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Translocation of Na+,K+-ATPase is induced by Rho small GTPase in renal epithelial cells
BBRC Biochemical and Biophysical Research Communications 297 (2002) 1231–1237 www.academicpress.com
Translocation of Na+,K+-ATPase is induced by Rho small GTPase in renal epithelial cellsq Akio Maeda, Mutsuki Amano, Yuko Fukata, and Kozo Kaibuchi* Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine, 65 Tsurumai, Showa, Nagoya, Aichi 466-8550, Japan Received 6 September 2002
Abstract The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Naþ ,Kþ -ATPase, one of the transmembrane proteins. In polarized renal epithelium, Naþ ,Kþ -ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoAVal14 ) into cultured renal epithelial cells, Naþ ,Kþ -ATPase was translocated to the spikelike protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoAVal14 was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Naþ ,Kþ -ATPase. We also found that Naþ ,Kþ -ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoAVal14 . Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: RhoA; Naþ ,Kþ -ATPase; Rho-kinase; ERM proteins
Transmembrane proteins, such as receptors, ion channels, ion pumps, and adhesion molecules, function as signal mediators between the inside and outside of cells. In polarized epithelial cells, the plasma membrane is functionally and morphologically divided into the apical and basolateral membranes by a tight junction. Water and electric gradients across the epithelial cell are essential to maintain homeostasis. In renal epithelium, sodium reabsorption from the apical membrane is regulated by a gradient caused mainly by Naþ ,Kþ -ATPase (NKA) in the basolateral membrane [1]. Many biologi-
q Abbreviations: C3, Botulinum C3 ADP-ribosyltransferase; GST, glutathione S-transferase; MBP, maltose-binding protein; RB/PH (TT), Rho-binding and Pleckstrin-homology domains of Rho-kinase in which 1036th asparagine and 1037th lysine are substituted to threonine; CAT, the catalytic domain of Rho-kinase; Y-27632, (R)(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; ERM proteins, ezrin, radixin, and moesin. * Corresponding author. Fax: +81-52-744-2083. E-mail address: [email protected] (K. Kaibuchi).
cal hormones are involved in the regulation of NKA. Among them, dopamine inhibits the NKA activity by causing endocytosis via its phosphorylation by protein kinase C [2,3]. Thus, the distribution of NKA is thought to be crucial for its function. Members of the Rho family GTPases have been implicated in the regulation of cytoskeletons [4–6]. They have both GTP-bound active and GDP-bound inactive forms. Rho plays pivotal roles in the regulation of various cellular functions such as stress fiber formation [7], smooth muscle contraction [8], cytokinesis [9], neurite retraction [10], microvilli-like structure formation [11], and endocytosis [12]. The downstream effectors of Rho are thought to cause these events. Among them, Rhokinase/ROCK/ROK plays pivotal roles in the regulation of cellular functions by phosphorylation of its effector proteins [13,14]. In this study, we investigated the role of Rho family GTPases in the regulation of NKA distribution and found that the activity of Rho affects the distribution of NKA.
