Article |
Address correspondence to Kathleen Kelly, Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, Building 10, Room 3B43, Bethesda, MD 20892. Tel.: (301) 435-4651. Fax: (301) 435-4655. E-mail: kkelly{at}helix.nih.gov
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: Gem; Rad; Rho kinase; myosin light chain; neuroblastoma
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although structural features of the RGK family have been known for many years, the physiological role of individual proteins has not been readily forthcoming. Recently, a role for Gem has been proposed in cells expressing voltage-gated calcium channels, such as endocrine and neuronal cells. Gem was reported to down-regulate channel activity as a result of binding the ß subunit and thereby inhibiting expression of the subunit at the plasma membrane (Beguin et al., 2001). However, it is likely that Gem has additional roles. Gem is expressed in cells such as T lymphocytes that do not contain voltage-gated calcium channels, and furthermore, Gem has been indirectly implicated in playing a role in cytoskeletal reorganization. Overexpression of mouse Gem was found to induce invasive pseudohyphal growth in Saccharomyces cerevisiae (Dorin et al., 1995). Although there is no apparent Gem orthologue in yeast, this assay most likely reflects the interaction of Gem with a protein common to yeast and mammalian cells. Recently, immunofluorescence and cell fractionation studies have localized a portion of Gem to microfilaments and microtubules (Piddini et al., 2001). Also, Gem expression stimulates cell flattening and neurite extension in human and mouse neuroblastoma cells (Leone et al., 2001).
Other RGK family members are implicated in cytoskeletal interactions as well. Ges, the likely human orthologue of mouse Rem1, and Rem1 were recently described to induce endothelial cell sprouting (Pan et al., 2000). Rad binds ß-tropomyosin in skeletal muscle and is associated partially with the cytoskeleton in C2C12 cells (Zhu et al., 1996; Bilan et al., 1998).
Rho family members regulate the dynamic organization of cytoskeletal proteins. As described herein, Gem and Rad bind Rho kinase (ROK), an effector of GTP-bound Rho that mediates a large proportion of the signals from Rho, leading to actinomyosin contractility. RhoA-dependent activation of ROK requires binding via the RhoA effector region and an additional activation function requiring the RhoA insert region (Zong et al., 2001). Two isoforms of ROK exist, referred to as either and ß or II and I, respectively, with an overall identity of 64% that is greatest in the kinase domain (90%) and least in the coiled-coil domain (55%) (Leung et al., 1995, 1996; Ishizaki et al., 1996; Nakagawa et al., 1996). Relatively few functional differences between the two isoforms are known presently. Both isoforms are ubiquitously expressed in tissues, although ROK
predominates in adult brain (Ishizaki et al., 1996; Leung et al., 1996; Matsui et al., 1996). In addition, ROKß, but not ROK
, is a substrate for caspase-3 during apoptosis, leading to a constitutively active kinase that participates in bleb formation (Coleman et al., 2001; Sebbagh et al., 2001).
Several substrates for ROK are known. ROK controls actinomyosin filament assembly and myosin contractile activity by inducing the phosphorylation of the regulatory myosin light chain (MLC). Increased MLC phosphorylation results directly from ROK-mediated phosphorylation of MLC and indirectly by the inactivation of myosin phosphatase through ROK-mediated phosphorylation of myosin binding subunit (MBS) (Amano et al., 1996; Kimura et al., 1996). MLC phosphorylation is detected after ROK activation and associated with the formation of stress fibers and focal adhesions (Amano et al., 1997, 1998; Chihara et al., 1997; Ishizaki et al., 1997), smooth muscle contraction (Kureishi et al., 1997), and neurite retraction (Amano et al., 1998; Hirose et al., 1998). Other ROK substrates include members of the ezrin/radixin/moesin family, adducin, LIM kinase (LIMK), Na-H exchanger 1, and intermediate filaments, and the phosphorylation state of these proteins appears to be associated with specific cell functions (for review see Amano et al., 2000).
We show here that Gem binds ROKß and inhibits ROK-mediated MLC phosphorylation. Ectopic Gem or Rad expression inhibits ROK-dependent functions such as formation of stress fibers and focal adhesions, neurite retraction, and Rho-dependent transformation. These data suggest that Gem and Rad perform regulatory functions in cytoskeletal remodeling, perhaps as spatially regulated inhibitors of ROK activity.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
An important question is the specificity of the GemROK interaction. Using the yeast two-hybrid system, the interactions of ROK and ROKß with the different RGK family members (Gem, Rad, Rem1, and Rem2) were determined. Gem binding appeared to be specific for ROKß, because various forms of Gem did not bind fragments spanning the length of ROK
. The length of the Rad construct that was assayed effected the binding specificity of Rad. Nearly full-length Rad (41308) bound the coiled-coil domain of ROK
(4391126) but did not bind various ROKß fragments or other ROK
fragments. By contrast, Rad containing a COOH-terminal deletion (41274) bound ROKß similarly to Gem (Table I). Neither full-length nor COOH-terminal truncated Rem1 or Rem2 bound either ROK isoform, but full-length Rem1 bound 14-3-3ß (unpublished data), as reported previously (Finlin and Andres, 1999).
