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© The Rockefeller University Press,
0021-9525/1997//1603 $5.00
The Journal of Cell Biology, Volume 137, Number 7,
, 1997 1603-1613
Article |
Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology
The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth.
The mechanisms by which neuronal cells regulate their complex morphology are poorly understood. Extracellular agonists as diverse as growth factors, neurotrophins, matrix components, secreted proteases, and bioactive phospholipids can exert dramatic effects on neural architecture, ranging from stimulation of neurite outgrowth to induction of growth cone collapse and neurite retraction. Studies on neuronal cell lines, including N1E-115, NG108-15, and PC12, have revealed that certain G protein–coupled receptor agonists, notably lysophosphatidic acid (LPA)1, thrombin, and sphingosine-1-phosphate (S1P), trigger rapid growth cone collapse, retraction of developing neurites, and transient rounding of the cell body (Jalink and Moolenaar, 1992; Jalink et al., 1993; Suidan et al., 1992; Tigyi and Miledi, 1992; Postma et al., 1996; see also Gurwitz and Cunningham, 1988). These dramatic shape changes are driven by actomyosin-based contraction of the cortical cytoskeleton, independently of known second messengers, rather than by a loss of adhesion (Jalink et al., 1993, 1994).
The small GTP-binding protein Rho regulates actin filament reorganization in response to extracellular stimuli (Machesky and Hall, 1996; Symons, 1996). In fibroblasts, active Rho-GTP mediates agonist-induced formation of focal adhesions and stress fibers (Ridley and Hall, 1992). In neuronal cells, Rho has been implicated in the control of neurite behavior, a notion largely based on the use of C3 toxin from Clostridium botulinum, which ADP-ribosylates and thereby inactivates Rho. In differentiated N1E-115 and PC12 cells, C3 toxin prevents agonist-induced cytoskeletal contraction and ensuing neurite retraction (Jalink et al., 1994; Tigyi et al., 1996). Furthermore, C3 treatment of undifferentiated N1E-115 or PC12 cells causes cell flattening followed by neurite extension and growth arrest (Nishiki et al., 1990; Jalink et al., 1994). The neurotrophic effects of C3 are not restricted to neuronal cell lines: in chick embryo sympathetic ganglia, C3 acts as effectively as nerve growth factor in promoting neurite outgrowth (Kamata et al., 1994). Together, these results support a model in which basal Rho activity is necessary to maintain cytoskeletal tension and cell shape; increased Rho activity (Rho-GTP accumulation) then drives neurite retraction, whereas Rho inactivation results in loss of contractility and induces neurite outgrowth (Jalink et al., 1994). Yet, direct evidence that Rho controls neurite behavior is still lacking, as studies with activated or dominant-negative Rho mutants have not yet been conducted in neuronal cells.
Little is known about how Rho is activated biochemically, although it is generally assumed that the mechanism is similar to Ras activation and mediated by specific GDP/ GTP exchange factors (Feig, 1994; Quilliam et al., 1995; Machesky and Hall, 1996), among which are the recently identified Lbc, Lfc, and Lsc oncoproteins (Zheng et al., 1995; Glaven et al., 1996). Other proteins involved in the regulation of Rho activity include GDP dissociation inhibitors (GDIs) that keep GDP-bound Rho in a cytoplasmic, inactive form (Hancock and Hall, 1993; Machesky and Hall, 1996). Several downstream targets of Rho have recently been identified (for review see Machesky and Hall, 1996; Ridley, 1996), including a protein kinase (Rho-kinase) that may regulate actomyosin contractility (Kimura et al., 1996).
In this study we set out to explore further the role of Rho in the control of neuronal morphology, using N1E-115 cells as a model. In addition to defining the effect of activated and dominant-negative RhoA on neuronal cell shape, we have used the two-hybrid system to identify RhoA-binding proteins that control neurite behavior. We report the identification of a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), and a 116-kD RhoA-binding protein, p116Rip, which are highly expressed in brain and N1E-115 cells. We show that RhoGEF, like activated RhoA, induces sustained cell rounding and suppresses neurite outgrowth. In contrast, overexpressed p116Rip promotes cell flattening and neurite extension in a similar way to dominant-negative RhoA or C3 toxin. Our results establish RhoGEF and p116Rip as critical determinants of RhoA-mediated neuronal shape changes.
