Gtpase Regulation: Getting Arnd Dispatch Rock and Rho Inhibition

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GTPase Regulation: Getting aRnd Dispatch Rock and Rho Inhibition

Pierre Chardin preferentially bind GDP. It is not yet clear whether they physiologically bind GTP, but they apparently possess very low, if any, intrinsic GTP hydrolysis activity. Rnd proteins are atypical members of the Rho small Expression of Rnd proteins or Gem disrupts acto- G protein family that inhibit the formation of actomyosin contractile fibers. Several mechanisms could myosin contractile fibers via activation of RhoGAPs possibly explain these antagonistic effects on Rho and inhibition of a Rho effector, the Ser/Thr kinase function: these proteins could inhibit Rho activation by Rock. These mechanisms might be used to fine-tune blocking exchange factors or inhibiting GDI dissocia- Rho GTPase inhibition locally at sites where particu- tion; they could possibly interact with some of the Rho lar actin structures need to be made. effectors, but with opposing effects; or they could stimulate GAPs. Because exchange factors are the best characterized regulators, the favorite model was that Within the past seven years several atypical members of Rnds would inhibit these factors; however our lab has the Rho small G protein family have been discovered, tested this hypothesis without finding such an inhibition including RhoE/Rnd3, Rnd1, Rnd2, RhoD, Rif and (our unpublished data). In fact, very recent findings by RhoH/TTF, to mention just the ones most closely related several labs [3,4] have now shown that Rnds and Gem to Rho. Rnd1, 2 and 3 represent a distinct sub-group use the other two proposed mechanisms to block Rho and, like other members of the Rho family, affect the function — inhibition of the Rho effector kinase Rock actin cytoskeleton, but with opposite effects, causing and stimulation of RhoGAPs. disruption of actomyosin contractile fibers (‘stress fibers’) and focal adhesions, leading to the ‘rounding’ of Regulation of Rock fibroblasts (hence the name Rnd). Rock is one of the most important Rho effectors and is The Rnd proteins have unusual biochemical proper- essential for the formation of contractile actomyosin ties compared with other Rho family GTPases. Canon- fibers. Rock phosphorylates and inhibits myosin light ical members of this family are activated by the loading chain (MLC) phosphatase, and may directly phospho- of GTP, triggered by exchange factors, and are inacti- rylate MLC itself, resulting in a significant increase in vated by GTPase-activating proteins (GAPs), which MLC phosphorylation and incorporation of myosin to stimulate GTP hydrolysis and the return to the inactive cross-link actin filaments and form contractile fibers. GDP-bound state. Most Rho family proteins are Rock has many other substrates, most of which also geranyl-geranylated at their carboxyl terminus, which control the cytoskeleton. The best known activator of promotes their membrane association in the GTP state. Rock is Rho, but Rock can also be activated by other In their resting GDP form, however, they are associated means, for instance the carboxyl terminus of ROCK I is with a guanine dissociation inhibitor (GDI) that masks cleaved by caspase 3 during apoptosis, resulting in the geranyl-geranyl lipid moiety and keeps them in the dramatic changes in membrane–cytoskeleton interac- cytosol. The GDI has to be displaced by membrane- tions, leading to membrane ‘blebbing’ [5,6]. Anne Ridley associated GDI displacement factors, which so far are and colleagues [3] have found that among Rho effec- still poorly characterized. In contrast, Rnd proteins are tors only Rock interacts with RhoE/Rnd3. However, like farnesylated and predominantly associated with mem- many other kinases, Rock seems to be auto-inhibited in branes, suggesting that they are not associated with a a closed conformation, with the binding of Rho to a GDI. Because they have no GTPase activity and a low central Rho-binding domain relieving this auto-inhibi- affinity for GDP, and because there is usually about 10 tion. In such a model it is difficult to imagine how the times more GTP than GDP in cells, it seems likely that binding of a related protein, RhoE, at the same site, pre- Rnd1, Rnd2 and Rnd3/RhoE are always bound to GTP sumably in a very similar way, could have the opposite and do not switch back to a GDP form [1]. Thus, Rnd effect (namely inhibiting Rock). proteins would not be regulated by exchange factors or An important breakthrough came with the discovery GAPs, instead they can be considered as constitutively that in fact RhoE/Rnd3 binds to Rock at a different active, and might be controlled exclusively by the site, close to the kinase domain, inhibiting its activity balance between transcription–translation and degra- [3]. It is therefore much easier to understand how dation. In addition, they could be regulated by post- RhoE/Rnd3 binding can inhibit Rock, whereas RhoA translational modifications