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Materials and methods Materials and chemicals. NRK-52E cells were purchased from ATCC (American Type Culture Collection, Manassas, VA). The expression plasmid of C3 was provided from Dr. A. Hall (University College London, London, UK). GST-RhoAVal14 , GST-Rac1Val12 , GST-Cdc42Val12 , and GST-C3 were produced and purified from Escherichia coli. For microinjection, GST-tagged proteins were cleaved by thrombin, purified to remove the GST, and concentrated [15]. MBP and MBP-RB/PH (TT) were also produced and purified from E. coli. GST-Rho-kinase-CAT was produced by baculovirus-infected Spodoptera frugiperda cells and purified [16]. Anti-NKA and anti-E-cadherin monoclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY) and BD Biosciences (San Jose, CA), respectively. A polyclonal antibody against moesin phosphorylated at 558th threonine (anti-pT558), which also recognizes other ERM proteins phosphorylated at 567th threonine (ezrin) and at 564th threonine (radixin), was raised [17]. Y-27632 was provided by Mitsubishi Pharma. Co. (Osaka, Japan). Other materials were obtained from commercial sources. Cell culture, microinjection, and immunostaining. NRK-52E cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma Chemical Co., St. Louis, MO) with 5% calf serum and 1% nonessential amino acids. NRK-52E cells were seeded at 5 103 cells onto 13-mm poly-D lysine-coated coverslips and cultured for 3 days. Recombinant GTPases were microinjected at 0.5 mg/ml with a marker protein (rabbit IgG at 1.5 mg/ml) into the cytoplasm of cells. MBP or MBP-RB/PH (TT) was injected at 5.0 mg/ml in combination with RhoAVal14 . Injected cells were cultured at 37°C for 2 h. For treatment with Y-27632, cells were pretreated with 10 lM Y-27632 for 30 min before the microinjection, and then incubated in the presence of the compound. For immunostaining of NKA, cells were fixed with ice-cold methanol on ice for 10 min and permeabilized with 0.3% Triton X-100. The cells were incubated with anti-NKA antibody for 12 h at 4°C and then with Cy3conjugated anti-mouse Ig antibody for 1 h at room temperature. For immunostaining of E-cadherin, cells were fixed in 3.7% formaldehyde for 10 min and permeabilized with 0.2% Triton X-100 for 10 min. The other procedures were the same as those for NKA. The injected cells were identified with immunostaining with flourescein isothiocyanate (FITC)-conjugated anti-rabbit mouse Ig antibody or by marking the locations on the coverslips with a grid. The observation was performed with a laser scanning confocal microscope LSM510 (Carl Zeiss, Oberkochen, Germany).
Results Constitutively active mutant of RhoA induces translocation of NKA To determine if Rho influences the distribution of NKA, microinjection of the constitutively active mutant of RhoA (RhoAVal14 ), which is supposed to stay in GTPbound active form in cells [18], in combination with rabbit IgG as a marker into NRK-52E cells was performed. In control cells, NKA was localized mainly at cell–cell contact sites (Fig. 1A). By microinjection of RhoAVal14 into NKR-52E cell, NKA was translocated to the fine spike-like protrusions over the cell surfaces (Fig. 1B), which may be consistent with the structures observed in Swiss 3T3 cells [19]. The formation of stress fibers in cells microinjected with RhoAVal14 was confirmed by staining of F-actin with phalloidin (data not shown). On the other hand, in cells microinjected with
constitutively active mutants of other GTPases of Rho family, Rac1Val12 and Cdc42Val12 , NKA was mainly localized at cell–cell contact sites (Figs. 1C and D). Compared with cells microinjected with buffer only, Rac1Val12 and Cdc42Val12 did not cause significant changes in the distribution of NKA. C3 is known to specifically ADP-ribosylate Rho and inactivate it [20]. We examined the distribution of NKA in NRK-52E cells microinjected with C3 and RhoAVal14 . Coinjection of C3 with RhoAVal14 partially inhibited the accumulation of NKA at the cell surface, compared with RhoAVal14 alone (data not shown). E-cadherin is also localized at the basolateral membrane and is a calcium-dependent cell– cell adhesion molecule that forms a complex with catenins [21]. Microinjection of RhoAVal14 did not show significant changes in the distribution of E-cadherin (Fig. 1F), compared