GemROK interaction in mammalian cells
We also investigated the physical association of Gem and Rad with ROK (Fig. 2) and determined that binding can be observed. As shown in Fig. 2 A, recombinant glutathione-S-transferase (GST)Gem and recombinant GSTRad bound ROKß and to a lesser extent ROK in whole-cell extracts derived from transfected COS7 cells. The presence of GTP-
S did not effect the binding efficiency (unpublished data). In addition, coprecipitation of ROKß with Gem was observed from extracts of either transfected COS7 or N1E-115 cells (Fig. 2 B), consistent with the interaction seen in the yeast two-hybrid analyses.
|
The functional effect observed after transient transfection of Gem or Rad suggested an inhibition of ROK activity (Fig. 3, A and B). That is, Gem or Rad individually stimulated flattening and neurite extension of N1E-115 cells, a phenotype that is observed after transfection of dominant-negative ROK or with the ROK inhibitor Y-27632 (see Fig. 8, A and B) (Hirose et al., 1998). Correlative with ROK binding in the yeast two-hybrid assay, Gem or Rad displayed functional activity but Rem1 and Rem2 did not. In addition, an interference assay was used to investigate the specificity of Gem and Rad for the ROK isoforms. Transfection of either ROK or ROKß into N1E-115 cells caused enhanced cell rounding (Fig. 3, A and B). Previous investigations have shown that transfected ROK is active in the absence of Rho binding (Leung et al., 1996). Cotransfection of Gem opposed the effects of ROKß but not ROK
. Surprisingly, cotransfection of Rad fully reversed the activity of ROK
and only weakly effected ROKß. Therefore, in N1E-115 cells, Rad appears to have functional specificity for full-length ROK
, as compared with ROKß. Western blots were used to verify equivalent expression levels for transfected Gem, ROKß, and ROK
in the various experimental conditions depicted in Fig. 3 (unpublished data). The Gem(S89N) mutant stimulated cell flattening and neurite extension, consistent with its ability to bind ROKß. Additionally, Gem and Rad expression in N1E-115 inhibited lysphosphatidic acidinduced cell rounding (unpublished data).
|
|
An important question is whether the inhibitory effects of Gem and Rad on ROK-mediated functions require the interaction of Gem/Rad with ROK, or, alternatively, are an indirect effect. To address this question, we have assayed the ability of Gem or Rad to interfere with cell rounding initiated by ROK mutants missing Gem/Rad binding domains (Fig. 4 A). Constitutively active ROKß or ROK truncated shortly after the kinase domain robustly stimulated rounding of N1E-115 cells that was unaffected by Gem or Rad expression (Fig. 4 A), suggesting that Gem/Rad binding to ROK is required for inhibition. An additional ROKß mutant, ROKß(
787906) was constructed by deleting the Gem binding domain but leaving other regulatory domains (including the Rho binding domain) intact. This mutant form of ROKß was stably expressed in COS and N1E-115 cells (unpublished data). As shown in Fig. 4 A, ROKß(
787-906) was effective at mediating neurite retraction that could not be reversed by Gem expression, further supporting the conclusion that Gem and ROKß interact directly.
|
We tested the possibility that these Gem and Rad binding domains in the coiled-coil regions of ROKß and ROK could act as dominant negatives for endogenous Gem and Rad. Expression of both protein fragments together in N1E-115 cells stimulated a small amount of cell rounding (Fig. 4 B), suggesting that Gem and Rad, in addition to other endogenous proteins, are responsible for maintaining the flattened morphology of these cells.
Recently, Gem was shown to bind the ß subunit of L-type Ca2+ channels, resulting in reduced channel activity due to decreased 1 subunit expression at the plasma membrane (Beguin et al., 2001). Because N1E-115 cells express L-type channels, we investigated whether inhibition of channel activity using nitrendipine would lead to the morphological alterations induced by Gem. Green fluorescence protein (GFP) or GFPGem-transfected N1E-115 cells were treated with nitrendipine (1, 5, 10, or 50 µM) for 24 h before being scored for neurite extension. Nitrendipine had no effect upon the distribution of morphological phenotypes in either GFP- or GFPGem-transfected cells (unpublished data), suggesting that inhibition of channel activity plays no role in the morphological differentiation described here.
