Actin monomers activate inverted formin 2 by competing with its autoinhibitory interaction.
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.
From the Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755.
Vinay
V
Ramabhadran
Anna L
AL
Hatch
Henry N
HN
Higgs
D000199:Actins / Q000378:metabolism*; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D000080865:Formins; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008958:Models, Molecular; D051304:Profilins / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D000080865:Formins; C546959:INF2 protein, human; D008840:Microfilament Proteins; D051304:Profilins; D020764:cdc42 GTP-Binding Protein
Actin; Cdc42; Endoplasmic Reticulum (ER); Formin; Rho