Actin monomers activate inverted formin 2 by competing with its autoinhibitory interaction

Abstract

INF2 is an unusual formin protein in that it accelerates both actin polymerization and depolymerization, the latter through an actin filament-severing activity. Similar to other formins, INF2 possesses a dimeric formin homology 2 (FH2) domain that binds filament barbed ends and is critical for polymerization and depolymerization activities. In addition, INF2 binds actin monomers through its diaphanous autoregulatory domain (DAD) that resembles a Wiskott-Aldrich syndrome protein homology 2 (WH2) sequence C-terminal to the FH2 that participates in both polymerization and depolymerization. INF2-DAD is also predicted to participate in an autoinhibitory interaction with the N-terminal diaphanous inhibitory domain (DID). In this work, we show that actin monomer binding to the DAD of INF2 competes with the DID/DAD interaction, thereby activating actin polymerization. INF2 is autoinhibited in cells because mutation of a key DID residue results in constitutive INF2 activity. In contrast, purified full-length INF2 is constitutively active in biochemical actin polymerization assays containing only INF2 and actin monomers. Addition of proteins that compete with INF2-DAD for actin binding (profilin or the WH2 from Wiskott-Aldrich syndrome protein) decrease full-length INF2 activity while not significantly decreasing activity of an INF2 construct lacking the DID sequence. Profilin-mediated INF2 inhibition is relieved by an anti-N-terminal antibody for INF2 that blocks the DID/DAD interaction. These results suggest that free actin monomers can serve as INF2 activators by competing with the DID/DAD interaction. We also find that, in contrast to past results, the DID-containing N terminus of INF2 does not directly bind the Rho GTPase Cdc42.

Keywords: Actin; Cdc42; Endoplasmic Reticulum (ER); Formin; Rho.

FIGURE 1.

INF2 domains and DAD interaction with DID or actin monomers. A, bar diagram showing INF2 domains. The N-terminal and FFC constructs used in this work are shown. B, alignments of INF2 DAD (human) with mDia1 DAD (mouse) and Spire WH2 domain 2 (Drosophila). Key hydrophobic residues mediating interaction with actin or DID are shown in red. C, model of INF2 DID/DAD interaction on the basis of the mouse mDia1 DID/DAD crystal structure (PDB code 2BAP). Gray, DID surface; black, DAD main chain. The position of the DID Ala-149 residue is shown in blue, illustrating why its mutation to D disrupts the DID/DAD interaction. Key interacting leucines in DAD are shown in red (also highlighted in B). D, model of INF2 DAD (black) bound to actin monomer (gray) on the basis of the Drosophila Spire WH2#2/actin crystal structure (PDB code 3MN5). Gray, actin surface; black, DAD main chain. Key interacting leucines in DAD are shown in red (also highlighted in B). ATP bound to actin is shown in blue.

FIGURE 2.

Disruption of the DID/DAD interaction causes constitutive actin polymerization by INF2 in cells. Shown is the effect of transfected GFP-INF2 constructs on actin organization in U2OS cells that were transfected with GFP-INF2 and the ER marker CFP-Sec61β (A and B) or GFP-INF2 alone (C and D) for 16–18 h and then fixed and stained with TRITC-phalloidin. All images were acquired with the same exposure times for actin and GFP and adjusted in the same manner. Enlarged images are single Z slices (0.2 μm) from confocal micrographs. A, INF2-CAAX WT showing ER localization but minimal actin filament accumulation on the ER (arrow). The lower panels denote the boxed region. Scale bars = 20 μm (upper panels) and 5 μm (lower panels). B, the INF2-CAAX A149D mutant showing ER localization and most of the cellular actin filament staining tracing the ER (compare the transfected cell to the non-transfected cell in the upper panels). Scale bars = 20 μm (upper panels) and 5 μm (lower panels). C, INF2-non-CAAX WT (upper panels) and A149D mutant (lower panels) showing normal actin filament staining for the WT but greatly increased actin filaments in A149D. Scale bars = 50 μm. D, Close-ups of the perinuclear regions of cells transfected with WT (upper panels) or A149D INF2-non-CAAX. Note the large increase in actin staining for the A149D mutant. Scale bars = 5 μm.

FIGURE 3.

Free actin monomers activate full-length INF2. Shown are pyrene-actin assays containing 1 μm actin monomer (10% pyrene label). A, Coomassie-stained SDS-PAGE of purified full-length INF2-non-CAAX used in these assays. Numbers represent molecular weight markers. B, effect of 20 or 200 nm INF2-FFC or full-length INF2 on actin polymerization in the absence of profilin. Note the rapid polymerization/depolymerization caused by 200 nm FFC but not by 200 nm full-length. AU, arbitrary units. C, effect of increasing profilin concentration (numbers indicate micromolar profilin) on 20 nm INF2-FFC polymerization activity. D, effect of increasing profilin concentration on 20 nm full-length INF2 polymerization activity. E, polymerization half-times (t½) for INF2-FFC and full-length INF2 as a function of profilin concentration (inset) or of calculated free actin monomer concentration (on the basis of a profilin:actin K_d of 0.5 μm (40)). F, effect of the WASp WH2 motif (48 μm) on FL-INF2 or INF2-FFC activity (20 nm INF2).

FIGURE 4.

The N terminus of IFN2 inhibits actin polymerization by INF2-FFC only when free actin monomer concentration is low. A, fluorescence anisotropy assay for actin monomer binding by INF2-Cterm. TMR-labeled INF2-Cterm (5 nm) was mixed with varying concentrations of LatB-stabilized actin (1.5 moles LatB:1 mole actin), and anisotropy was recorded. Inset, competition assay in which 5 nm TMR-INF2-Cterm and 200 nm LatB/actin were mixed with varying concentrations of unlabeled INF2-C, and anisotropy was recorded. B and C, pyrene-actin polymerization assays (1 μm actin monomer, 10% pyrene labeled) using 20 nm INF2-FFC and 50 μm INF2-Nterm (wild-type or A149D mutant) in the absence of profilin (B) or in the presence of 12 μm profilin (C). a.u., arbitrary units. D, concentration dependence of INF2-Nterm (wild-type and A149D mutant) on INF2-FFC inhibition as measured by the half-time to full polymerization.

FIGURE 5.

The N terminus of INF2 does not display a high-affinity direct interaction with Cdc42. Cdc42 was precharged with either GMP-PNP or GDP and then mixed (3 μm) with 9 μm of GST-fusion of the N terminus of IFN2 (amino acids 1–420) bound to glutathione-Sepharose beads in actin polymerization buffer. Beads were isolated by centrifugation, and supernatant and pellet were analyzed by Coomassie-stained SDS-PAGE (equal volumes loaded). Positive controls were the CRIB of WASp for verification of Cdc42 binding ability and INF2-FFC for verification of INF2-N binding ability.

FIGURE 6.

Competition with DID/DAD binding activates full-length INF2. A, pyrene-actin assays containing 1 μm actin monomer (10% pyrene), 12 μm profilin, and 20 nm full-length INF2. After preincubation with anti-INF2-Nterm antibody (0.1 mg/ml, 650 nm), INF2 is active. In contrast, GMP-PNP-charged Cdc42 (1 μm, bacterially expressed) has no effect on INF2 activity. AU, arbitrary units. B, model for actin monomer activation of INF2. In the absence of actin, INF2 is autoinhibited through the DID/DAD interaction. Actin monomer binding to DAD competes with DID binding, freeing the FH2 domain. Profilin sequesters actin monomers from DAD binding, preventing activation.