Localization and Activation of Ras-like GTPases
Function and regulation of RhoE K. Riento*†, P. Villalonga*†, R. Garg* and A. Ridley*†1 *Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House Street, London W1W 7BS, U.K., and †Department of Biochemistry and Molecular Biology, University College London, London, U.K.
Abstract The three Rnd proteins, Rnd1, Rnd2 and RhoE/Rnd3, are a subset of Rho family proteins that are unusual in that they bind but do not hydrolyse GTP, and are therefore not regulated by the classical GTP/GDP conformational switch of small GTPases. Increased expression of each Rnd protein induces loss of stress fibres in cultured fibroblasts and epithelial cells, acting antagonistically to RhoA, which stimulates stress fibre formation. RhoE is farnesylated and localizes partly on membranes, including the Golgi and plasma membrane, and in the cytosol. RhoE inhibits RhoA signalling in part by binding to the RhoA-activated serine/threonine kinase ROCK I (Rho-associated kinase I), thereby preventing it from phosphorylating its targets. RhoE activity is itself regulated by phosphorylation by ROCK I on multiple sites. RhoE phosphorylation enhances its stability, leading to an increase in RhoE levels. In addition, phosphorylation reduces its association with membranes and correlates with its ability to induce loss of stress fibres. RhoE also acts independently of ROCK to inhibit cell cycle progression, in part by preventing translation of cyclin D1 , and to inhibit transformation of fibroblasts by oncogenic H-Ras. RhoE is therefore a multifunctional protein whose localization and actions are regulated by phosphorylation.
Introduction The Rho family of proteins is part of the Ras superfamily of GTPases. There are 22 members of the Rho family in humans, of which the best characterized are Rho (A, B and C), Rac (1, 2 and 3) and Cdc42. Paralogues of Rho, Rac and Cdc42 are present in Drosophila melanogaster and Caenorhabditis elegans, but several other members of the family have evolved more recently. In particular, the Rnd subfamily, Rnd1, Rnd2 and RhoE/Rnd3, are present in fish, birds and mammals, but not in invertebrates. RhoE was the first member of the family to be identified, and was isolated through its ability to interact with p190RhoGAP, a GAP (GTPase-activating protein) for RhoA [1]. Unlike RhoA, RhoE does not bind to the GAP domain of p190RhoGAP, but to the central region of the protein [2]. Independently, Rnd1, Rnd2 and RhoE/Rnd3 were cloned by Pierre Chardin and co-workers [3]. Rnd proteins are unusual in that they do not hydrolyse GTP, and RhoE does not detectably bind to GDP [1,3,4]. This is due to changes in key amino acids involved in catalysing GTP hydrolysis [1]. The crystal structure of RhoE has been solved, and overall is similar to RhoA, but there are critical differences that explain why RhoE is not able to hydrolyse GTP and suggest why its affinity for GDP is very low [5,6]. Rho proteins have a variety of localizations within the cell, and this is often regulated by sequences close to their C-termini [7,8]. For example, RhoB is localized on late endosomes and lysosomes, whereas Cdc42 is primarily localized to Key words: cell cycle, RhoE, Rho GTPase, phosphorylation, signal transduction. Abbreviations used: GAP, GTPase-activating protein; MYPT1, regulatory subunit of myosin lightchain phosphatase; ROCK I, Rho-associated kinase I. 1 To whom correspondence should be addressed (email
[email protected]).
the Golgi. Most Rho family proteins are modified by addition of a 20-carbon chain geranylgeranyl group at their C-termini, promoting their interaction with membranes. In contrast, RhoE has been shown to be farnesylated [1], and Rnd1 and Rnd2 C-terminal sequences indicate that they too will be farnesylated. This suggests that Rnd protein interaction with membranes is likely to be different from other family members.
Rnd protein expression pattern and localization RhoE is widely expressed, although its expression levels vary significantly between different cell types [3,4]. RhoE is found in both membrane and cytosolic fractions of cells, and localizes at least partly to the Golgi complex [9] as well as to the plasma membrane [4]. Rnd1 is highly expressed in neurons in the brain [10], although it is also expressed in the liver and in endothelial cells [11], and is up-regulated in myometrial cells of the uterus during pregnancy [12]. In the brain, Rnd1 localizes to a synaptosomal membrane fraction [10]. Rnd2 is highly expressed in spermocytes and spermatids in the testis, and localizes to the Golgi-derived pro-acrosomal vesicle [13]. Overexpressed Rnd2 was reported to localize to early endosomes in HeLa cells [14].