with that of buffer alone (Fig. 1E). This is consistent with the observation of MDCK cells stably expressing constitutively active RhoA [22]. Z-axis scanning revealed that NKA was localized in spike-like protrusions at the apical membrane (Fig. 2A) in the RhoAVal14 -injected cells. In the basal membrane, NKA was detected aroud cell periphery (Fig. 2B). The vertical image confirmed that NKA was mainly localized in spike-like protrusions over the apical surfaces, which seemed to be microvilli-like structures (Fig. 2C). Involvement of Rho-kinase in the translocation of NKA induced by RhoA We next examined the involvement of Rho-kinase/ ROCK/ROK, the effector of Rho, in the translocation of NKA induced by RhoAVal14 . RB/PH (TT) is the carboxyl terminal portion of Rho-kinase and it serves as a dominant negative mutant of Rho-kinase [23]. Coinjection of MBP with RhoAVal14 induced the translocation of NKA in about 60% of total injected cells (Figs. 3A and E), whereas that of MBP-RB/PH (TT) effectively reduced the ratio of the translocation to about 30% of cells (Figs. 3B and E). Y-27632 is a compound that specifically inhibits the activity of Rho-kinase [24]. When the cells were pretreated with 10 lM Y-27632 for 30 min and incubated in the presence of the compound after injection, the RhoAVal14 -induced translocation of NKA was inhibited (Figs. 3C and E). These results indicate the involvement of Rho-kinase as a downstream molecule of RhoA in the translocation of NKA. We next examined the effect of GST-Rho-kinase-CAT (CAT), a constitutively active mutant of Rho-kinase [25]. When purified CAT was microinjected into NRK-52E cells at a concentration up to 10 mg/ml, the typical translocation of NKA was not induced. NKA seemed to be aggregated in large dot-like structures (Fig. 3D) in which actin filaments accumulated. The nature of the large dot-like structures was unclear. By the staining of F-actin, stress fiber formation in cells microinjected with CAT was confirmed.
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Fig. 1. The immunostaining images of NKA in cells microinjected with buffer (A), or constitutively active mutants of Rho family GTPases at 0.5 mg/ ml, RhoAVal14 (B), Rac1Val12 (C), and Cdc42Val12 (D). The localization of NKA in cells microinjected with buffer was not changed from that of noninjected cells (A). NKA was localized in spike-like protrusions in the cells microinjected with RhoAVal14 (B). In cells microinjected with Rac1Val12 (C) and Cdc42Val12 (D), NKA was mainly localized at cell–cell contact sites. The distribution of E-cadherin in cells injected with RhoAVal14 (F) was not changed from that in cells injected with buffer (E). The arrows indicate microinjected cells. The arrowheads denote the localization of E-cadherin at cell–cell contact sites. Bar, 10 lm.
Fig. 2. Z-axis scanning of NRK-52E cells microinjected with RhoAVal14 . All the cells displayed here were microinjected with RhoAVal14 at 0.5 mg/ml. NKA was localized in spike-like protrusions at the apical membrane (A). In the basal membrane, NKA was detected aroud cell periphery (B). The vertical image confirmed that NKA was mainly localized in spike-like protrusions over the apical surfaces, which seemed to be microvilli-like structures (C). Bar, 10 lm.
NKA is localized at microvilli-like structures in cells injected with RhoAVal14 The spike-like protrusions in which NKA was localized in cells microinjected with RhoAVal14 seemed to be
microvilli-like structures. Our previous study indicated that the Rho/Rho-kinase pathway is involved in the formation of microvilli-like structures through phosphorylation of moesin at 558th threonine [17,26]. We next performed double immunostaining of NRK-52E
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at microvilli-like structures in cells microinjected with RhoAVal14 (Fig. 4E). The merged image revealed that NKA and phosphorylated ERM proteins seemed to be colocalized at microvilli-like structures (Fig. 4F). The pretreatment of RhoAVal14 -microinjected cells with Y27632 canceled the accumulation of phosphorylated ERM proteins in microvill-like structures (Fig. 4H). In the cells pretreated with Y-27632, the colocalization of NKA and phosphorylated ERM proteins was weakly observed at cell–cell contact sites but not at microvillilike structures (Fig. 4I).
