Activation of the transcription factor SRF by extracellular signals is mediated by Rho GTPases and requires alterations in actin dynamics (Hill et al., 1995
; Sotiropoulos et al., 1999
). The Diaphanous Related Formins (DRFs) are candidate effectors of RhoA in this signaling pathway (Sotiropoulos et al., 1999
; Tominaga et al., 2000
). In this study we performed a detailed analysis of the mDia1 DRF to investigate the relationship between mDia1-induced actin polymerization and SRF activation. Our results show that the ability of mDia1 derivatives to activate SRF strictly correlates with their ability to promote F-actin accumulation and reveal an important role for the mDia1 FH2 region in these processes. The quantitative transcription assay allowed the identification of inactive mDia1 derivatives whose expression can interfere with both SRF activation and F-actin assembly, whether induced by extracellular signals or expression of activated DRF proteins. Our findings suggest a model whereby SRF activation occurs as a consequence of mDia induced F-actin assembly (Figure A).
We previously proposed that SRF is activated in response to depletion of the cellular G-actin pool (Sotiropoulos et al., 1999
). Consistent with this model, mDia1-induced SRF activation, but not F-actin assembly, is inhibited by expression of the nonpolymerizable actin mutant G13R. This places actin downstream of mDia1 in the signal pathway to SRF and strongly suggests that it is the ability of mDia1 to regulate the level of G-actin, or a subpopulation of it, that controls SRF activity (Figure A). We have not detected direct interaction between activated mDia1 and G-actin, suggesting that SRF activation by mDia1 is likely to occur as a consequence of its effects on F-actin assembly rather than through direct interaction with actin. In contrast to a previous proposal (Tominaga et al., 2000
), our data indicate that the Src tyrosine kinase does not appear essential for SRF activation by serum-induced signals, instead appearing to act upstream or in parallel to mDia1.
Deletion of the N-terminal Rho binding domain (RBD) activates the mDia DRF proteins by relieving inhibitory interactions between the RBD and the C-terminal Diaphanous autoregulatory domain (DAD; Watanabe et al., 1999
; Alberts, 2001
). In agreement with previous studies of DAD point mutations, we found that a C-terminal deletion that removes conserved sequences within the mDia1 DAD, while retaining the core homology, also strongly activates SRF. Our experiments revealed no requirement for the DAD domain in SRF activation or F-actin assembly suggesting that, like the RBD, DAD has primarily a regulatory function (see below).
SRF activation and cytoskeletal reorganization by mDia1 derivatives lacking either the FH1 domain itself, or sequences N-terminal to it, occurred independently of functional RhoA, placing the DRFs downstream of Rho in the signaling pathway. Our studies thus concur with previous findings (Nakano et al., 1999
; Watanabe et al., 1999
; Tominaga et al., 2000
) and do not support the recent proposal that formins function to activate Rho by recruiting GEFs (Habas et al., 2001
). Induction of SRF activity and F-actin accumulation by the mDia1 FH2 region alone was partially dependent on functional Rho, however, suggesting that either the FH3/coiled-coil domain or the FH1 domain is required to render mDia1 function completely independent of Rho activity. Functional Rho is required for subcellular localization of proteins such as Src (Fincham et al., 1996
), and it may be that in the absence of both FH1 and FH3 domains appropriate subcellular localization of the FH2 region becomes Rho dependent. Indeed, previous studies have shown that the FH3 domain mediates subcellular localization of DRFs (Petersen et al., 1998
; Ozaki-Kuroda et al., 2001
; Sharpless and Harris, 2002
The integrity of virtually the entire FH2 region, which exhibits substantial sequence conservation throughout the formin family, is required for mDia1 function. Even in the presence of the FH1 domain, deletions that impinge on the N- and C-termini of the FH2 region, or the core FH2 motif, completely abolish both SRF activation and actin polymerization. The C terminus of the FH2, which is disrupted by the inactivating deletion of residues 1130–1150, contains a conserved EEFF motif reminiscent of the DDW motif mediating interaction of ActA, N-Wasp, and Cortactin with the Arp2/3 complex (Weed et al., 2000
; Uruno et al., 2001
). The possibility that the mDia1 FH2 region functions by recruiting Arp2/3 is made less likely, however, by the recent demonstration that genetically the yeast DRF Bni1 functions independently of the Arp2/3 complex (Evangelista et al., 2002
). Instead, we favor the notion that the FH2 region of mDia1 induces F-actin assembly directly by nucleating actin polymerization. Indeed, while this article was under review, it was shown that the FH2 domain of Bni1 is sufficient to nucleate actin polymerization in vitro (Pruyne et al., 2002
). The N-terminal inactivating deletion (amino acids 750–770) of mDia1 removes another conserved motif, corresponding to the binding site in Bni1 for the translation elongation factor EF1α (Umikawa et al., 1998
). EF1α interacts with actin (Demma et al., 1990
; Yang et al., 1990
) and binds to the ends of stress fibers, where it is thought to block actin polymerization (Murray et al., 1996
). We are currently addressing the possibility that mDia1 may function in part by relieving such inhibition in vivo.