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Materials and Methods |
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Yeast Two-Hybrid Analysis
Yeast strain Y190 (Harper et al., 1993) was transformed with plasmids encoding wild-type or mutant RhoA cDNAs fused to the Gal4 DNA-binding domain, and two-hybrid screens with a day 14.5 CD1 mouse embryo library (Chevray and Natans, 1992) and a mouse brain library (obtained from Dr. L. Van Aelst, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) were performed using the lithium acetate method (Schiestl et al., 1989). Transformants were selected for growth on plates lacking histidine and supplemented with 25 mM 3-aminotriazole. His+ colonies were subsequently tested for β-galactosidase activity as previously described (Durphee et al., 1993). Binding specificity of cDNAs obtained from positive yeast colonies was analyzed by retransformation with the different RhoA mutants and with an irrelevant protein fused with the Gal4 DNA-binding domain.
cDNA Cloning and Analysis
To obtain full-length cDNAs for Rho-interacting protein (RIP) 2 and RIP3, a day 16 mouse embryo library (Novagen, Madison, WI), a mouse brain library (Stratagene, La Jolla, CA), and a mouse lung library (Stratagene) were used initially with probes derived from the clones isolated in the two-hybrid screen. Subsequently, probes derived from these libraries were used until overlapping clones, representing full-length cDNAs, were isolated. Of each cDNA, at least two independent clones of the same region were analyzed to avoid mistakes due to possible cloning artifacts during library construction. Restriction analysis, subcloning, and other standard DNA manipulations and procedures were performed to obtain complete sequence information of the clones. DNA sequencing was done with the T7 sequencing kit (Pharmacia, Uppsala, Sweden); sequences were analyzed using Genetics Computer Group software (Devereux et al., 1984).
Truncated HA-tagged mutant RhoGEF, termed HA-
RhoGEF, was generated by cloning the entire two-hybrid cDNA of RIP2 (see Results) into pMT2SM-HA and subsequently in pcDNA3. Therefore, a BglII–KpnI fragment (bp 2,783–end) of RIP2 was cloned into pMT2SM, resulting in pMT2SM-RIP2a, and an EcoRI (bp 2,160–2,955) fragment was cloned into pMT2SM-HA, resulting in pMT2SM-HA-RIP2b. Subsequently, a BglII fragment from pMT2SM-HA-RIP2b was cloned into pMT2SM-RIP2a, resulting in the final expression construct, pMT2SM-HA-RhoGEF, which encodes the COOH-terminal amino acids starting from amino acid 685. pcDNA3-HA-RhoGEF was generated in two steps, resulting in cloning of the entire HA-RhoGEF as a PstI (filled in with Klenow)–XbaI fragment in pcDNA3 digested with HindIII (filled in with Klenow) and XbaI. Full-length RhoGEF was generated by cloning an EcoRI–SphI fragment (bp 1–1,343), derived from pBSKML4 and an SphI–NotI fragment (1,343– end) from pBSK-ML3 into pcDNA3 digested with EcoRi and NotI. Both pBSK-ML3 and pBSK-ML4 are clones obtained from the mouse cDNA libraries.
We constructed two NH2-terminal deletion mutants of p116Rip, termed I-p116Rip and II-p116Rip, both with an NH2-terminal HA tag. For I-p116Rip, the EcoRI fragment containing almost the entire insert, including the RhoA-binding region, of the two-hybrid clone was cloned into pMT2SMHA, resulting in pMT2SM-HA-Ip116Rip. Subsequently, the entire fragment, including the NH2-terminal HA tag, was subcloned in two steps into the HindIII–EcoRI sites of pcDNA3, resulting in pcDNA3A-Ip116Rip. Plasmid pcDNA3-Ip116Rip was obtained by cloning an EcoRI– HindIII (filled in with Klenow) fragment (bp 1,824–end) from clone RP20 in pcDNA3-Ip116Rip digested with EcoRI and EcoRV. pcDNA3-Ip116Rip encodes amino acids 545–824, and pcDNA3-IIp116Rip encodes amino acids 545–1,024 (end). A full-length p116Rip construct, termed pcDNA3-p116Rip, was generated by cloning the EcoRI–HindIII (filled in with Klenow) from RP20 in pcDNA3 digested with EcoRI and EcoRV, followed by insertion of an EcoRI fragment from ML27 that contains the 5' end. Clones RP20 and ML27 were obtained from the mouse cDNA libraries.