Discussion
Fig. 3. Involvement of Rho-kinase in the RhoAVal14 -induced translocation of NKA. Coinjection of MBP (5.0 mg/ml) with RhoAVal14 (A) had no effects on the translocation of NKA induced by RhoAVal14 alone. Coinjection of MBP-RB/PH (TT) (5.0 mg/ml) with RhoAVal14 suppressed the translocation of NKA (B). RhoAVal14 -injected cells pretreated with and incubated with 10 lM Y-27632 (C) did not exhibit typical changes of NKA distribution. Microinjection of CAT (D) could not induce the translocation of NKA observed in cells microinjected with RhoAVal14 . The arrows and arrowhead indicate microinjected cells and large dot-like structure, respectively. Bar, 10 lm. (E) The ratio of the cells displaying NKA translocation into microvilli-like structures. Bars represent means SEM for at least three indepedent experiments.
cells with anti-NKA and anti-phosphorylated ERM antibodies. In control cells, phosphorylated ERM proteins were localized at cell–cell contact sites and diffusely in the cytoplasm (Fig. 4B). The apparent colocalization of NKA and phosphorylated ERM proteins was partly observed at cell–cell contact sites in the merged image (Fig. 4C). Phosphorylated ERM proteins accumulated
The restricted distribution of NKA in the basolateral membrane is important for its function in sodium homeostasis. In this paper, we found that RhoAVal14 induced translocation of NKA into microvilli-like structures. Neither Rac1Val12 nor Cdc42Val12 induced this translocation. The inhibition of Rho-kinase canceled the effects of RhoAVal14 . These results indicate that Rho pathway is supposed to participate in the regulation of sodium and water homeostasis through the translocation of NKA. In the microvilli-like structures, NKA was colocalized with phosphorylated ERM proteins. In this study, because of technical restriction by the microinjection we did not observe the ratio of phosphorylation in total ERM proteins by the biochemical methods. Previous data demonstrated that RhoAVal14 induced phosphorylation of ERM proteins in COS7 cells [17] or NIH3T3 cells [11]. We speculate that ERM proteins should be phosphorylated through Rho pathway rather than phosphorylated ERM proteins were relocated to the microvilli-like structures. Although the precise interaction between the translocation of NKA and the phosphorylation of ERM proteins remains to be clarified, phosphorylation of ERM proteins might be involved in this translocation. The following possibilities may account for this translocation. (i) ERM proteins recruit NKA to microvilli-like structures in an activated Rho-dependent manner. In gastric parietal cells, proton secretion is mediated by the translocation of Hþ ; Kþ -ATPase (HKA) to apical membrane in microvilli [27]. Ezrin is one of the candidates to connect HKA with the actin cytoskeleton [28]. HKA was co-precipitated with ezrin by anti-ezrin antibody from the lysate of gastric parietal cells [29]. Because both HKA and NKA belong to Ptype ATPase and share a 65% identity in their amino acid sequences, it is possible that NKA also interacts with ERM proteins. Our previous data indicate that phosphorylation of moesin by Rho-kinase is crucial for the formation of microvilli-like structures in the signaling pathway from RhoA [17,26]. The role of phosphorylation of ERM proteins in the translocation of
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Fig. 4. Colocalization of NKA with phosphorylated ERM proteins. In cells injected with buffer, NKA was localized at cell–cell contact sites and cell periphery (A). Phosphorylated ERM proteins were detected at cell–cell contact sites and diffusely in the cytoplasm (B). In the merged image (C), the apparent colocalization of NKA and phosphorylated ERM proteins was partly observed at cell–cell contact sites. Both NKA (D) and phosporylated ERM proteins (E) were localized at microvilli-like structures in RhoAVal14 -microinjected cells. The colocalization of NKA and phosphorylated ERM proteins in microvilli-like structures is observed in the merged image (F). The pretreatment of RhoAVal14 -microinjected cells with Y-27632 canceled accumulation of phosphorylated ERM proteins in microvilli-like structures (H). In the cells pretreated with Y-27632, colocalization of NKA and phosphorylated ERM proteins was weakly observed at cell–cell contact sites (I). The arrows indicate microinjected cells. The arrowheads denote the colocalization of NKA and phosphorylated ERM proteins at cell–cell contact sites. Bar, 10 lm.
