The Formin/Diaphanous-Related Protein, FHOS, Interacts with Rac1 and Activates Transcription from the Serum Response Element

represent the mean of three independent transfections ± the standard error of the mean (SEM). Immunofluorescence. NIH3T3 were grown on coverslips and transfected with the indicated FHOS expression plasmids with Superfect (Qiagen). C2C12 cells were transduced with the indicated MSCV -HA-FHOS vector using conventional methods (38). Forty hours later, cells were fixed with 2% paraformaldehyde, lysed in PBS containing 0.3% Triton X-100, and blocked for 30 min in IF buffer (3% BSA, 20mM MgCl2, 0.3% Tween20, 1X PBS). Cells were incubated sequentially in IF buffer containing anti-HA mAb (clone 12CA5, Sigma), anti-mouse IgG-Alexa 546 (Molecular Probes), and anti-Rac1-FITC (UBI). Cells were mounted in 90% glycerol containing 0.4% N-propyl-gallate. Endogenous FHOS was detected in HeLa cells with FHOS antiserum (30). DNA was counterstained for 5 min with Hoescht’s dye (5µg/ml) as indicated.


INTRODUCTION
Formin homology (FH) proteins are highly structured proteins and components of Rho family GTPase signaling pathways that affect cytoskeletal organization and induce transcriptional activation of the serum response element (SRE) (1)(2)(3)(4)(5)(6). Eukaryotic FH proteins are characterized by several conserved domains that are organized in a precise order. From the N-terminus, FH proteins contain a loosely conserved FH3 domain, a GTPase binding domain (GBD), highly conserved FH1 and FH2 domains, a coiled-coil and an autoregulatory domain (1,2,(7)(8)(9). In a subset of FH proteins, the Diaphanous-Related Formin (DRF) proteins, the autoregulatory domain forms intramolecular interactions with residues within and/or between the FH3 and GBD (3). This self-interaction(s) is predicted to mask the conserved FH1 and FH2 domains, which associate with kinase signaling pathways, actin-binding proteins and microtubules (2,4,6,(10)(11)(12)(13)(14). Inhibition of the intramolecular interactions by deleting the GBD or cell cycle progression, adhesion, actin cytoskeletal organization, cytokinesis and motility, by cycling between inactive GDP-and active GTP-bound states and relaying signals from membrane receptors to downstream effector molecules (21,22). DRF proteins are downstream effectors of Rho GTPases. Activated Rho GTPases bind to the GBDs of DRFs and relieve intramolecular interactions. At the present time, four FH proteins are known to interact with Rho GTPases. DRF1 and DRF2 bind to RhoA, RhoB and RhoC (2,3). DRF2 also interacts with Cdc42Hs, but neither DRF1 nor DRF2 associate with Rac1 (2,3,6). The S. cerevisiae DRF, Bni1p, associates with Rho1p and Cdc42p (13,23). DRFs preferentially interact with GTPbound Rho proteins and are inhibited by Rho inactivation (2,23). The product of formin related gene in leukocytes (FRL) was recently shown to interact with Rac1, but not with RhoA or Cdc42Hs (15). Thus, FH proteins appear to interact with specific Rho family GTPases.
One effect of Rho family GTPase and DRF activation is the induction of gene transcription from the SRE (5,6). The SRE is a short gene-regulatory sequence that is sufficient to activate transcription of immediate-early genes, such as c-fos and ß-actin, in response to growth factor stimulation (24). Maximal activation of the c-fos SRE requires the formation of a complex between the ubiquitous transcription factor, SRF (25), and certain Ets-domain proteins, such as Elk1 and SAP1, which are collectively referred to as ternary complex factors (TCF).
TCFs potentiate SRE transcriptional activation when they are phosphorylated by ERKs in response to Ras/Raf or Rac1/Cdc42/PAK signals (21,(26)(27)(28). SRF activity is controlled independently of TCF activation, and is induced by activated forms of RhoA, Rac1, and Cdc42 (29). RhoA-induced activation is essential for SRE-serum responsiveness and requires the FH proteins, DRF1 (mDia1) and DRF2 (mDia2) (5,6,9). Rho activation however is not required for FHOS Interacts with Rac1 and Activates the SRE Rac1 or Cdc42-induced SRF activation (29). Thus, multiple pathways target the SRF and TCF complex at the c-fos SRE and distinct Rac1 signals affect both subunits of the complex.