RNA Isolation and Northern Blot Analysis
Total RNA was prepared using TRIzol Reagent (GIBCO BRL, Gaithersburg, MD) according to the instructions of the manufacturer. RNA was separated on a 1.5% agarose-formaldehyde gel and blotted onto Hybond-N membrane (Amersham Intl., Little Chalfont, UK). The membrane was hybridized with 
[32P]dCTP-labeled (Amersham Intl.) probes generated with the Prime-It RmT random primer labeling kit (Stratagene).
Antibodies
A polyclonal anti-RhoGEF serum was made by immunizing rabbits with a synthetic peptide corresponding to amino acids 1,529–1,547 (sequence NKHSRQRSLPAVFSPGSKEV) coupled to a lysine backbone (Posnett et al., 1988). A polyclonal anti-RIP3 serum was raised by immunizing rabbits with a glutathione-S-transferase (GST) fusion protein containing amino acid residues 545–824 of RIP3 (EcoRI fragment derived from two-hybrid screen).
Cos Cell Transfections
Cos-7 cells were transfected using the DEAE-dextran method as described (Zondag et al., 1996). After 48 h, the cells were washed and lysed in SDS sample buffer followed by boiling for 5 min. Samples were analyzed on 10% SDS-PAGE gels followed by immunoblotting. Immunoblots were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 8.8, 150 mM NaCl, 0.05% Tween-20) and incubated with anti-RhoGEF or anti-p116Rip polyclonal antibodies followed by chemiluminescence detection (ECL; Amersham Intl.) using HRP-conjugated secondary antibodies.
Transfection of N1E-115 Cells
N1E-115 cells were transiently transfected with pcDNA3-p116Rip, pcDNA-Rho, pcDNA-GEF, together with pCMVlacZ in a 1:5 ratio, using the calcium phosphate precipitation method as described (Kranenburg et al., 1995). After 14–16 h, the cells were washed and either incubated for 24–36 h in DME containing 10% FCS or in serum-free DME to induce morphological differentiation. Cells were fixed in 3.5% formaldehyde/ PBS and assayed for β-galactosidase activity. For each transfection, the percentage of undifferentiated (rounded without neurites) and neurite-bearing cells (flattened) was calculated from at least 300 positive cells counted. An average percentage was calculated from three dishes per transfection and three independent transfections.
GST-RhoA Fishing Experiments
Purified GST–RhoA (wild-type) fusion protein (45 µg) was incubated with either GDPβS or GTP
S (40 µM) for 30 min at 30°C in 500 µl reaction mixture containing 20 mm Tris/HCl (pH 7.5), 10 mM EDTA, 1 mM DTT, 5 mM MgCl2. N1E-115 cell lysates were prepared in a buffer containing 50 mM Tris, 250 mM NaCl, 1 mM EDTA, 0.5% NP-40, supplemented with protease and phosphatase inhibitors. Preloaded GST–RhoA proteins, or GST alone, were then added to the lysates and incubated for 1 h on ice. Glutathione-Sepharose beads were used to collect the GST fusion proteins. The beads were washed three times in lysis buffer and subjected to 8% SDS-PAGE followed by immunoblot analysis using anti-p116Rip antibody.