such as phosphorylation. binding at a different site activates it. In fact this group Another small G protein named Gem also affects has data suggesting that the activation of Rock by stress fiber formation. Gem belongs to a distinct sub- RhoA and the generation of the ‘open’ conformation is group of the Ras family, which also comprises Kir and required before RhoE/Rnd3 can bind and subse- Rad [2]. In contrast to Rnds, Gem proteins seem to quently inhibit, most likely in a second step [3]. There are two highly related Rocks, ROCK I/ROKβ and Dynamique moléculaire des compartiments cellulaires, Institut ROCK II/ROKα/Rho kinase, with similar structures and de Pharmacologie du CNRS, 660 route des Lucioles, 06560 mechanisms of activation, but probably different Sophia Antipolis, France. E-mail: [email protected] modes of regulation. Ward and colleagues [7] have Current Biology R703

shown that Gem preferentially binds and inhibits β α A pY1105 ROK whereas Rad preferentially binds ROK . The FF ArsB RhoGAP site of interaction is located in the central region of p190 Rock between the amino-terminal kinase domain and the Rho-binding site at the carboxy-terminal end of GBD GBD the central coiled-coil domain (Figure 1). Rnd3-binding domain Those discoveries provide a new level of under- B Kinase α-helical PHRBDCRD standing of small G protein function, showing for the Rock first time that a single protein, Rock, can bind three different small G proteins on different sites. Rock is a Rnd3-binding Gem Rho major Rho effector involved in bundling actin filaments domain into stress fibers. However, Rho has several other Current Biology effectors, such as the Diaphanous-related formins (Dia Figure 1. or DRF proteins), which, when activated, induce the formation of many thin, non-bundled actin filaments. If (A) Schematic representation of p190 RhoGAP conserved domains. A GTP-binding domain (GBD) is found in the amino the only function of RhoE was to inhibit Rock, one terminus and possibly a second one around position 700, with would expect to see other Rho effectors such as DRFs four FF domains in between. These domains are followed by an still active, whereas RhoE/Rnd3 expression results in a ArsB domain, and the RhoGAP domain is at the carboxyl termi- massive loss of F-actin, greater than that seen by inhi- nus. The major Src tyrosine phosphorylation site (Y) is in posi- bition of Rock alone, suggesting that RhoE/Rnd3 has tion 1105 of p190-A. Rnd3/RhoE binds in the central domain additional effects. This finding indicates the complex- between positions 382 and 1007 of p190-B. (B) Schematic representation of Rock, with the kinase domain in the amino termi- ity of the regulation of Rho and its effectors in the mod- nus which also contains the RhoE/Rnd3 binding site, the central ulation of the actin cytoskeleton. α-helical domain which harbors the Gem-binding site and the Rho-binding site (RBD) followed by a PH domain and a cys- Regulation of RhoGAPs teine-rich region (CRD) at the carboxyl terminus. Another addition to the story has now been provided by the group of Steen Hansen, in collaboration with of the RhoGAP activity in fibroblasts [10]. MgcRac- Jeff Settleman and Channing Der. Wennerberg et al. [4] GAP also plays a major role in cytokinesis (as demon- identified p190 RhoGAP as a binding partner for strated in Caenorhabditis elegans by the MgcRacGAP Rnd3/RhoE by yeast two-hybrid analysis — not a com- homologue, CYK-4). In mammalian cells, the RhoGAP plete surprise because mouse RhoE had originally activity of MgcRacGAP is controlled by the cytokine- been isolated in a two-hybrid screen using p190 sis kinase Aurora [11], but it is not clear whether RhoGAP as bait. Similar to the Rock story, the enlight- MgcRacGAP’s role in cytokinesis also involves an enment came when the region of p190 RhoGAP that interaction with Rnds. This is clearly not the case in interacts with RhoE was mapped and was found to lie flies and worms, which have no Rnd ortholog: aside in a central domain of p190 RhoGAP with unknown from mammals (man, mice, pig), an Rnd1 ortholog has function and not in the RhoGAP domain, as first only been found in Xenopus. assumed. Again, it was much easier to imagine how This range of findings showing that Rnd3/RhoE, RhoE/Rnd3 binding upstream of the RhoGAP domain Rnd2, and Gem affect actomyosin fibers by activating could recruit and activate this RhoGAP, and therefore different RhoGAPs (p190, MgcRacGAP and GMIP, induce Rho inactivation. The authors went on to show respectively) highlights the importance of GAPs in the that Rnd3/RhoE expression in fibroblasts deficient in regulation of Rho activity. Little was known about the either p190-A or p190-B induced a much weaker dis- mechanisms that control the ~60 different RhoGAPs ruption of actomyosin contractile fibers than in cells encoded in our genome. The best understood is p190 expressing normal levels of these two isoforms of RhoGAP, which associates with p120RasGAP and is p190. This evidence