The effect of Gem upon the actin cytoskeleton
ROK has been shown to play a fundamental role in the regulation of the actinomyosin cytoskeleton, including the formation of stress fibers and focal adhesions. Therefore, we analyzed the effect of Gem and Rad expression in epithelial cells and fibroblasts on the cytoskeleton as judged by staining for F-actin and vinculin (Fig. 5). As shown for HeLa cells in Fig. 5, transient Gem or Rad overexpression inhibited the presence of focal adhesions in the main cell body while leaving peripheral focal complexes intact, in agreement with previous reports that ROK activity is required for the maintenance of central but not peripheral focal contacts (Totsukawa et al., 2000). Gem or Rad overexpression in fibroblasts often induced an unusual dendritic morphology (Fig. 5) characterized by abnormal cellular elongation or the presence of branching filopodial structures and rounding or retraction of the cell body. Gem or Rad expression was accompanied by loss of central but not peripheral focal contacts and loss of stress fibers. In addition, enhanced lamellipodia formation was evident in Gem-transfected cells (unpublished data). Interestingly, a dendritic morphology is induced in BALB/c 3T3 cells after prolonged inhibition of RhoA or ROK (Hirose et al., 1998). Low levels of Gem expression generally did not result in loss of focal adhesions or stress fibers or induction of a dendritic morphology, possibly as a result of residual ROK activity.
|
|
Biochemical analyses
An important question is the mechanism of action whereby Gem functionally opposes ROKß. We considered the possibilities that Gem (a) inhibits ROK kinase activity or (b) redirects ROK localization and/or substrate specificity. We have obtained no evidence suggesting a direct effect of Gem upon the kinase activity of ROKß. For example, no change in the level of in vitro kinase activity was observed in immunoprecipitated ROKß relative to the presence or absence of coexpressed Gem (unpublished data). Therefore, in order to test the second possibility, the effect of Gem expression upon the in vivo activity of ROKß was investigated in COS cells for the substrates MLC, MBS, and LIMK. ROK-dependent phosphorylation of MLC and MBS was assayed with phosphospecific antibodies. LIMK phosphorylation was measured indirectly by an immune complex kinase activity assay using cofilin as the substrate. As shown in Fig. 7 A, ROKß stimulated increased phosphorylation of MLC, which was reversed in the presence of coexpressed Gem. ROK-mediated phosphorylation was unaffected by Gem as was phosphorylation mediated by the kinase domain of ROKß in the absence of the Gem binding region. Similarly, ROKß-dependent phosphorylation of MBS was inhibited by Gem coexpression (Fig. 7 B). By contrast, as shown in Fig. 7 C, immunoprecipitated LIMK demonstrated a ROK-dependent increase in cofilin-directed kinase activity, which was essentially unaffected by coexpressed Gem. Therefore, Gem had a selective effect on ROK-mediated phosphorylation, inhibiting MLC and myosin phosphatase phosphorylation, consistent with the opposition by Gem of ROK-activated actinomyosin contractility. These data suggest that Gem most likely differentially modifies the access of ROK to its substrates.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are many parallel functional effects of Gem and Rad expression, such as the stimulation of neurite extension and loss of stress fibers and focal adhesions, that suggest inhibition of endogenous ROK-dependent actinomyosin contractility. Although a COOH-terminal truncated Rad was found to bind ROKß in the yeast two-hybrid assay, interference assays with full-length Rad and ROK or ROKß indicated a specificity of Rad for ROK
in the neurite retraction assay (Fig. 3 B) or in an actin fiber bundling assay in HeLa cells (unpublished data). The interference data suggest that the interaction of Rad with ROK isoforms in mammalian cells may be tightly regulated.
The specificity of Gem for ROKß was observed with regard to binding in the yeast two-hybrid system, biochemical assays, and functional assays. The significance of this specificity is currently unknown. To date, relatively few distinctions between potential ROK and ROKß functions have been described. One exception is that ROKß is sensitive to apoptosis-induced caspase-3 cleavage and subsequent constitutive activation, leading to membrane blebbing (Coleman et al., 2001; Sebbagh et al., 2001). The caspase cleavage site in ROKß is located at positions 11101113, COOH-terminal to the Gem binding region, suggesting that Gem could influence the outcome of caspase-3 regulation of ROKß.
