis elegans (6); Dictyostelium (10); budding yeast (2); and fission yeast (3).

Phylogenetic analysis of FH2 domains shows that

The FH1 domain and profilin

Almost all formins contain an FH1 domain just N-terminal to FH2 (Figure 1). The main feature of the FH1 is high proline content. FH1 domains are highly variable in length (15–229 residues), proline content (35–100%) and number of potential profilin-binding sites (0–16). The high proline content and non-conserved nature of other amino acids suggest that FH1 domains are unstructured.

Apart from the potential for binding SH3 or WW domain-containing signaling proteins [1], the FH1 is a binding site

Actin nucleation by formins

Nucleation by formins might occur by stabilization of an unfavorable nucleation intermediate (Figure 3a), possibly by FH2 domains binding monomers in the same manner that they bind to barbed ends. Kinetic modeling of Bni1-mediated actin polymerization reactions suggests that one nucleation mechanism might be dimer stabilization [13], although other mechanisms are not excluded.

Although formins clearly accelerate actin nucleation, several points must be made about this activity. First, there is

Auto-regulatory domains for Dia formins

Cellular studies suggest that the mammalian formins, mDia1 and mDia2, are auto-inhibited, with the N terminus inhibiting actin assembly by the FH2 domain-containing C terminus 35, 36. Expression of mDia1 or mDia2 constructs deleted in portions of the N terminus (ΔN constructs) cause constitutive formation of actin bundles in cells, suggesting disruption of auto-inhibition 35, 37. A C-terminal region important for this inhibitory interaction is the diaphanous auto-regulatory domain (DAD), which

Rho GTPase-binding regions for formins

Several formins interact with Rho family GTPases (Table 1), the best studied being between mammalian mDia1 and RhoA. Two-hybrid and GST pull-down assays have narrowed the RhoA-binding region to amino acids 60–260 of mDia1 [44]. This region partially overlaps with the auto-inhibitory DID [8]. Cellular studies suggest that RhoA binding might relieve auto-inhibition of mDia1 [35] and biochemical assays support this 8, 20. Thus, the current thinking is that RhoA competes with DAD for binding the

Other domains in formins

Outside the FH1 and FH2 domains, formins diverge considerably. The most divergent formin group in this sense is the metazoan INF (inverted formin) group [3]. All other formins have their FH2 domain positioned in the C-terminal half of the protein, whereas INF formins have N-terminal FH2 domains. The significance of this difference is unknown.

Several formins contain predicted coiled-coil sequences N-terminal to FH1 [3] (Figure 1). For mDia1, a region including the predicted coiled-coil mediates

Physiological roles of formins

The in vitro effects of formins on actin polymerization suggest possibilities for cellular function. Perhaps the most intriguing possibility is that formin-bound barbed ends might be protected from capping protein. Capping protein is present at micromolar concentrations in cells and caps with nanomolar affinity [25]. In the absence of inhibitory factors, newly-assembled filaments would be capped in <1 second, enabling a maximum filament length of <500 nm [27]. Several mammalian actin-based

Concluding remarks

Most eukaryotes possess multiple formin isoforms. The general mechanisms by which formins affect actin polymerization are beginning to be understood. It is likely that these mechanisms, centered around activity of the FH2 domain, are qualitatively similar for all formins but differ quantitatively. Formins exert multiple effects on actin polymerization in vitro (nucleation, elongation, severing, block capping protein), therefore, these quantitative differences between formin isoforms might

Acknowledgements

Many thanks to those who helped immensely with this review by their helpful discussions, including: Art Alberts, Fred Chang, Michael Eck, Hans Faix, Ron Fim, Bruce Goode, Dave Kovar, Sophie Martin, Takanori Otomo, Tom Pollard and Mike Rosen. This work was supported by the National Institutes of Health (NIH) grant GM069818 and by a Pew Biomedical Scholars award.

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