RhoE interacts with and inhibits ROCK I (Rho-associated kinase I) To determine how RhoE inhibits stress fibre formation, we investigated whether it interacts with known RhoA targets that affect actin cytoskeletal organization (Figure 1A). RhoE specifically interacted with ROCK I [9], which is C 2005
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Figure 1 RhoE binds ROCK I
Figure 2 Location of phosphorylation sites on RhoE
(A) Targets for RhoA that are involved in RhoA-induced actin reorganization. (B) Localization of RhoE-binding site on ROCK I.
ROCK I phosphorylates seven Ser/Thr residues in RhoE, located in the Nand C-terminal extensions outside of the core GTP-binding domain.
RhoE regulation by phosphorylation
known to stimulate actin reorganization by phosphorylating a number of actin-associated proteins [15]. Interestingly, RhoE did not interact with ROCK II [16], providing the first indication that these two closely related kinases are not functionally redundant. RhoE also did not bind to any other RhoA targets tested [9]. ROCK I consists of an N-terminal kinase domain, followed by an extended region predicted to form a coiled coil, a Rho-binding domain and a C-terminal PH (pleckstrin homology)/cysteine-rich domain (Figure 1B). The minimal region that binds to RhoE was mapped to the N-terminal 420 amino acids [9]. Deletion of amino acids either from the N-terminus or the C-terminus of the kinase domain (residues 375–420) resulted in loss of RhoE binding. This suggests that RhoE binds to sequences both N-terminal and C-terminal to the kinase domain. Given that RhoE interacts with ROCK I around the kinase domain, we investigated whether it could inhibit ROCK Iinduced responses. In Swiss 3T3 fibroblasts, ROCK I induces an increase in stress fibres, and this response is reduced when RhoE is co-expressed. One of the best-characterized targets for ROCKs is MYPT1 (the regulatory subunit of myosin light-chain phosphatase). Consistent with its ability to bind to the kinase domain of ROCK I, RhoE expression inhibited ROCK I-induced MYPT1 phosphorylation in cells [9]. C 2005
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Since RhoE binds to ROCK I, it was postulated that RhoE could itself be a target for ROCK I. Indeed, RhoE is phosphorylated on seven sites by ROCK I in vitro [16], all of which are located in N-terminal and C-terminal extensions of RhoE, outside of the core GTP-binding domain (Figure 2). One of these, Ser-240, is immediately adjacent to the Cterminal cysteine residue that is modified by farnesylation. Two of the sites, Ser-7 and Ser-11, have been shown to be phosphorylated by ROCK I in vivo. In contrast to RhoE, Rnd1 and Rnd2 do not bind to ROCK I and are very weak substrates for ROCK I in vitro, and there is so far no evidence that they are phosphorylated in vivo. How does phosphorylation affect RhoE activity? First, cell fractionation indicated that RhoE phosphorylated on Ser-11 (pSer11-RhoE) is localized exclusively in the cytosol, whereas unphosphorylated RhoE is present on cell membranes [16]. Immunostaining indicated that pSer11-RhoE is predominantly cytoplasmic and not membrane-associated. This suggests that ROCK I-induced phosphorylation of RhoE reduces its affinity for membranes, possibly because phosphorylation of Ser-240 will affect the insertion of the farnesyl group into membranes. A similar site in RhoA has been reported to be phosphorylated by protein kinase A and to reduce RhoA binding to membranes [17]. In addition to regulating RhoE localization, ROCK I phosphorylation of RhoE increases its stability [16]. In fibroblasts, PDGF (platelet-derived growth factor) and the phorbol ester PMA increase phosphorylation of RhoE on Ser-11 at approx. 10 min after stimulation. Each of these stimuli induces a decrease in stress fibres, coinciding with an increase in RhoE protein levels, consistent with RhoE phosphorylation increasing its stability.
