FHOD1 coordinates actin filament and microtubule alignment to mediate cell elongation

Diaphanous-related formins (DRFs) are actin nucleators that mediate rearrangements of the actin cytoskeleton downstream of specific Rho GTPases. The DRF Formin Homology 2 Domain containing 1 (FHOD1) interacts with the Rac1 GTPase and induces the formation of and associates with bundled actin stress fibers. Here we report that active FHOD1 also coordinates microtubules with these actin stress fibers. Expression of a constitutive active FHOD1 variant in HeLa cells not only resulted in pronounced formation of FHOD1-actin fibers but also caused marked cell elongation and parallel alignment of microtubules without affecting cytokinesis of these cells. The analysis of deletions in the FH1 and FH2 functional regions revealed that the integrity of both domains was strictly required for FHOD1's effects on the cytoskeleton. Dominant-negative approaches demonstrated that filament coordination and cell elongation depended on the activity of the Rho-ROCK cascade, but did not involve Rac or Cdc42 activity. Experimental depolymerization of actin filaments or microtubules revealed that the formation of FHOD1-actin fibers was a prerequisite for the polarization of microtubules. However, only simultaneous disruption of both filament systems reversed the cell elongation induced by activated FHOD1. Thus, sustained cell elongation was a consequence of FHOD1-mediated actin-microtubule coordination. These results suggest filament coordination as a conserved function of mammalian DRFs.     

The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin–based motility

Vaccinia virus dissemination relies on the N-WASP–ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP–ARP2/3 and Rac1–FHOD1 pathways.     

The Diaphanous-related Formin FHOD1 associates with ROCK1 and promotes Src-dependent plasma membrane blebbing

Diaphanous-related formins (DRFs) mediate GTPase-triggered actin rearrangements to regulate central cellular processes, such as cell motility and cytokinesis. The DRF FHOD1 interacts with the Rho-GTPase Rac1 and mediates formation of actin stress fibers in its deregulated form; the physiologically relevant activities and molecular mechanisms of endogenous FHOD1, however, are still unknown. Here we report that FHOD1 physically associates via the N-terminal part of its FH2 domain with the central domain of ROCK1. Although FHOD1 does not affect the kinase activity of ROCK1, the DRF is an efficient substrate for phosphorylation by ROCK1. Co-expression of FHOD1 and ROCK1 results in the generation of nonapoptotic plasma membrane (PM) blebs, to which the DRF is efficiently recruited. Blebbing induced by FHOD1 and ROCK1 depends on F-actin integrity, the Rho-ROCK cascade, and Src activity and is reminiscent of the recently described PM blebs triggered by expression of Src homology 4 (SH4) domain PM targeting signals. Consistently, endogenous FHOD1 is required in SH4 domain expressing cells for efficient PM blebbing and rounded cell morphology in two-dimensional cultures or three-dimensional matrices, respectively. Efficient association of FHOD1 with ROCK1, as well as recruitment of the DRF to blebs, depends on Src activity, suggesting that the functional interaction between both proteins is regulated by Src. These results define a role for endogenous FHOD1 in SH4 domain-induced blebbing and suggest that its activity is regulated by ROCK1 in a Src-dependent manner.     

The human formin FHOD1 contains a bipartite structure of FH3 and GTPase-binding domains required for activation

Formins induce the nucleation and polymerization of unbranched actin filaments. They share three homology domains required for profilin binding, actin polymerization, and regulation. Diaphanous-related formins (DRFs) are activated by GTPases of the Rho/Rac family, whose interaction with the N-terminal formin domain is thought to displace a C-terminal Diaphanous-autoregulatory domain (DAD). We have determined the structure of the N-terminal domains of FHOD1 consisting of a GTPase-binding domain (GBD) and the DAD-recognition domain FH3. In contrast to the formin mDia1, the FHOD1-GBD reveals a ubiquitin superfold as found similarly in c-Raf1 or PI3 kinase. This GBD is recruited by Rac and Ras GTPases in cells and plays an essential role for FHOD1-mediated actin remodeling. The FHOD1-FH3 domain is composed of five armadillo repeats, similarly to other formins. Mutation of one residue in the predicted DAD-interaction surface efficiently activates FHOD1 in cells. These results demonstrate that DRFs have evolved different molecular solutions to govern their autoregulation and GTPase specificity.     