FHOS is a member of the formin homology (FH) family of proteins that is expressed at high levels in human splenic cells (30). FHOS contains all the conserved domains, including FH1, FH2, and FH3 domains, and a coiled-coil, that are characteristic of FH proteins. In this report, the identification of a C-terminal autoregulatory domain and a GTPase binding domain are described. The role of FHOS in Rho GTPase signaling pathways is also examined. In contrast to DRFs (mDia1 and mDia2) which are down-stream effectors of GTP-bound RhoA, FHOS binds to Rac1 in a guanine nucleotide-independent manner. Deletion of either the N-or C-termini created active forms of FHOS that stimulated transcription from the SRE. The Nterminal deletion mutant was localized to membrane ruffles and its ability to activate the SRE was blocked by overexpression of several Rac1 proteins. These data identify novel functions for FH proteins and Rac1 in the cell.

EXPERIMENTAL PROCEDURES.
Plasmids-The pCMV5-HA-FHOS full-length and HA-FHOS(1-1010) expression plasmids were previously described (30). For in vitro transcription reactions, these FHOS sequences were excised from pCMV5 with Asp718 and BamHI or Asp718 and SmaI, respectively, and    Luciferase activity was measured with the Luciferase Assay Systems (Promega) as instructed by the manufacturer. SEAP activity was measured as previously described (36,37).
Luciferase activity was normalized for transfection efficiency with the SEAP values for each sample. Fold activation was determined relative to samples transfected with pCMV5. Values  FHOS interacts with the carboxy-terminus of Rac1. The interaction between FHOS and Rac1, but not with Rac2 or Rac3 was somewhat surprising given the extensive sequence similarity between the three proteins (31,32). The extreme C-termini of Rac proteins however are poorly conserved (Figure 2A Figure 4B). To identify the region of FHOS that interacted with the Cterminus, several deletion mutants ( Figure 3A) were generated and tested in vitro ( Figure 4A) and in a yeast-two-hybrid binding assay ( Figure 4B). FHOS proteins lacking the C-terminus  Figure 4C. FHOS Interacts with Rac1 and Activates the SRE FHOS co-localizes with Rac1. To begin to understand the mechanism by which Rac1 preferentially represses the N-terminal FHOS mutant (469-1165), the localization of these FHOS proteins was determined. Cellular fractionation experiments indicated that FHOS is predominantly cytoplasmic (30). Immunofluorescence imaging demonstrated that full-length FHOS (1-1165) is concentrated in the perinuclear region, although light staining is also observed in cellular extensions and membrane edges (Figures 1D and 7A). This staining pattern was observed for both endogenous ( Figure 1D) and ectopic FHOS ( Figure 7A) in multiple cell lines, does not appear to effect Rac1 localization ( Figure 7B), and resembles that of mDia1 (2). The N-terminal mutant (469-1165) is concentrated in the perinuclear region like the wild-type protein and is detected at the cellular edges and membrane ruffles ( Figure 7A). In contrast, the C-terminal truncation mutants, FHOS (1-1010) and , are diffusely localized throughout the cell ( Figure 7A). Thus, the C-terminus may regulate the cellular localization of FHOS.

DISCUSSION
FHOS was previously characterized as a cytoplasmic protein with significant similarity to Formin and Diaphanous proteins (30). RNA blot analysis revealed that FHOS mRNA levels are very high in the spleen, skeletal muscle, and lung (30) (JJW, unpublished data). In this report, the first insights into the cellular functions and molecular regulation of FHOS are described. The data reported here demonstrate that FHOS may be regulated in a similar manner as other FH proteins but also identify novel mechanisms of regulation. The similarities between FHOS and other FH proteins include autoregulation by associations between N-and C-terminal residues, a specific interaction with a Rho family GTPase, and the ability of truncated forms to stimulate SRE-mediated transcriptional activation. Unlike what has been described for DRFs, FHOS associated with Rac1 in a guanine-nucleotide-independent manner in vitro. Overexpression of either dominant-negative or constituitively-active Rac1 proteins blocked SRE induction by an N-terminal FHOS truncation mutant but not by a C-terminal deletion mutant. These data suggest that FHOS may activate immediate early gene transcription from the SRE by multiple mechanisms, and that Rac1 may be a negative regulator of at least one FHOS signaling pathway.