Other mechanisms for regulating RhoE Several bacterial toxins and enzymes covalently modify Rho family members, and either activate or inactivate them [18]. The best known is the Clostridium botulinum exoenzyme C3 transferase, which ADP-ribosylates RhoA, RhoB and RhoC on amino acid Asn-41, thereby inhibiting their activity [19]. C3 transferase does not modify RhoE, but unexpectedly is ADP-ribosylated by a C3 transferase relative from Staphylococcus aureus, C3(Stau), which also modifies RhoA. Whether this alters RhoE localization or activity, or
Localization and Activation of Ras-like GTPases
contributes to cellular responses to C3(Stau), is not known. It will also be interesting to determine whether Rnd1 and Rnd2 are substrates for C3(Stau). RhoE is highly regulated transcriptionally, and several microarray gene expression analyses have picked up RhoE as a transcript altered under various conditions. For example, RhoE/Rnd3 is up-regulating in the myometrium at midpregnancy, and this correlates with inhibition of RhoA/ ROCK-induced contractility [20].
RhoE and cell cycle progression Rnd1, Rnd2 and RhoE have all been implicated in responses involving actin cytoskeletal reorganization. Recently, RhoE was found to have a novel function in regulating cell cycle progression, independent of its ability to inhibit ROCK I [21]. Increased RhoE expression in fibroblasts inhibits cell proliferation, and prevents serum-starved cells from entering the cell cycle in response to growth factor stimulation. RhoE does not prevent many early signalling responses to growth factors, including activation of ERKs (extracellularsignal-regulated kinases) and PKB (protein kinase B)/Akt, RhoA and Rac1. However, it does prevent accumulation of cyclin D1 , which normally occurs between 4 and 6 h after stimulation. Since cyclin D1 is important for cell cycle progression, this could explain the actions of RhoE. Cyclin D1 protein levels are regulated by transcription, translation and degradation [22]. RhoE does not affect cyclin D1 mRNA levels, but predominantly affects translation of this mRNA [21]. Interestingly, cyclin D1 expression is unable to rescue the growth arrest induced by RhoE, suggesting that RhoE may affect the translation of other mRNAs, as well as that of cyclin D1 . However, expression of the viral oncogenes adenoviral E1A and papilloma viral E7 does rescue cells from RhoE-induced growth arrest, indicating that the effects of RhoE are reversible. RhoE levels do not change in response to serum stimulation, but are increased by the DNA-damaging agent cisplatin [21]. Cisplatin also induces cyclin D1 accumulation, and is known to lead to cell cycle arrest. It is therefore possible that RhoE contributes to DNA-damage-mediated effects on cell cycle progression. Consistent with its ability to inhibit cell proliferation, RhoE expression reduces the ability of oncogenic Ras to induce fibroblast transformation, as measured by focus formation. Interestingly, a mutant RhoE that has all seven ROCK I phosphorylation sites changed to alanines and is thus unable to be phosphorylated by ROCK I, is less effective at inhibiting Ras-induced focus formation. It is possible that phosphorylated RhoE is more active in this assay because it is more stable [16].
Conclusions and future directions Studies on RhoE indicate that it has two distinct functions, one in regulating the actin cytoskeleton and the other in cell cycle progression. RhoE appears to modulate the actin cytoskeleton primarily through its effects on ROCK I, and also through its interactions with p190RhoGAP and Socius
[2,23]. So far, a target for RhoE that explains its ability to inhibit translation of cyclin D1 mRNA has not been identified, and this will be key to understanding how it acts at a molecular level. Analysis of RhoE effects on the translation of other mRNAs associated with cell growth, such as those for ribosomal proteins, may provide further insight into how it affects cell cycle progression. It will also be interesting to determine whether Rnd1 and Rnd2 affect the cell cycle in a similar way to RhoE. RhoE is regulated at multiple levels, from transcription to protein stability and localization. Central to its actions is its ability to be phosphorylated by ROCK I. Rnd1 and Rnd2 also appear to be regulated transcriptionally, but whether they too are regulated by phosphorylation remains to be determined. So far, no kinases have been identified as partners for Rnd1 and Rnd2, and the ROCK I phosphorylation sites in RhoE are not conserved in Rnd1 or Rnd2. Finally, it will be interesting in the future to determine how phosphorylation of RhoE regulates its stability and association with membranes.
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