After the introduction of exogenous ROK, Gem inhibits ROK-dependent phosphorylation of MLC and myosin phosphatase in situ, but does not appear to inhibit ROK-dependent activation of LIMK, implying substrate specificity to the inhibitory function of Gem. Such specificity suggests that Gem is not affecting a generalized regulatory function for ROK, such as Rho binding. Thus, the effects of Gem on transfected ROK appear independent of Rho binding. Although it seems unlikely, we have not formally demonstrated that the loss of actinomyosin contractility, which occurs in the presence of Gem after activation of the Rho pathway, could not involve an effect of Gem upon Rho binding to endogenous ROK.
The in vitro kinase activity of immunoprecipitated ROK directed against purified MLC or meromyosin is unaffected by Gem coexpression (unpublished data), suggesting that Gem does not induce an inhibitory covalent modification of subsequently purified ROK. Gem may selectively effect the substrate specificity of ROK as a result of being localized in the cytoskeletal fraction (Piddini et al., 2001), a possibility supported by the association of ROK and myosin phosphatase with isolated stress fibers containing phosphorylation-competent MLC (Kawano et al., 1999; Katoh et al., 2001). Alternatively, Gem binding may obscure a region in ROK that plays a role in substrate-specific binding. Inhibition of ROK activity toward selective substrates by Gem provides a means for fine tuning the response of cytoskeletal components to ROK and by extension, Rho activation.
Although Rad expression mimics the functional effects of Gem with regard to inhibiting actinomyosin contractility, it has not been possible to assign a biochemical mechanism similar to that of Gem. Cotransfection of ROK and MLC with Rad resulted in the rapid turnover of MLC protein (unpublished data), the physiological significance of which merits further investigation.
Ectopic expression of Gem or Rad in fibroblasts or epithelial cells resulted in a loss of stress fibers and focal adhesions but not peripheral focal complexes, consistent with previous reports of the cytoskeletal organization observed after treatment with the ROK inhibitor Y27632 (Rottner et al., 1999). Peripheral focal complex formation has been shown to be regulated by Rac activation (Nobes and Hall, 1995) and dependent upon myosin 2 contractility, whereas focal complex maturation into focal adhesions is ROK dependent (Rottner et al., 1999). MLC phosphorylation at the cell periphery appears to be regulated by MLC kinase (Totsukawa et al., 2000) but not ROK. Thus, it has been suggested that ROK plays an important role in maintaining cytoplasmic or tonic tension within both smooth muscle (Katoh et al., 2001) and nonmuscle cells (Totsukawa et al., 2000). The dendritic morphology of fibroblasts induced by Gem or Rad probably results in part from a loss of cytoplasmic tension and rounding within the cell body while maintaining adhesion along the cell periphery.
We have observed increased lamellipodia formation in Gem-expressing cells, indicating that Rac activity may be increased. A mutual antagonism between Rac and Rho pathways has been previously proposed in studies on the regulation of neurite extension in neuronal cells (Leeuwen et al., 1997; Hirose et al., 1998) and actin filament reorganization in fibroblasts (Moorman et al., 1999). It will be interesting to determine whether the inhibition of ROK by Gem is accompanied by an increase in Rac activity.
What is the expected biological role of Gem and/or Rad with regard to regulating actinomyosin contractility? The actin cytoskeleton is central to such cellular processes as neurite extension, substrate adhesion, motility, secretion, cellular polarization, and cell cleavage (Carpenter, 2000). Dynamic processes such as motility and secretion cycle through periods of assembly and disassembly of the actin cytoskeleton. For example, in some cells, movement and positioning of exocytotic granules requires an intact cytoskeleton, whereas cortical F-actin disassembly appears to be a prerequisite for juxtamembrane apposition of granules and exocytosis (Muallem et al., 1995; Lang et al., 2000).
The data presented here demonstrating that Gem(S89N) is functional in ROK inhibition suggest that the effect of Gem upon ROKß function is regulated by a mechanism other than differential GTP or GDP binding. Gem expression is highly responsive to various signaling pathways (Leone et al., 2001), and both Gem and Rad proteins are potentially regulated by not only GTP binding but phosphorylation (Maguire et al., 1994; Moyers et al., 1998; Finlin and Andres, 1999) and binding to other proteins such as 14-3-3 (Finlin and Andres, 1999) and calmodulin (Fischer et al., 1996; Moyers et al., 1997). In summary, Gem and Rad provide a mechanism for localized signal-responsive regulation of the RhoROK-mediated assembly and contraction of the actin cytoskeleton.