The mammalian formin FHOD1 interacts with the ERK MAP kinase pathway

Formin homology 2 domain containing protein (FHOD1), a mammalian formin, regulates cytoskeletal architecture, enhances cell migration, and induces gene expression from the serum response element. In this study, we describe co-precipitation of FHOD1 with components of the ERK MAP kinase pathway while co-precipitation of FHOD1 with p38 MAP kinase and JNK was not observed. In addition, FHOD1 co-localized to lamellipodia with Raf-1 and to stress fibers with MEK. FHOD1-induced gene expression from the serum response element was dependent on ERK MAP kinase activation, and the native skeletal actin promoter were activated by FHOD1 through the SRF site. However, FHOD1-induced stress-fiber formation and gene expression from the skeletal actin promoter was independent of ERK activation. These novel data demonstrate that FHOD1-ERK MAP kinase interaction regulates key aspects of FHOD1 biology.     

Mechanisms of activation and action of mDial in the formation of parallel stress fibers in MDCK cells

mDial is a downstream target molecule of Rho small G protein and regulates the formation of parallel stress fibers in MDCK cells. mDial consists of at least one Rho-binding domain (RBD), one FH3 domain (FH3D), one coiled-coil domain (CCD), one FH1 domain (FH1D), one FH2 domain (FH2D), and another CCD in this order from the N-terminus to the C-terminus. We constructed various deletion mutants of mDial and investigated the mechanisms of its activation and action by measuring the formation of parallel stress fibers in MDCK cells. We show here that at least FH1D and second CCD are essential for the formation of parallel stress fibers. Furthermore, we present the evidence suggesting that mDial has another domain which interacts with RBD, that this interaction masks FH1D and second CCD and keeps mDial inactive, and that the binding of Rho to RBD opens this folded structure, resulting in the activation of mDial.     

Formin homology domain protein (FHOD1) is a cyclic GMP-dependent protein kinase I-binding protein and substrate in vascular smooth muscle cells

Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration.     

Nadrin, a novel neuron-specific GTPase-activating protein involved in regulated exocytosis

It has been proposed that the cortical actin filament networks act as a cortical barrier that must be reorganized to enable docking and fusion of the synaptic vesicles with the plasma membranes. We identified a novel neuron-associated developmentally regulated protein, designated as Nadrin. Expression of Nadrin is restricted to neurons and correlates well with the differentiation of neurons. Nadrin has a unique structure; it contains a GTPase-activating protein (GAP) domain for Rho family GTPases, a potential coiled-coil domain, and a succession of 29 glutamines. In vitro the GAP domain activates RhoA, Rac1, and Cdc42 GTPases. Expression of Nadrin in NIH3T3 cells markedly reduced the number of the actin stress fibers and the formation of the ruffled membranes, suggesting that Nadrin regulates actin filament reorganization. In PC12 cells, Nadrin colocalized with synaptotagmin in the neurite termini and also with cortical actin filaments in the subplasmalemmal regions. Expression of Nadrin or its mutant composed of the coiled-coil and GAP domain enhanced Ca(2+)-dependent exocytosis of PC12 cells, but a mutant lacking the GAP domain inhibited exocytosis. These results suggest that Nadrin plays a role in regulating Ca(2+)-dependent exocytosis, most likely by catalyzing GTPase activity of Rho family proteins and by inducing the reorganization of the cortical actin filaments.     

Specificity of interactions between mDia isoforms and Rho proteins

Formins are key regulators of actin nucleation and polymerization. They contain formin homology 1 (FH1) and 2 (FH2) domains as the catalytic machinery for the formation of linear actin cables. A subclass of formins constitutes the Diaphanous-related formins, members of which are regulated by the binding of a small GTP-binding protein of the Rho subfamily. Binding of these molecular switch proteins to the regulatory N-terminal mDia(N), including the GTPase-binding domain, leads to the release of auto-inhibition. From the three mDia isoforms, mDia1 is activated only by Rho (RhoA, -B, and -C), in contrast to mDia2 and -3, which is also activated by Rac and Cdc42. Little is known about the determinants of specificity. Here we report on the interactions of RhoA, Rac1, and Cdc42 with mDia1 and an mDia1 mutant (mDia(N)-Thr-Ser-His (TSH)), which based on structural information should mimic mDia2 and -3. Specificity is analyzed by biochemical studies and a structural analysis of a complex between Cdc42.Gpp(NH)p and mDia(N)-TSH. A triple NNN motif in mDia1 (amino acids 164-166), corresponding to the TSH motif in mDia2/3 (amino acids 183-185 and 190-192), and the epitope interacting with the Rho insert helix are essential for high affinity binding. The triple N motif of mDia1 allows tight interaction with Rho because of the presence of Phe-106, whereas the corresponding His-104 in Rac and Cdc42 forms a complementary interface with the TSH motif in mDia2/3. We also show that the F106H and H104F mutations drastically alter the affinities and thermodynamics of mDia interactions.     