Inactive derivatives of mDia1 containing deletions within the FH2 region strongly inhibit SRF activation and reorganization of the actin cytoskeleton, whether induced by extracellular signals such as serum or LPA or by expression of activated mDia1 and mDia2 derivatives. In contrast, an mDia1 derivative containing the FH3 domain, which interferes with F-actin structures in MDCK epithelial cells (Nakano et al., 1999
), was merely inactive in NIH3T3 fibroblasts, perhaps reflecting differences in mDia1 function in these different cell types. Our interfering mDia1 proteins act specifically, because they do not affect activity of the MAP kinase-regulated SRF accessory protein Elk-1. Moreover, experiments in PC12 cells indicate that they do not act as nonspecific Rho inhibitors because their expression does not affect Rho-dependent cofilin phosphorylation (Geneste et al., 2002
). The interfering mDia1 proteins must either interact nonproductively with downstream DRF effectors or interact with endogenous DRFs to generate nonfunctional complexes. Whatever the mechanism, the interactions involved must be mediated by the FH2-containing region of the mDia C terminus, because neither the DAD nor the FH1 domain is required for interference. Although interfering mDia1 derivatives interfere with DRF-induced F-actin accumulation, they do not abolish formation of F-actin bundles, which is likely mediated by ROCK (Nakano et al., 1999
; Watanabe et al., 1999
; Tominaga et al., 2000
), and their effect on cytoskeletal morphology is thus much less marked than that observed upon inactivation of Rho by C3 transferase expression. In keeping with our proposal that G-actin depletion and SRF activation are linked, our interfering mDia1 derivatives inhibit both LIM kinase-induced F-actin formation and SRF activation in PC12 cells (Geneste et al., 2002
In contrast to previous studies (Watanabe et al., 1999
; Alberts, 2001
), our results indicate that the mDia1 FH1 domain is not required for SRF activation or F-actin assembly by overexpressed mDia1 derivatives, nor is it required for inhibition of SRF activation by interfering mDia1 proteins. This apparent discrepancy may reflect our use of fibroblast rather than epithelial cells and the sensitivity of our assays for SRF and F-actin. Our results suggest that mDia1 function is not strictly dependent on direct binding to poly-proline binding cofactors such as profilin and Src. Profilin binds to the FH1 domain of the formins Bni1, cdc12, mDia1, and mDia2 (Chang et al., 1997
; Evangelista et al., 1997
; Imamura et al., 1997
; Watanabe et al., 1997
) and in yeast profilin is required with the Bni1 FH1 domain, for Bni1-mediated assembly of actin cables (Evangelista et al., 2002
). We have confirmed that deletion of the mDia1 FH1 abolishes the interaction with profilin in two-hybrid assays (J.C., unpublished observations), in agreement with a biochemical study (Krebs et al., 2001
). It remains possible, however, that profilin might be recruited to mDia derivatives lacking FH1 through their interaction with other actin remodeling proteins. Alternatively, if profilin enhances actin polymerization without being absolutely required for it, overexpression of mDia1 derivatives might be sufficient to bypass FH1-mediated profilin recruitment.
The mDia1 FH1 domain also binds the Src tyrosine kinase, and it has been proposed that Src mediates Dia-dependent signaling to SRF (Tominaga et al., 2000
; Alberts, 2001
). Our findings, which suggest that activation by both mDia1 and Src involves alterations in actin dynamics, do not support this view. Activation of SRF by active Src is completely dependent on functional Rho, suggesting that the kinase either induces activation of Rho, perhaps via effects on p190RhoGAP (Chang et al., 1995
; Fincham et al., 1999
), or that functional Rho is required for its activity, perhaps through involvement of Rho in subcellular targetting of Src (Fincham et al., 1996
). Moreover, serum-induced SRF activation was not blocked upon inhibition of Src, whether by expression of the inactivating C-terminal Src kinase (CSK) or kinase-inactive Src, or by treatment of cells with Src inhibitor PP2, suggesting that Src is not required for SRF activation.
Two recent reports have implicated the mDia proteins in regulation of the microtubule cytoskeleton, both in its polarization (Ishizaki et al., 2001
) and in the generation of the stable glu-MT population (Palazzo et al., 2001
). It remains unclear whether these properties are controlled by the functional domains identified here. We have found that an FH2 domain mutation reported to selectively affect MT polarization (Ishizaki et al., 2001
) is also severely defective in SRF activation and F-actin assembly in NIH 3T3 cells (J.C. and R.T., unpublished data). Our preliminary data indicate that expression of our interfering mDia1 proteins does not inhibit serum- or LPA-induced glu-MT assembly. We are currently investigating the role of the DRFs in MT organization.
Our results establish a tight correlation between the ability of mDia1 derivatives to promote actin polymerization and to activate SRF. Both processes require the same sequences in and around the FH2 domain but do not require the FH1 domain, suggesting that they do not involve obligatory direct interaction of mDia1 with FH1 ligands such as profilin. We have obtained similar results with both mDia2 and mouse formin (J.C., unpublished data). We used the transcriptional assay to identify inactive mDia1 derivatives that interfere with signal-induced SRF activation and cytoskeletal reorganization and showed that actin itself appears to lie downstream of mDia in the Rho-SRF signaling pathway. Future work will focus on how mDia1 interacts with the actin polymerization machinery and how our deletions affect other known mDia functions.