Increased Gem protein levels have been shown to be associated with ganglionic differentiation of neuroblastoma in vivo (Leone et al., 2001), and ectopic Gem expression stimulates neurite extension in vitro (Fig. 3 A and Fig. 7 B), consistent with a potential role for Gem in morphological regulation of neurites/dendrites. Also, recently, binding of the ß subunits of L-, P/Q-, and N-type voltage-gated calcium channels to Gem was shown to inhibit their transport to the plasma membrane (Beguin et al., 2001). Interestingly, Rho and ROK have been reported to control the intracellular localization of the water channel aquaporin-2 via regulation of the F-actin cytoskeleton (Klussmann et al., 2001). Inhibition of Rho or ROK induces translocation of aquaporin-2 to the plasma membrane, a process that is normally stimulated by vasopressin and cAMP production. It will be interesting to determine whether actin filament dynamics and/or regulation of ROK play a role in Gem-regulated transport of the ß subunit.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pEGFPN1-MLC and pCMV-flagMLC constructs were made using T7-7-MLC from Kathy Trybus (University of Vermont, Burlington, VT) as a template. Quik Change site-directed mutagenesis (Stratagene) was used to generate the pEGFPN1-MLC(18D,19D) mutant. PGex2T-Rad was a gift from Ron Kahn (Joslin Diabetes Center, Boston, MA) and pEFBos-Rad was from James Lenhard (Glaxo Wellcome Inc., Research Triangle Park, NC). PMT2T-Rem1 and -Rem2 were generated by PCR cloning using pGexKG-Rem1 and -Rem2 (Douglas Andres, University of Kentucky, Lexington, KY) as templates. pLEGFPN1-M133 was a gift from David Hartshorne (University of Arizona, Phoenix, AZ), and pUCD2-3xHALIMK1 and pQE60Ampr-Hiscofilin were from Kensaku Mizuno (Tohoku University, Sendai, Japan). pCEV-RhoA63L, -RhoA19N, and -Rac17N were obtained from Silvio Gutkind (National Cancer Institute, Bethesda, MD) and CTV-Dbl was from Geoff Clark (National Cancer Institute).
Yeast two-hybrid analysis
All Gal4 DNA binding domain fusions were generated by cloning into pGBT9 (CLONTECH Laboratories, Inc.). S. cerevisiae strain Y190 (obtained from Stephen Elledge, Baylor College of Medicine, Houston, TX) was sequentially transformed with the pGBT9 full-length Gem bait vector and a human Raji cDNA library in the GAL4 activation domain vector pACT1 (CLONTECH Laboratories, Inc.) according to the protocols described for the MATCHMAKER yeast two-hybrid system (CLONTECH Laboratories, Inc.). Transformants were plated on synthetic complete (SC) plates lacking Trp, Leu, and His for 3 d. Colonies were rescreened for expression of the lacZ marker after lifting onto nitrocellulose filters. 5 million colonies were plated, and 3/27 clones that specifically interacted with Gem were identical to ROKß. For further two-hybrid analyses, DNA fragments were inserted into derivatives of pGBT9 and pGAD424. Yeast were cotransformed by pairs of binding and activation domain plasmids, selected on Trp-, Leu-deficient SC plates, and subsequently patched to Trp-, Leu-, His-deficient SC plates containing 2, 25, or 50 mM 3-aminotriazole. Interactions were assessed by growth after 48 h, and ß-galactosidase activity was measured after transfer to nitrocellulose filters.
Immunofluorescent staining and confocal microscopy
Exponentially growing cells were plated on glass coverslips (A. Daigger & Co.) in 24-well cell culture dishes and incubated overnight at 37°C and 5% CO2. The next day, cells on each coverslip were transfected with 0.05 µg pEGFP-N1 and 0.5 µg pEFBos-Rad or 0.5 µg PMT2T-Gem using Lipofectamine Plus (Invitrogen). 24 h later, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed three times with PBS, and permeabilized with 1% Triton X-100 in 0.02% BSAPBS for 2 min at room temperature. Cells were blocked in 20% goat serum containing 2% BSAPBS for 20 min at 37°C. Cells transfected with Gem were incubated for 1 h at room temperature with polyclonal anti-Gem antibody. One half of the coverslips transfected with Gem or Rad were then incubated with monoclonal antivinculin antibody (Sigma-Aldrich) for 1 h at room temperature, rinsed three times with PBS, and incubated at room temperature for 30 min with Texas red-Xconjugated goat antimouse antibody (Molecular Probes). The other half of the coverslips were incubated with rhodaminephalloidin (Molecular Probes) for 30 min at room temperature. All antibodies were diluted in 2% goat serum in 2% BSAPBS. After three more rinses with PBS, coverslips were inverted into 7 µl of mounting medium containing antifade agents (Biomeda Corp.) and were allowed to dry at room temperature in the dark. Stained cells were examined on a ZEISS Axioplan microscope equipped with a 100x/1.4 oil immersion objective. Confocal images were generated using an LSM 510 scanning laser microscope (ZEISS).