ArhGAP15, a novel human RacGAP protein with GTPase binding property

We have previously described a partial cDNA sequence encoding a RhoGAP protein, GAP25 that is homologous to the recently reported ArhGAP9 and ArhGAP12. We now describe a related new member ArhGAP15 that shares a number of domain similarities, including a pleckstrin homology (PH) domain, a RhoGAP domain and a novel motif N-terminal to the GAP domain. This novel motif was found to be responsible for nucleotide-independent Rac1 binding. Using swop mutants of Rac/Cdc42, we have established that the binding is through the C-terminal half of Rac1. The GAP domain of ArhGAP15 showed specificity towards Rac1 in vitro. The PH domain is required for ArhGAP15 to localize to cell periphery and over-expression of the full-length ArhGAP15, but not the mutant with a partial deletion of the PH domain, resulted in an increase in actin stress fibers and cell contraction. These morphological effects can be attenuated by the co-expression of dominant negative Rac1(N17). HeLa cells expressing ArhGAP15 were also resistant to phorbol myristatate acetate treatment, suggesting that ArhGAP15 is a potential regulator of Rac1.     

Kank proteins: a new family of ankyrin-repeat domain-containing proteins

The human Kank gene was found as a candidate tumor suppressor for renal cell carcinoma, and encodes an ankyrin-repeat domain-containing protein, Kank. Here, we report a new family of proteins consisting of three Kank (Kank1)-associated members, Kank2, Kank3 and Kank4, which were found by domain and phylogenetic analyses. Besides the conserved ankyrin-repeat and coiled-coil domains, there was a conserved motif at the N-terminal (KN motif) containing potential motifs for nuclear localization and export signals. Gene expression of these genes was examined by RT-PCR at the mRNA level and by Western blotting and immunostaining at the protein level. Kank family genes showed variations in the expression level among tissues and kidney cell lines. Furthermore, the results of overexpression of these genes in NIH3T3 cells suggest that all of these family proteins have an identical role in the formation of actin stress fibers.     

Actin Filament Bundling and Different Nucleating Effects of Mouse Diaphanous-Related Formin FH2 Domains on Actin/ADF and Actin/Cofilin Complexes

Mouse Diaphanous-related formins (mDias) are members of the formin protein family that nucleate actin polymerization and subsequently promote filamentous actin (F-actin) elongation by monomer addition to fast-growing barbed ends. It has been suggested that mDias preferentially recruit actin complexed to profilin due to their proline-rich FH1 domains. During filament elongation, dimeric mDias remain attached to the barbed ends by their FH2 domains, which form an anti-parallel ring-like structure enclosing the filament barbed ends. Dimer formation of mDia-FH2 domains is dependent on their N-terminal lasso and linker subdomains (connector). Here, we investigated the effect of isolated FH2 domains on actin polymerization using mDia1-FH2 domain plus connector, as well as core mDia1, mDia2, and mDia3 missing the connector, by cosedimentation and electron microscopy after negative staining. Analytical ultracentrifugation showed that core FH2 domains of mDia1 and mDia2 exhibited a low degree of dimer formation, whereas mDia3-FH2 minus connector and mDia1-FH2 plus connector readily dimerized. Only core mDia3-FH2 was able to nucleate actin polymerization. However, all tested core FH2 domains decorated and bundled F-actin, as demonstrated by electron microscopy after negative staining. Bundling activity was highest for mDia3-FH2, decreased for mDia2-FH2, and further decreased for mDia1-FH2. The mDia1-FH2 domain plus connector induced actin polymerization also in the absence of profilin, but failed to induce F-actin deformation and bundling. We also tested whether mDia1-FH2 was able to repolymerize actin in complex with different proteins that stabilize globular actin. The data obtained demonstrated that mDia1-FH2 induced actin repolymerization only from the actin/cofilin-1 complex, but not when complexed to actin depolymerizing factor, gelsolin segment 1, vitamin D binding protein, or deoxyribonuclease I.     