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Binding to Wild-Type and Mutant RhoA Proteins
We tested the various RIP clones for their ability to interact with wild-type and activated RhoA. Since the two- hybrid assay detects interactions only when both proteins translocate to the nucleus, we also used mutant versions of RhoA and V14RhoA in which the cysteine at codon 190 in the RhoA CAAX lipid modification motif was replaced by an arginine to prevent membrane localization (Katayama et al., 1991; Adamson et al., 1992a). The results of the two-hybrid screens are shown in Fig. 1. RIP1, encoding the novel RhoGDI, binds strongly to wild-type RhoA, but only poorly to V14RhoA; further details of RIP1 will be reported elsewhere. RIP2, encoding RhoGEF (see below), binds equally well to both wild-type and activated RhoA, suggesting similar affinities for the GDP- and GTP-bound forms of RhoA. In contrast, RIP3 and RIP4 interact only with activated RhoA, at least under two-hybrid conditions, and thus represent potential targets of RhoA-GTP. We note that, in the case of RIP2, RIP3, and RIP4, the interaction with RhoA was critically dependent on the C190R mutation that disrupts the membrane localization signal (Fig. 1). In the absence of this mutation, lacZ staining with RIP2, RIP3, and RIP4 was only observed after very long incubations. In contrast, binding between RhoA and RIP1 (RhoGDI2) apparently requires the CAAX lipid modification motif (Fig. 1), as one would expect for Rho–GDI interactions (Hancock and Hall, 1993).
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Upstream of its conserved DH/PH domain, RhoGEF contains a leucine-rich region and a cysteine-rich zinc finger–like motif, which is also found in the Lfc exchangers (Fig. 3 B); such a motif is thought to be involved in protein–lipid interactions (Ahmed et al., 1991). Downstream of the DH/PH domain, RhoGEF contains a region with weak homology to cytoskeleton-associated proteins such as plectin and myosin heavy chain. This highly charged region is supposed to form an -helical coiled-coil structure mediating protein–protein interactions (Lupas et al., 1991).
Transfection of an expression vector for RhoGEF into COS cells yields a protein with an apparent size of 
200 kD in Western blot (Fig. 3 D, which also shows expression of a truncated RhoGEF protein, RhoGEF). That RhoGEF migrates somewhat slower on SDS-PAGE than predicted is presumably due to the charged coiled-coil region.
RhoGEF, Like V14RhoA, Induces Cytoskeletal Contraction and Inhibits Neurite Outgrowth
To examine the possible role of RhoGEF and RhoA in controlling neurite behavior, we determined their effect on the morphology of N1E-115 cells. These cells acquire a flattened morphology and begin to extend neurites after serum removal. Readdition of serum, LPA (the active ingredient of serum; Moolenaar, 1995), a thrombin receptor agonist peptide, or S1P triggers rapid retraction of developing neurites and transient rounding of the cell body (Jalink and Moolenaar, 1992; Jalink et al., 1993, 1994; Postma et al., 1996). We used a transient transfection protocol, in which transfected N1E-115 cells are identified by blue staining (Kranenburg et al., 1995; see Materials and Methods).
The majority of the V14RhoA-expressing cells are fully rounded and fail to extend neurites upon serum removal, whereas untransfected or control transfected cells remain flattened and undergo morphological differentiation (Fig. 4 A and Table I). Correct protein expression of the transfected cDNAs was verified by immunoblot analysis (Fig. 4 B). Thus, activation of RhoA is sufficient to prevent neurite outgrowth, consistent with active RhoA maintaining the cortical actomyosin system in a fully contracted state (Jalink et al., 1993, 1994). As shown in Fig. 4 A and Table I, the effect of RhoGEF expression on cell morphology is indistinguishable from that observed with V14RhoA. These results strongly suggest that RhoGEF activates RhoA in vivo to promote cytoskeletal contraction and to inhibit neurite outgrowth. Expression of NH2-terminally truncated RhoGEF (Figs. 3 D and 4 B), lacking amino acids 1–684, yielded the same phenotype (Fig. 4 A and Table 1). This implies that the leucine-rich region and the zinc finger motif of RhoGEF are dispensable for its activity.
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125 kD and exactly comigrates with the endogenous protein in N1E-115 cells (Fig. 5 C). As with RhoGEF, the discrepancy between the apparent and predicted size of p116Rip may be due to the charged coiled-coil region.
