Regulation of the Formin for3p by cdc42p and bud6p

Abstract

Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6⌬ cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition. INTRODUCTIONFormins are key regulators of the actin cytoskeleton and form a large family conserved in all eukaryotes Faix and Grosse, 2006). These proteins are necessary for the formation of numerous actin structures, including stress fibers, filopodia, cytokinetic actin rings, junctional actin structures, or actin cables. The proper regulation of formins is likely to be critical for cellular processes such as cell migration, cytokinesis, cell adhesion, and cell polarity.A well characterized biochemical activity of formins is to nucleate and elongate linear actin filaments Sagot et al., 2002b;Kovar et al., 2003;Li and Higgs, 2003;Moseley et al., 2004). This activity occurs through the formin-homology (FH) 2 domain, which dimerizes to form a doughnut-shaped structure containing multiple actin-binding sites in its core Otomo et al., 2005b). This dimer is thought to stabilize otherwise unstable intermediates in the assembly of new actin filaments, and it binds processively to the fast-elongating barbed end of existing actin filaments (Pring et al., 2003;Kovar and Pollard, 2004).The adjacent FH1 domain binds profilin-actin and helps accelerate the elongation of actin filaments (Chang et al., 1997;Evangelista et al., 1997;Watanabe et al., 1997;Romero et al., 2004;Kovar et al., 2006). The budding yeast formin Bni1p also binds the actin monomer-binding protein Bud6p, which, similar to profilin, stimulates the activity of the FH2 domain (Moseley and Goode, 2005). In addition to their activity in actin filament nucleation and elongation, some formins have been suggested to also function in actin bundling and severing, further contributing to the remodeling of actin structures (Harris et al., 2004Moseley and Goode, 2005;Harris et al., 2006...

Pob1 Participates in the Cdc42 Regulation of Fission Yeast Actin Cytoskeleton

“…Error-prone PCR for obtaining cdc42 ORF mutants was performed as described previously (Martin et al, 2007). cdc42-879 strain identification and isolation were carried out as described previously (Martin et al, 2007).…”

“…Fission yeast Cdc42 is an essential protein that has been implicated in the regulation of the cell shape and growth (Miller and Johnson, 1994). It interacts with the PAKs, Shk1 and Shk2 (Yang et al, 1998;Chang et al, 1999), and it is the main Rho GTPase required for actin cable assembly (Martin et al, 2007). Actin cables are important structures that contribute to polarized cell growth, serving as tracks for delivery of secretory vesicles to cell tips.…”

“…Similar to other formins, For3 is controlled by an autoinhibitory interaction between the N-terminal Dia inhibitory domain (DID) with the C-terminal Dia autoregulatory domain (DAD) (Goode and Eck, 2007). The proper localization of For3 depends on relief of this autoinhibition (Martin et al, 2007), and so far the only proteins involved in this process are the actin-interacting protein Bud6 and the Rho GTPase Cdc42 (Martin et al, 2007).…”

Structure of the formin-interaction domain of the actin nucleation-promoting factor Bud6

“…3B); blue datasets indicate mutations in c-Bud6 that were pseudo-WT for stimulating Bni1. 23,38). This region corresponds to the Bud6 core domain and a portion of the flank (residues 517-753) that is 22% identical and ∼46% similar to a 234-residue region of p160 rock .…”

Cytoskeletal dynamics in fission yeast: A review of models for polarization and division

“…With other proteins, it activates actin-cable-nucleator For3p and therefore affects cytoskeletal organization (Chang and Martin, 2009;Martin et al, 2007). If a reaction-diffusion model best explains NETO, some substrate such as a signaling molecule may limit growth.…”