RhoE is known to induce loss of actin stress fibers, but the molecular basis for this effect has not been elucidated. Here, we show that RhoE binds to ROCK I, a kinase required for stress fiber formation and contractility. We found that RhoE binds to the N-terminal region of ROCK I (amino acids 1 to 420) and not to the previously mapped RhoA-binding site (amino acids 934 to 1015, RBD in Fig. ) (11
). The RhoE-binding region includes the N-terminal amino acids 1 to 76, the kinase domain (amino acids 76 to 338), and amino acids 338 to 420 of ROCK I. This explains why the ROCK I construct used in the yeast two-hybrid analysis (amino acids 349 to 1025), which lacks the N terminus and kinase domain, did not interact with RhoE. Interestingly, deletion of the N-terminal 78 amino acids from ROCK II/Rho-kinase/ROKα has previously been reported to prevent ROCK-induced stress fiber formation (24
), suggesting that the N-terminal region upstream of the kinase domain is critical for ROCK function. Even though RhoE does not bind to the C-terminal RhoA-binding site, RhoA and RhoE are unable to bind ROCK I simultaneously and compete for ROCK I binding. This suggests that the binding site for RhoE on ROCK I is masked when RhoA binds (and vice versa), either because of the conformational change induced or because RhoA binds to part of the RhoE-binding site in addition to the C-terminal RBD. Considering that the effector domain sequences of RhoA and RhoE are highly similar, it is perhaps surprising that RhoE does not bind to the RhoA-binding site of ROCK I. However, ROCK I binding to RhoA requires a second region (amino acids 75 to 92) of RhoA in addition to the effector domain (12
), and this region is less conserved between RhoE and RhoA.
Both ROCK I and RhoE localize to the trans-Golgi network in COS-7 cells. Recently, ROCK I has been reported to be bound to centrosomes (5
), which are localized in the Golgi area. Localization of ROCK II by using different antibodies has yielded contradictory results, but some reports show ROCK II to partially associate with stress fibers (4
). COS-7 cells have very few actin stress fibers, and we cannot exclude the possibility that ROCK I would associate with them in other cell types. Unfortunately, immunofluorescence studies with the commercial ROCK I antibodies gave only very weak staining in Swiss 3T3 cells. Nevertheless, other GTPases localize to the Golgi complex, including Cdc42, TC10, and RhoG (3
), and Ras proteins have been shown elsewhere to be active on the Golgi complex (6
). In addition, it is known that a Golgi complex-associated actin network is important for protein trafficking (10
), and myosins can be found on the Golgi complex (40
). Golgi complex-to-endoplasmic reticulum trafficking is affected by Cdc42 and TC10 (20
). Interestingly, we found that actin filaments were organized around the perinuclear region in the RhoE-overexpressing cells and colocalized with the trans-Golgi network. RhoE may therefore function primarily on the Golgi complex, and one possibility is that it acts to sequester ROCK I, and/or ROCK I may act on Golgi complex-associated myosins.
Of the tested RhoA effectors ROCK I is the only one that binds RhoE. Similarly, Rnd1 did not interact with several Rho/Rac effectors in yeast two-hybrid assays (44
). On the other hand, RhoE shows weak interaction with a kinase homologous to ROCK I, namely, citron kinase (27
), but this interaction is barely detectable in comparison to RhoA binding. The fact that overexpression of RhoE has a major inhibitory effect on stress fibers is explained by our observation that it binds ROCK I, and prevents ROCK I-induced phosphorylation of MLCP, a major downstream target of ROCKs involved in stress fiber formation (13
). In this respect it acts similarly to ROCK I inhibitors, such as Y27632, and our results are therefore in concordance with ROCK playing a central role in stress fiber formation (43
). We cannot rule out the possibility that other RhoE-binding proteins also contribute to RhoE-mediated effects on cell morphology, especially as multiple RhoA effectors contribute to stress fiber assembly. Nevertheless, by binding around the kinase domain, RhoE could sterically inhibit ROCK I from interacting with its targets. Recently, Ward et al. (46
) reported that overexpressed Gem, another small GTPase, inhibited ROCK-mediated functions, but unlike RhoE, Gem bound adjacent to the RhoA-binding site of ROCK I. Here we have shown that endogenous RhoE interacts with ROCK I, and further studies are required to reveal if other GTPases operate in different areas of the cell to negatively control ROCK I activity.