The Interaction of p116Rip with RhoA in N1E-115 Cells Is Nucleotide Independent
Although RIP3 binds only to activated RhoA under yeast two-hybrid conditions (Fig. 1), it remains to be examined whether the interaction between p116Rip and RhoA is GTP specific under physiological conditions. To this end, N1E-115 cell lysates were incubated with either the GDPβS- or GTPS-bound forms of GST–RhoA, or with GST alone. The fusion proteins were pulled down with glutathione beads and analyzed for interaction with endogenous p116Rip by immunoblotting. As shown in Fig. 5 E, both the GTP- and the GDP-bound forms of RhoA are able to bind p116Rip. Thus, whereas p116Rip binds only to activated RhoA in yeast, the interaction of p116Rip with RhoA is nucleotide independent in N1E-115 cells. One explanation for this discrepancy would be that the p116–RhoA interaction requires an additional protein not present in the yeast system.
Overexpressed p116Rip Induces Cell Flattening and Neurite Outgrowth Similar to Dominant-Negative N19RhoA and C3 Toxin
We next examined the effects of p116Rip on N1E-115 cell morphology. As can be seen from Fig. 6 A and Table I, overexpression of p116Rip (Fig. 6 B) induces cell flattening and neurite outgrowth in the presence of serum, similar to the action of C3 (Jalink et al., 1994). The same morphological effects were observed after transfection of dominant-negative RhoA, N19RhoA (Fig. 6, A and B, and Table I). By analogy to dominant-negative Ras mutants (Feig, 1994; Quilliam et al., 1995), N19RhoA is thought to form stable, inactive complexes with Rho-specific exchange factors, thus titrating out RhoGEF activity. An NH2-terminally truncated form of p116Rip (p116Rip), which lacks amino acids 1–545 encompassing the PH domain and both proline-rich regions, had no detectable effect on N1E-115 cell morphology, even when it was expressed at much higher levels than full-length p116Rip (Fig. 6, A and B, and Table I). This suggests that the PH domain and/or the SH3-binding sites are essential for p116Rip to exert its effect.
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RhoGEF
RhoGEF is a large protein (190 kD), whose catalytic DH/ PH region is 54% identical to those of the Rho-specific exchangers Lbc and Lfc (Whitehead et al., 1995; Glaven et al., 1996). RhoGEF mRNA is abundant in brain and N1E-115 cells, whereas Lbc transcript is prominent in skeletal muscle and hematopoietic cells but not detectable in brain (Tokosz and Williams, 1995). Therefore, Lbc is unlikely to participate in Rho-dependent neuronal morphogenesis. RhoGEF expression is not restricted to neuronal tissue and cell lines but is also found in kidney, lung, and, to a much lesser extent, heart (Fig. 2).
In addition to its catalytic DH/PH region, RhoGEF contains a zinc finger motif, a leucine-rich domain, and a coiled-coil region; the latter two domains may be involved in mediating protein–protein interactions. Experiments with an NH2-terminally truncated version of RhoGEF reveal that the leucine-rich region and the zinc finger motif are dispensable for the catalytic activity of RhoGEF in vivo. We find that RhoGEF binds equally well to wild-type and activated RhoA, at least under two-hybrid conditions. This finding supports a model in which RhoGEF not only stimulates GDP release but also actively participates in promoting GTP accumulation. In contrast, Ras-specific GDP/GTP exhanger factors, such as yeast CDC25 and mammalian SOS, predominantly recognize the inactive, GDP-bound form of their target GTPase (e.g., Munder and Fuerst, 1992; Quilliam et al., 1995). That RhoGEF acts specifically on Rho is further supported by the finding that overexpression of Rac or Cdc42 fails to mimic RhoGEF action in N1E-115 cells: both activated Rac and Cdc42 promote cell flattening instead of cell rounding (van Leeuwen, F., and J. Collard, unpublished observations).