N1E-115 neuroblastoma morphology assay
Neurite remodeling was assayed as previously described (Leone et al., 2001). ROK and Gem expression were assayed by Western blots to determine relative levels in samples compared for morphology. Data presented are the average of at least four independent experiments. ROK inhibitor Y-27632 was obtained from Hiroyuki Sueoka (Welfide Corp, Osaka, Japan). Cells were treated with 10 µM of Y-27632 for 30 min to inhibit ROK. Transfections were done using Lipofectamine Plus.
Cosedimentation of recombinant Gem and Rad with ROK
Cos7 cells were plated on 10-cm cell culture dishes (2.0 x 106 cells/plate) and the next day were transfected with pCAG-mycROKß (4 µg) or pEFBos-mycROK (4 µg) using Lipofectamine Plus. Cell lysates were prepared as described below and precleared at 4°C on glutathione-Sepharose 4B (Amersham Pharmacia Biotech) for 30 min. Precleared Cos cell extracts were incubated at 4°C for 1 h with glutathione-Sepharose beads that were prebound to 50 µg GSTGem or Rad and blocked with 500 µl Cos cell lysate from nontransfected cells. The presence of ROK that had formed a physical complex with Rad or Gem on glutathione-Sepharose was revealed by Western blot analysis as outlined below.
Coimmunoprecipitation and Western blot analysis
Cos7 or N1E-115 cells (2.0 x 106 cells/10-cm cell culture plate) were transfected with PMT2T-Gem (2 µg) and pCAG-mycROKß (2 µg) using Lipofectamine Plus. Soluble protein extracts were prepared as previously described (Leone et al., 2001) and precleared on 50 µl of recombinant protein G agarose beads (Invitrogen) for 30 min at 4°C. Gem was immunoprecipitated by incubating 1 ml of precleared lysate (one plate of cells) with 25 µl of packed recombinant protein G agarose beads and 25 µg of anti-Gem monoclonal antibody P7G4 for 2 h at 4°C. Beads were then washed three times in 40 volumes of lysis buffer. ROKß that cosedimented with Gem was visualized by Western blot analysis using anti-myc polyclonal antibody (Upstate Biotechnology) and chemiluminescence (Pierce Chemical Co.).
Soft agar colony forming assay
NIH 3T3 cells were permanently transfected with CTV vector or CTV-hemagglutinin (HA)Dbl and polyclonal populations were selected with hygromycin (300 µg/ml). Selected cells were checked for Dbl expression using Western blot analysis with monoclonal anti-HA antibody (Roche Molecular Biochemicals) and were then transfected with pRCCMV vector, pRCCMV-Gem, or pRCCMV-Gem(S89N). These cells were selected with geneticin (400 µg/ml) and expression of Gem and/or Dbl was visualized by Western blot. Dbl expression was the same in cells with empty pRCCMV vector and those transfected with pRCCMV-Gem or -Gem(S89N). Cells were assayed for their ability to form colonies in soft agar using the method of Cox and Der (1994).
In vitro invasion assay
Invasion capability of cells was determined as described previously (Ward et al., 2001). 10% FBS was used as the chemoattractant.
Phosphorylation of MLC and MLC phosphatase (MBS)
Cos7 cells were cotransfected with 2 µg pCEV-flagMLC and empty vector or 2 µg PMT2T-Gem and/or 1 µg pEFBos-ROK, pCAG-ROKß, or pCAG-ROKß(
4) using Lipofectamine Plus. Transfected cells were TCA precipitated with 5% TCA (2 mM DTT) and MLC was extracted with urea sample buffer (20 mM Tris, 22 mM glycine, 10 mM DTT, 8.3 mM urea, 0.1% bromophenol blue). Extract was filtered through a 0.45-µm centrifugal filter (Millipore), and proteins were resolved on a 15% SDS-polyacrylamide gel. Phosphorylated and total MLC were detected by Western blot analysis using antiphosphoserine 19 MLC polyclonal antibody and antiflag M5 (Sigma-Aldrich) respectively. The effect of Gem on MBS phosphorylation was determined using the same procedure except that cells were transfected with pLEGFPN1-M133 instead of MLC, and pM1333T695 polyclonal and antimyosin phosphatase polyclonal (Berkeley Antibody Company) antibodies were used to detect phosphorylated and total MBS, respectively.