strongly suggests that p190 is regulated by tyrosine phosphorylation. Until recently, indeed a major effector of Rnd3/RhoE induced stress- however, the large central domain of p190 had no fiber disruption [4]. known function, although, as described above the Interestingly, other groups using different systems binding of Rnd3/RhoE to this region has just been dis- have reached similar conclusions regarding the regu- covered [4]. Local activation of p190 has already been lation of RhoGAPs. De Gunzburg and colleagues [8] suggested in the case of insulin-like growth factor 1, found that Gem interacts and most likely activates a which directs tyrosine phosphorylation of p190-B RhoGAP named GMIP. Another GAP, MgcRacGAP, is RhoGAP and its re-localization to lipid rafts where it mostly expressed in male germ cells (Mgc), sperma- may encounter and inactivate Rho-GTP. In contrast, the tocytes and early spermatids, but Gacon and col- tyrosine phosphatase SHP-2 might dephosphorylate leagues [9] could not detect any expression of Rho, p190-B RhoGAP and lead to its release from lipid rafts, Rac or Cdc42 in these germ cells. In fact they found which would favor a sustained activation of Rho [12]. that Rnd2, which is highly expressed in the testis, The recent discoveries provide us with new and interacts with MgcRacGAP and co-localizes with this important insights into the regulation of GAP activity. It GAP in male germ cells. Rnd2 does not induce the is tempting to speculate that the recruitment of these disruption of actomyosin fibers in Swiss 3T3 cells, GAPs on membrane-associated Rnd3/RhoE or Gem possibly because MgcRacGAP is expressed at much will induce a local inhibition of Rho. Then, how is the lower levels than p190 which accounts for almost half localization of Gem or Rnds controlled? In the case of Dispatch R704

Gem, which also binds ezrin (J. de Gunzburg, personal A cell moves by reptation (crawling), like a slug, and communication), it might be the membrane recruitment the extension at the front has to be tightly coordinated of ezrin that brings associated Gem and Gmip in the temporally, with contraction in the middle and retrac- vicinity of their substrate, whereas in the case of Rnds tion at the back, to make the movement effective. an interaction with cadherins might be involved [1]. Because the requirement for such coordination is quite One can imagine that a cell segregates proteins at obvious, the underlying molecular mechanisms have its membrane to build a ‘mosaic’ with some places attracted little attention, and not much is known on the where Rho activation will occur, and others where Rho spatial and temporal coordination mechanisms. Recent will be inhibited. Large ‘molecular machines’, or protein work suggests that a major Rac effector, Pak, might complexes, are built at these sites. The complexes phosphorylate some Rho exchange factors and inhibit responsible for activation contain at least a transmem- them (A. Alberts and J. Frost, personal communication). brane protein, a GDI displacement factor (possibly In addition to these few known examples, it seems ERM proteins), an exchange factor and frequently likely that localized inhibition might be due to the some pre-recruited effectors (such as Pak), whereas expression and specific targeting of other family the inactivation complexes contain at least a trans- members, such as Rnds or Gem, which could recruit membrane protein, a small G protein (such as Rnd or and activate specific GAPs. Gem), and a GAP. The complexity of these molecular machines emphasizes the precise regulation of small References 1. 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J. 372, 105–112. tion only one type of actin structure is made: for 10. Vincent, S. and Settleman, J. (1999). Inhibition of RhoGAP activity is example, local recruitment and activation of Rac pro- sufficient for the induction of Rho-mediated actin reorganization. motes lamellipodial formation (seen as membrane ruf- Eur. J. Cell Biol. 78, 539–548. 11. Minoshima, Y., Kawashima, T., Hirose, K., Tonozuka, Y., Kawajiri, fling), but it is important too that a stress fiber is not A., Bao, Y.C., Deng, X., Tatsuka, M., Narumiya, S., May, W.S., Jr., et formed at the same place, where it would induce al. (2003). Phosphorylation by aurora B converts MgcRacGAP to a retraction. In filopodia or microspikes generated by RhoGAP during cytokinesis. Dev. Cell 4, 549–560. 12. Sordella, R., Jiang, W., Chen, G.C., Curto, M. and Settleman, J. activated Cdc42, it seems that Rac and Rho activities (2003). 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Cell Biol. 160, vated receptors, together with p190 RhoGAP, which 17–23. could explain Rho inhibition in the context of Rac- mediated ruffling. At present it is not known whether another small G protein, such as Rnd3, also partici- pates in p190 RhoGAP activation in this context. Thus, it seems to be a common theme that in the region where one Rho family member is activated the others are usually inhibited.