A major challenge for future studies is to delineate how receptor stimulation may couple to the RhoGEF-RhoA pathway. We previously showed that the Src tyrosine kinase is involved in LPA- and thrombin-induced neurite retraction, but cause–effect relationships are not clear (Jalink et al., 1993). We are currently investigating whether RhoGEF may be activated, either directly or indirectly, via a protein tyrosine kinase.
p116Rip
The amino acid sequence of p116Rip shows little homology to known proteins. While the protein specifically interacts with activated RhoA under two-hybrid conditions, our GST fishing experiments indicate that in N1E-115 cells p116Rip interacts with both RhoA–GDP and RhoA–GTP. Although such a nucleotide-independent interaction seems unusual for molecular "switches" like RhoA, there is a precedent for proteins to interact with Rho in a nucleotide-independent manner. For example, both the GDP- and GTP-bound forms of RhoA are capable of binding phosphatidylinositol-5 kinase type 1 (Ren et al., 1996). Furthermore, the interaction between the p140 PRK2 kinase and RhoA is nucleotide independent, at least in vitro (Vincent and Settleman, 1997). Our failure to detect an association between p116Rip and wild-type RhoA under two-hybrid conditions may suggest that this association requires an additional protein (possibly RhoGDI) that is not present in yeast. We are currently investigating this possibility.
Although their primary sequences are highly divergent, p116Rip shares some characteristics with the Rho-binding protein citron (Madaule et al., 1995), particularly a PH domain and a large coiled-coil region. In p116Rip, as in citron, the coiled-coil region largely overlaps with the putative Rho-binding domain. While the biochemical function of p116Rip is as yet unknown, it is likely that p116Rip participates in a multimolecular signaling complex that regulates Rho action. Overexpression of p116Rip stimulates neurite formation and, furthermore, renders the newly formed neurites resistant to receptor stimulation, similar to what is observed with dominant-negative N19RhoA or C3 toxin. This suggests that p116Rip is a negative regulator of RhoA signaling that inhibits, either directly or indirectly, RhoA-stimulated actomyosin contractility. Importantly, overexpression of truncated p116Rip (to higher levels than the full-length protein) has no morphological effects, while p116Rip does bind to activated RhoA (Fig. 6 A and Table I). This rules out the formal possibility that overexpressed p116Rip would inhibit RhoA signaling by sequestering active RhoA–GTP. Studies to elucidate the normal biochemical function and protein–protein interactions of p116Rip are underway.
Concluding Remarks
The present study indicates that RhoA, RhoGEF, and p116Rip are critical determinants of neuronal cell shape. The available evidence supports a model in which certain G protein–coupled receptors stimulate RhoGEF-RhoA "activation" and consequent actomyosin contraction mediated by Rho-kinase (Kimura et al., 1996). Rho inactivation would then abrogate RhoGEF/RhoA/Rho-kinase signaling, thereby inducing cytoskeletal relaxation followed by neurite formation (see also Jalink et al., 1994). This model is probably not unique for neuronal cells, as Rho-stimulated contractility is also observed in serum-deprived fibroblasts (Chrzanowska-Wodnicka and Burridge, 1996), albeit to a much lesser extent than in N1E-115 cells. While our present work focuses on the role RhoA, there can be little doubt that other members of the Rho family, particularly Rac and Cdc42, also play a critical role in several aspects of neuronal morphogenesis, such as growth cone formation and axonal pathfinding; in fact, genetic evidence points to a role for both Rac and Cdc42 in neurite formation during Drosophila embryogenesis (see Mackay et al., 1995, and references therein).
Finally, at the molecular level, a number of key questions are outstanding. What is the nature of the G protein subunit(s) that couple(s) the receptors for LPA, S1P, and thrombin to RhoGEF activation? What are the intermediate biochemical steps? And, what is the normal function of p116Rip in RhoA signaling? Future experiments should provide answers to these questions.
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Acknowledgments |
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This work was supported by the Dutch Cancer Society.
Submitted: 2 December 1996
Revised: 18 April 1997
1. Abbreviations used in this paper: DH, Dbl homologous; GDI, GDP dissociation inhibitor; GST, glutathione-S-transferase; HA, hemagglutinin; LPA, lysophosphatidic acid; PH, pleckstrin homology; RIP, Rho-interacting protein; S1P, sphingosine-1-phosphate; SH3, Src homology 3.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Adamson P, Marshall CJ, Hall A & Tilbrook PA. Post-translational modifications of p21rho proteins, J Biol Chem, 1992a, 267, 20033–20038.