The Diaphanous-related formin dDia2 is required for the formation and maintenance of filopodia

Formins have important roles in the nucleation of actin and the formation of linear actin filaments, but their role in filopodium formation has remained elusive. Dictyostelium discoideum Diaphanous-related formin dDia2 is enriched at the tips of filopodia and interacts with profilin II and Rac1. An FH1FH2 fragment of dDia2 nucleated actin polymerization and removed capping protein from capped filament ends. Genetic studies showed that dDia2 is important for cell migration as well as the formation, elongation and maintenance of filopodia. Here we provide evidence that dDia2 specifically controls filopodial dynamics by regulating actin turnover at the barbed ends of actin filaments.     

Characterization of a novel GTPase-activating protein associated with focal adhesions and the actin cytoskeleton

In the present study we characterize a novel RhoGAP protein (RC-GAP72) that interacts with actin stress fibers, focal adhesions, and cell-cell adherens junctions via its 185-amino acid C-terminal region. Overexpression of RC-GAP72 in fibroblasts induces cell rounding with partial or complete disruption of actin stress fibers and formation of membrane ruffles, lamellipodia, and filopodia. RC-GAP72 mutant truncated downstream of the GTPase-activating protein (GAP) domain retains the ability to stimulate membrane protrusions but fails to affect stress fiber integrity or induce cell retraction. A mutant protein consisting of the C terminus of RC-GAP72 and lacking the GAP domain does not exert any visible effect on cellular morphology. Inactivation of the GAP domain by a point mutation does not abolish the effect of RC-GAP72 on actin stress fibers but moderates its capability to induce membrane protrusions. Our data imply that the cytoskeletal localization of RC-GAP72 and its interaction with GTPases are essential for its effect on the integrity of actin stress fibers, whereas the induction of lamellipodia and filopodia depends on the activity of the GAP domain irrespective of binding to the actin cytoskeleton. We propose that RC-GAP72 affects cellular morphology by targeting activated Cdc42 and Rac1 GTPases to specific subcellular sites, triggering local morphological changes. The overall physiological functions of RC-GAP72 are presently unknown, yet our data suggest that RC-GAP72 plays a role in regulating cell morphology and cytoskeletal organization.     

INF1 is a novel microtubule-associated formin

Formin proteins, characterized by the presence of conserved formin homology (FH) domains, play important roles in cytoskeletal regulation via their abilities to nucleate actin filament formation and to interact with multiple other proteins involved in cytoskeletal regulation. The C-terminal FH2 domain of formins is key for actin filament interactions and has been implicated in playing a role in interactions with microtubules. Inverted formin 1 (INF1) is unusual among the formin family in having the conserved FH1 and FH2 domains in its N-terminal half, with its C-terminal half being composed of a unique polypeptide sequence. In this study, we have examined a potential role for INF1 in regulating microtubule structure. INF1 associates discretely with microtubules, and this association is dependent on a novel C-terminal microtubule-binding domain. INF1 expressed in fibroblast cells induced actin stress fiber formation, coalignment of microtubules with actin filaments, and the formation of bundled, acetylated microtubules. Endogenous INF1 showed an association with acetylated microtubules, and knockdown of INF1 resulted in decreased levels of acetylated microtubules. Our data suggests a role for INF1 in microtubule modification and potentially in coordinating microtubule and F-actin structure.     

Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2

The rapid turnover of actin filaments and the tertiary meshwork formation are regulated by a variety of actin-binding proteins. Protein phosphorylation of cofilin, an actin-binding protein that depolymerizes actin filaments, suppresses its function. Thus, cofilin is a terminal effector of signaling cascades that evokes actin cytoskeletal rearrangement. When wild-type LIMK2 and kinase-dead LIMK2 (LIMK2/KD) were respectively expressed in cells, LIMK2, but not LIMK2/KD, phosphorylated cofilin and induced formation of stress fibers and focal complexes. LIMK2 activity toward cofilin phosphorylation was stimulated by coexpression of activated Rho and Cdc42, but not Rac. Importantly, expression of activated Rho and Cdc42, respectively, induced stress fibers and filopodia, whereas both Rho- induced stress fibers and Cdc42-induced filopodia were abrogated by the coexpression of LIMK2/KD. In contrast, the coexpression of LIMK2/KD with the activated Rac did not affect Rac-induced lamellipodia formation. These results indicate that LIMK2 plays a crucial role both in Rho- and Cdc42-induced actin cytoskeletal reorganization, at least in part by inhibiting the functions of cofilin. Together with recent findings that LIMK1 participates in Rac-induced lamellipodia formation, LIMK1 and LIMK2 function under control of distinct Rho subfamily GTPases and are essential regulators in the Rho subfamilies-induced actin cytoskeletal reorganization.