The kinase domain of inactive ROCK is highly inaccessible, as the protein forms inter- and intramolecular interactions. As the C-terminal region of ROCK can bind to the N-terminal kinase region to form an autoinhibited structure (1
), it would be expected that in this inactive conformation the RhoE-binding site around the kinase domain would be masked. In cells transfected with wt myc-ROCK I, we consistently observed a lower-molecular-weight form of ROCK I that interacts much better with RhoE than does full-length ROCK I. C-terminal ROCK I cleavage by caspase 3 has been shown elsewhere to yield a constitutively active ROCK I protein, which is similar in size to the lower-molecular-weight form of ROCK I (7
). This implies that ROCK I has to be in an open conformation for RhoE to bind. Although RhoE can bind both full-length ROCK I and a ROCK I mutant that is resistant to caspase cleavage (7
), much less full-length ROCK I is pulled down by RhoE than is truncated ROCK I. Possibly the full-length ROCK I pulled down by RhoE represents the small fraction of activated (but not RhoA-bound) ROCK I in cells. Presumably, RhoA-GTP binding to ROCK I induces this open conformation, and RhoE can bind to this once RhoA has dissociated. Our observations that RhoE inhibited stress fiber formation and MLCP phosphorylation induced by either wt or activated ROCK I further support a model where RhoE functions by binding to activated ROCK I.
As RhoE is constitutively in an active, GTP-bound form (9
), regulation of RhoE function must be controlled differently from cycling Rho GTPases. Interestingly, we showed that the expression of RhoE is transiently upregulated upon PDGF treatment of Swiss 3T3 cells and that this correlates with the levels of actin stress fibers and cell morphology. In addition, RhoE expression has been shown elsewhere to be upregulated upon Raf activation in MDCK cells (17
) and by hepatocyte growth factor, which stimulates cell migration (41
). Raf is a Ras effector, and there are several similarities between the phenotypes of Ras-transformed cells and those of cells overexpressing RhoE. RhoE overexpression increases the motility of cells, including hepatocyte growth factor-stimulated MDCK cells, and causes a loss of stress fibers and focal contacts (15
). Similarly, Ras-transformed cells are highly motile, and they lack stress fibers and focal contacts. Ras-transformed cells frequently show an upregulation of RhoA activity, which is important for cell proliferation (38
). However, to allow the increased motility of Ras-transformed cells, RhoA needs to be uncoupled from inducing actin stress fibers and focal contacts, and this has been attributed to a reduction in Rho to ROCK signaling (38
) and extracellular signal-regulated kinase-induced downregulation of ROCK I and/or II expression (31
). Reduction in ROCK function by increased RhoE expression could contribute together with ROCK downregulation to the Ras-transformed motile phenotype. Upregulation of RhoE expression may also explain why wt ROCK cannot reverse the phenotype of Ras-transformed cells (31
). In the future it will be important to determine whether a sustained increase in RhoE expression alters the level of ROCK I activity.
Our data suggest that the relative expression levels and activities of Rho(A/B/C) and RhoE will determine the final readout of ROCK I. Other studies indicate that similar competition may occur between other Rho family proteins. For example, Xenopus laevis
Rnd1 prevents RhoA-induced cell adhesion (48
) and RhoH reduces Rho-mediated activation of NF-κB and p38 (25
). In addition, RhoD inhibits RhoA-activated stress fiber formation (42
) and Rnd1-induced plexin-A1-mediated cytoskeletal collapse (49
). The GTPase-deficient Rho family members particularly may employ competition as their mode of action to affect cellular responses. We propose that controlling the expression level of RhoE and thus its binding to ROCK I provides a mechanism to regulate cell behavior in addition to altering the GTP/GDP ratio on conventional Rho GTPases.