Adamson P, Paterson HF & Hall A. Intracellular localization of the p21rhoproteins, J Cell Biol, 1992b, 119, 617–627.
Ahmed S, Kozma R, Lee J, Monfries C, Harden N & Lim L. The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc, Biochem J, 1991, 280, 233–241.[Medline]
Chevray PM & Natans D. Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of jun, Proc Natl Acad Sci USA, 1992, 89, 5789–5793.
Chrzanowska-Wodnicka M & Burridge K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions, J Cell Biol, 1996, 133, 1403–1415.
Devereux J, Haeberli P & Smithies O. A comprehensive set of sequence analysis programs for the VAX, Nucleic Acids Res, 1984, 12, 387–395.
Durphee T, Becherer K, Chen P-L, Yeh S-H, Yang Y & Kilburn AE. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit, Genes & Dev, 1993, 7, 555–569.
Feig LA. Guanine-nucleotide exchange factors: a family of positive regulators of Ras and related GTPases, Curr Opin Cell Biol, 1994, 6, 204–211.[Medline]
Glaven JA, Whitehead IP, Nomanbhoy T, Kay R & Cerione RA. Lfc and Lsc oncoproteins represent two new guanine nucleotide exchange factors for the Rho GTP-binding protein, J Biol Chem, 1996, 272, 27374–27381.[Medline]
Gurwitz D & Cunningham DD. Thrombin modulates and reverses neuroblastoma neurite outgrowth, Proc Natl Acad Sci USA, 1988, 85, 3440–3444.
Habets G, Scholtes E, Zuydgeest D, van der Kammen RA, Stam J, Berns A & Collard JG. Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins, Cell, 1994, 77, 537–549.[Medline]
Hancock JF & Hall A. A novel role for RhoGDI as an inhibitor of GAP proteins, EMBO (Eur Mol Biol Organ) J, 1993, 12, 1915–1921.[Medline]
Harper JW, Adami GR, Wei N, Keyomarsi K & Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases, Cell, 1993, 75, 805–816.[Medline]
Jalink K & Moolenaar WH. Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers, J Cell Biol, 1992, 118, 411–419.
Jalink K, Eichholtz T, Postma FR, van Corven EJ & Moolenaar WH. Lysophosphatidic acid triggers neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action, Cell Growth Differ, 1993, 4, 247–255.[Abstract]
Jalink K, van Corven EJ, Hengeveld T, Morii N, Narumiya S & Moolenaar WH. Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho, J Cell Biol, 1994, 126, 801–810.
Kamata Y, Nishiki T, Matsumura K, Hiroi T & Kozaki S. Morphological effects, rate of incorporation, and the enzymatic action of Botulinum ADP-ribosyltransferase, known as C3 exoenzyme, on human neuroblastoma GOTO cells, Microbiol Immunol, 1994, 38, 421–428.[Medline]
Katayama M, Kawata M, Yoshida Y, Horichi T, Yamamoto T, Matsura Y & Takai Y. The posttranslationally modified C-terminal structure of rhoAp21, J Biol Chem, 1991, 266, 12639–12645.
Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A & Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase), Science (Wash DC), 1996, 273, 245–248.[Abstract]
Kranenburg O, Scharnhorst V, Van der Eb AJ & Zantema A. Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells, J Cell Biol, 1995, 131, 227–234.
Lupas A, Van Dyke M & Stock J. Predicting coiled coils from protein sequences, Science (Wash DC), 1991, 252, 1162–1164.