FHOS is the second FH protein family member identified that interacts with Rac1. FRL also interacts with Rac1 but not with RhoA or Cdc42Hs (15). Rac1 interacted with N-terminal regions FHOS and FRL in the presence of either GDP or GTP ( Figure 2) and (15). This is unlike the GTP-dependent Rho interactions with DRFs (2,3,6). The guanine-nucleotide-independent binding of Rac1 to FHOS and the inhibition of FHOS (469-1165)-induced SRE activation by active and inactive Rac1 proteins suggest that Rac1 binding may inhibit FHOS signaling pathways. Alternatively, FHOS may affect Rac1 activity. Because FHOS does not contain any FHOS Interacts with Rac1 and Activates the SRE conserved domains found in GTPase-activating proteins (GAPs) or guanine nucleotide exchange factors (GEFs), it is unlikely that FHOS directly effects Rac1 activation. Moreover, inhibition of FHOS activity by both dominant-negative and constituitively active Rac1 proteins indicate that FHOS does not lie upstream of Rac1. One possibility is that FHOS acts as a scaffold to link Rac1 to other signaling molecules that associate with the FH1, FH2 and C-terminal domains.
FHOS activation could thus affect the course Rac1 signaling pathways and the activation of effectors. The polybasic residues in the Rac1 C-terminus that are necessary for FHOS interaction are also required for maximum PAK1 activation and Rac1 homodimerization (33,46). Rac1 homodimerization potentiates the activity of PAK1 and other effectors (46). In preliminary studies, FHOS does not appear to associate with PAK1 (unpublished data). Thus by interacting with FHOS, Rac1 may activate PAK1-independent pathways (47). Additional studies are necessary to determine if any Rac1 effector molecules associate with FHOS and to identify the factors that regulate Rac1 binding to FHOS. It will also be important to determine if FHOS effects Rac1-dependent cytoskeletal organization and to identify the factors or signals that activate FHOS in vivo.
The C-termini of FHOS and other FH proteins contain several important signaling and autoregulatory domains. Alberts recently described a Dia-autoregulatory domain (DAD) in the C-termini of DRF subfamily of FH proteins that interact with N-terminal sequences of the same molecule (9). FHOS shares limited sequence similarity with Dia proteins within the FH2 domain (30), but it does not appear to be a member of the DRF subfamily based on comparative sequence alignments of the full-length proteins (data not shown). A specific search for a DAD however identified twenty-six residues in the C-terminus of FHOS that aligns with 46% identity FHOS Interacts with Rac1 and Activates the SRE to DADs in human DRF proteins (data not shown). The major difference in these DADs is a seven to nine amino acid spacer region in FHOS that separates the polybasic residues from the remainder of the motif. The DAD of the putative FH protein, KIAA1695, also contains a nonconserved spacer. Interestingly, Alberts' data indicate that the DAD domain may have an effector function in addition to an autoinhibitory function (9). Biochemically, this model would seem intuitive, as releasing the DAD domain from intramolecular interactions should expose a protein-protein interaction surface. Our data indicate that region may also target FHOS to specific sites in the cell and thereby stimulate a specific signaling pathway(s).
The importance of the C-terminus is highlighted by the fact that the only known penetrant genetic mutations in FH proteins alter their extreme C-termini (16)(17)(18)(19)(20). Moreover, a C-terminal deletion mutant of DRF1 (mDia1) altered cytoskeletal organization in a manner consistent with Rho inactivation (3). It was proposed that defective intramolecular binding and the exposure of the FH1 and FH2 domains caused this phenotype. Our data suggest that Cterminal mutations may also effect FH protein localization and that the mislocalization could alter SRE regulation. Although FHOS (1-1010) appeared to be localized differently than the wild-type or N-terminal truncation mutant, it was still capable of activating the SRE. Rac1 however did not effect the activity of FHOS . Preliminary data indicate that MEK inhibitors specifically block this pathway. Thus, the mechanisms by which mutations in the Ctermini of FH proteins alter cellular and developmental phenotypes may involve differential protein localization and usage of differentially regulated signaling pathways.