LIMK activity assay
Cos7 cells were cotransfected with 1 µg pUCD2-3xHALIMK1 and empty vector or 3 µg PMT2T-Gem and/or 1 µg pCAG-mycROKß. The effect of Gem on ROK-dependent phosphorylation of LIMK1 was determined using the in vitro kinase assay of Ohashi et al. (2000).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported in part by a grant-in-aid to M. Ito from the Ministry of Education, Science, Technology, Sports, and Culture of Japan and a National Institutes of Health grant (CA42742) to F. Matsumura.
Submitted: 8 November 2001
Revised: 11 March 2002
Accepted: 12 March 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amano, M., M. Ito, K. Kimura, Y. Fukata, K. Chihara, T. Nakano, Y. Matsuura, and K. Kaibuchi. 1996. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 271:2024620249.
Amano, M., K. Chihara, K. Kimura, Y. Fukata, N. Nakamura, Y. Matsuura, and K. Kaibuchi. 1997. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science. 275:13081311.
Amano, M., K. Chihara, N. Nakamura, Y. Fukata, T. Yano, M. Shibata, M. Ikebe, and K. Kaibuchi. 1998. Myosin II activation promotes neurite retraction during the action of Rho and Rho-kinase. Genes Cells. 3:177188.
Beguin, P., K. Nagashima, T. Gonoi, T. Shibasaki, K. Takahashi, Y. Kashima, N. Ozaki, K. Geering, T. Iwanaga, and S. Seino. 2001. Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem. Nature. 411:701706.[CrossRef][Medline]
Carpenter, C.L. 2000. Actin cytoskeleton and cell signaling. Crit. Care Med. 28:N94N99.[CrossRef][Medline]
Chihara, K., M. Amano, N. Nakamura, T. Yano, M. Shibata, T. Tokui, H. Ichikawa, R. Ikebe, M. Ikebe, and K. Kaibuchi. 1997. Cytoskeletal rearrangements and transcriptional activation of c-fos serum response element by Rho-kinase. J. Biol. Chem. 272:2512125127.
Cohen, L., R. Mohr, Y.Y. Chen, M. Huang, R. Kato, D. Dorin, F. Tamanoi, A. Goga, D. Afar, N. Rosenberg, et al. 1994. Transcriptional activation of a ras-like gene (kir) by oncogenic tyrosine kinases. Proc. Natl. Acad. Sci. USA. 91:1244812452.
Cox, A.D., and C.J. Der. 1994. Biological assays for cellular transformation. Methods Enzymol. 238:277294.[Medline]
Feng, J., M. Ito, K. Ichikawa, N. Isaka, M. Nishikawa, D.J. Hartshorne, and T. Nakano. 1999. Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J. Biol. Chem. 274:3738537390.
Finlin, B.S., and D.A. Andres. 1997. Rem is a new member of the Rad- and Gem/Kir Ras-related GTP-binding protein family repressed by lipopolysaccharide stimulation. J. Biol. Chem. 272:2198221988.
Finlin, B.S., H. Shao, K. Kadono-Okuda, N. Guo, and D.A. Andres. 2000. Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases. Biochem J. 347:223231.[CrossRef][Medline]
Fischer, R., Y. Wei, J. Anagli, and M.W. Berchtold. 1996. Calmodulin binds to and inhibits GTP binding of the ras-like GTPase Kir/Gem. J. Biol. Chem. 271:2506725070.
Hirose, M., T. Ishizaki, N. Watanabe, M. Uehata, O. Kranenburg, W.H. Moolenaar, F. Matsumura, M. Maekawa, H. Bito, and S. Narumiya. 1998. Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells. J. Cell Biol. 141:16251636.
Ikebe, M., and D.J. Hartshorne. 1985. Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. J. Biol. Chem. 260:1002710031.
Ishizaki, T., M. Maekawa, K. Fujisawa, K. Okawa, A. Iwamatsu, A. Fujita, N. Watanabe, Y. Saito, A. Kakizuka, N. Morii, and S. Narumiya. 1996. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 15:18851893.[Abstract]
Itoh, K., K. Yoshioka, H. Akedo, M. Uehata, T. Ishizaki, and S. Narumiya. 1999. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat. Med. 5:221225.[CrossRef][Medline]
Katoh, K., Y. Kano, M. Amano, H. Onishi, K. Kaibuchi, and K. Fujiwara. 2001. Rho-kinasemediated contraction of isolated stress fibers. J. Cell Biol. 153:569584.