Machesky LM & Hall A. Rho: a connection between membrane signalling and the cytoskeleton, Trends Cell Biol, 1996, 6, 304–310.[Medline]
Mackay DJG, Nobes CD & Hall A. The Rho's progress: a potential role during neuritogenesis for the Rho family of GTPases, Trends Neurosci, 1995, 18, 496–501.[Medline]
Madaule P, Furuyashiki T, Reid T, Ishizaki T, Watanabe G, Morii N & Narumiya S. A novel partner for the GTP-bound forms of rho and rac, FEBS Lett, 1995, 377, 243–248.[Medline]
Moolenaar WH. Lysophosphatidic acid, a multifunctional phospholipid messenger, J Biol Chem, 1995, 270, 8944–8951.
Munder T & Fuerst P. The S. cerevisiae CDC25gene product binds specifically to catalytically inactive Ras proteins in vivo, Mol Cell Biol, 1992, 12, 2091–2099.
Nishiki T, Narumiya S, Morii N, Yamamoto M, Fujiwara M, Kamata Y, Sakaguchi G & Kozaki S. ADP-ribosylation of the rho/racproteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC12 cells, Biochem Biophys Res Commun, 1990, 167, 265–272.[Medline]
Paterson HF, Self A, Garrett MD, Just I, Aktories K & Hall A. Microinjection of recombinant p21 rho induces rapid changes in cell morphology, J Cell Biol, 1990, 111, 1001–1007.
Posnett DN, McGrath H & Tam JP. A novel method of producing anti-peptide antibodies, J Biol Chem, 1988, 263, 1719–1725.
Postma FR, Jalink K, Hengeveld T & Moolenaar WH. Sphingosine-1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor, EMBO (Eur Mol Biol Organ) J, 1996, 15, 2388–2395.[Medline]
Quilliam LA, Khosravi-Far R, Huff SY & Der CJ. Guanine nucleotide exchange factors: activators of the Ras superfamily of proteins, Bioessays, 1995, 17, 395–404.[Medline]
Ren X-D, Bokoch GM, Traynor-Kapln A, Jenkins GH, Anderson RA & Schwartz MA. Physical association of the small GTPase Rho with a 68-kda phoshatidylinositol-5 kinase in Swiss 3T3 cells, Mol Biol Cell, 1996, 7, 435–442.[Abstract]
Ridley AJ. Rho: themes and variations, Curr Biol, 1996, 6, 1256–1264.[Medline]
Ridley AJ & Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors, Cell, 1992, 70, 389–399.[Medline]
Schiestl RH & Gietz RD. High efficiency transformation of intact yeast cells using single stranded nucleic acid as a carrier, Nucleic Acids Res, 1989, 18, 1068.
Suidan HS, Stone SR, Hemmings BA & Monard D. Thrombin causes neurite retraction in neuronal cells through activation of cell surface receptors, Neuron, 1992, 8, 363–375.[Medline]
Symons M. Rho family GTPases: the cytoskeleton and beyond, Trends Biochem Sci, 1996, 21, 178–181.[Medline]
Tigyi G & Miledi R. Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochromocytoma cells, J Biol Chem, 1992, 267, 21360–21367.
Tigyi G, Fischer D, Sebok S, Yang C, Dyer DL & Miledi R. Lysophosphatidic acid-induced neurite retraction in PC12 cells: control by phosphoinositide-Ca2+signaling and Rho, J Neurochem, 1996, 66, 537–548.[Medline]
Toksoz D & Williams DA. Novel oncogene lbcdetected by transfection with distinct homology regions to signal transduction products, Oncogene, 1995, 9, 621–628.
Vincent S & Settleman J. The PRK2 kinase is a potential effector target of both Rho and Rac GTPases and regulates actin cytoskeletal organization, Mol Cell Biol, 1997, 17, 2247–2256.[Abstract]
Whitehead I, Kirk H, Tognom C, Trigo-Gonzalez G & Kay R. Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C, J Biol Chem, 1995, 270, 18388–18395.
Zheng Y, Olson MF, Hall A, Cerione RA & Toksoz D. Direct involvement of the small GTP-binding protein Rho in lbconcogene function, J Biol Chem, 1995, 270, 9031–9034.
Zondag GCM, Moolenaar WH & Gebbink MFBG. Lack of association between receptor tyrosine phosphatase RPTPµ and cadherins, J Cell Biol, 1996, 134, 1513–1517.
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