Kawano, Y., Y. Fukata, N. Oshiro, M. Amano, T. Nakamura, M. Ito, F. Matsumura, M. Inagaki, and K. Kaibuchi. 1999. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J. Cell Biol. 147:10231038.
Kimura, K., M. Ito, M. Amano, K. Chihara, Y. Fukata, M. Nakafuku, B. Yamamori, J. Feng, T. Nakano, K. Okawa, et al. 1996. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science. 273:245248.[Abstract]
Klussmann, E., G. Tamma, D. Lorenz, B. Wiesner, K. Maric, F. Hofmann, K. Aktories, G. Valenti, and W. Rosenthal. 2001. An inhibitory role of Rho in the vasopressin-mediated translocation of aquaporin-2 into cell membranes of renal principal cells. J. Biol. Chem. 276:2045120457.
Kureishi, Y., S. Kobayashi, M. Amano, K. Kimura, H. Kanaide, T. Nakano, K. Kaibuchi, and M. Ito. 1997. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J. Biol. Chem. 272:1225712260.
Lang, T., I. Wacker, I. Wunderlich, A. Rohrbach, G. Giese, T. Soldati, and W. Almers. 2000. Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells. Biophys. J. 78:28632877.
Leeuwen, F.N., H.E. Kain, R.A. Kammen, F. Michiels, O.W. Kranenburg, and J.G. Collard. 1997. The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; opposing roles for the small GTPases Rac and Rho. J. Cell Biol. 139:797807.
Leung, T., E. Manser, L. Tan, and L. Lim. 1995. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J. Biol. Chem. 270:2905129054.
Leung, T., X.Q. Chen, E. Manser, and L. Lim. 1996. The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16:53135327.[Abstract]
Matsui, T., M. Amano, T. Yamamoto, K. Chihara, M. Nakafuku, M. Ito, T. Nakano, K. Okawa, A. Iwamatsu, and K. Kaibuchi. 1996. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J. 15:22082216.[Abstract]
Matsumura, F., S. Ono, Y. Yamakita, G. Totsukawa, and S. Yamashiro. 1998. Specific localization of serine 19phosphorylated myosin II during cell locomotion and mitosis of cultured cells. J. Cell Biol. 140:119129.
Moyers, J.S., P.J. Bilan, J. Zhu, and C.R. Kahn. 1997. Rad and Rad-related GTPases interact with calmodulin and calmodulin-dependent protein kinase II. J. Biol. Chem. 272:1183211839.
Muallem, S., K. Kwiatkowska, X. Xu, and H.L. Yin. 1995. Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J. Cell Biol. 128:589598.[Abstract]
Nobes, C.D., and A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 81:5362.[Medline]
Pan, J.Y., W.E. Fieles, A.M. White, M.M. Egerton, and D.S. Silberstein. 2000. Ges, a human GTPase of the Rad/Gem/Kir family, promotes endothelial cell sprouting and cytoskeleton reorganization. J. Cell Biol. 149:11071116.
Piddini, E., J.A. Schmid, R. de Martin, and C.G. Dotti. 2001. The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9. EMBO J. 20:40764087.
Rottner, K., A. Hall, and J.V. Small. 1999. Interplay between Rac and Rho in the control of substrate contact dynamics. Curr. Biol. 9:640648.[CrossRef][Medline]
Sahai, E., A.S. Alberts, and R. Treisman. 1998. RhoA effector mutants reveal distinct effector pathways for cytoskeletal reorganization, SRF activation and transformation. EMBO J. 17:13501361.
Sebbagh, M., C. Renvoize, J. Hamelin, N. Riche, J. Bertoglio, and J. Breard. 2001. Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat. Cell Biol. 3:346352.[CrossRef][Medline]
Totsukawa, G., Y. Yamakita, S. Yamashiro, D.J. Hartshorne, Y. Sasaki, and F. Matsumura. 2000. Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J. Cell Biol. 150:797806.
Ward, Y., W. Wang, E. Woodhouse, I. Linnoila, L. Liotta, and K. Kelly. 2001. Signal pathways which promote invasion and metastasis: critical and distinct contributions of extracellular signal-regulated kinase and Ral-specific guanine exchange factor pathways. Mol. Cell. Biol. 21:59585969.
Zhu, J., P.J. Bilan, J.S. Moyers, D.A. Antonetti, and C.R. Kahn. 1996. Rad, a novel Ras-related GTPase, interacts with skeletal muscle beta-tropomyosin. J. Biol. Chem. 271:768773.
Zong, H., K. Kaibuchi, and L.A. Quilliam. 2001. The insert region of RhoA is essential for Rho kinase activation and cellular transformation. Mol. Cell. Biol. 21:52875298.