NKA remains to be investigated. Among ERM proteins, ezrin and moesin were equally detected in the lysate of NRK-52E cells by immunoblotting with antiERM antibody (date not shown). Perturbation of moesin by antisense phosphorothioate oligonucleotide affected the formation of microvilli in thymoma cells,
whereas that of ezrin or radixin did not [30]. Taken together these results, it is plausible that among ERM proteins moesin should be involved in the translocation of NKA into microvilli-like structures in NRK-52E cells. (ii) Activated RhoA relocates NKA through reorganization of cytoskeletons. It has been shown that
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NKA is delivered to both apical and basolateral membranes equally and is stabilized at the basolateral membrane by the membrane cytoskeleton [31]. NKA is known to interact with ankyrin and spectrin, which are members of the cortical actin meshwork. RhoAVal14 might stabilize the cortical actin cytoskeleton both at the apical and basolateral sites and hence enhance the interaction of NKA with these binding partners. To address the downstream effectors participating in the translocation, we examined the involvement of Rho-kinase by a Rho-kinase inhibitor or the dominant negative mutant of Rho-kinase. The translocation of NKA by RhoAVal14 was canceled by the inhibition of Rho-kinase. On the other hand, microinjection of the constitutively active mutant of Rho-kinase did not induce the translocation. These results indicate that Rho-kinase is considered to be necessary but not sufficient for the translocation of NKA. Effectors of RhoA other than Rho-kinase may be involved. (iii) Activated RhoA alters NKA localization by modulating membrane trafficking. Introduction of the constitutively active mutant of RhoA inhibits transferrin internalization in HeLa cells [12]. In polarized MDCK cells, RhoAVal14 inhibits recycling of transferrin receptors at the basolateral membrane [32]. It has been demonstrated that newly synthesized NKA is delivered both to apical and basolateral membranes. The inhibition of endocytosis of delivered NKA from the apical membrane by RhoAVal14 might be the cause of this translocation. The precise mechanism of the translocation of NKA by Rho is still to be clarified, but these findings will provide insights into the function of Rho in the regulation of vectorial water and sodium transport.
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Acknowledgments We thank Dr. A. Hall (University College London, UK) for providing pGEX-C3 and Dr. T. Urushidani (University of Tokyo, Japan) for helpful discussion. We are also grateful to Mrs. T. Ishii for secretarial assistance. This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Society for the Promotion of Science Research for the Future Science.
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Translocation of Na+,K+-ATPase is induced by Rho small GTPase in renal epithelial cellsq Akio Maeda, Mutsuki Amano, Yuko Fukata, and Kozo Kaibuchi* Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine, 65 Tsurumai, Showa, Nagoya, Aichi 466-8550, Japan Received 6 September 2002
Abstract The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Naþ ,Kþ -ATPase, one of the transmembrane proteins. In polarized renal epithelium, Naþ ,Kþ -ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoAVal14 ) into cultured renal epithelial cells, Naþ ,Kþ -ATPase was translocated to the spikelike protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoAVal14 was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Naþ ,Kþ -ATPase. We also found that Naþ ,Kþ -ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoAVal14 . Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: RhoA; Naþ ,Kþ -ATPase; Rho-kinase; ERM proteins
Transmembrane proteins, such as receptors, ion channels, ion pumps, and adhesion molecules, function as signal mediators between the inside and outside of cells. In polarized epithelial cells, the plasma membrane is functionally and morphologically divided into the apical and basolateral membranes by a tight junction. Water and electric gradients across the epithelial cell are essential to maintain homeostasis. In renal epithelium, sodium reabsorption from the apical membrane is regulated by a gradient caused mainly by Naþ ,Kþ -ATPase (NKA) in the basolateral membrane [1]. Many biologi-
q Abbreviations: C3, Botulinum C3 ADP-ribosyltransferase; GST, glutathione S-transferase; MBP, maltose-binding protein; RB/PH (TT), Rho-binding and Pleckstrin-homology domains of Rho-kinase in which 1036th asparagine and 1037th lysine are substituted to threonine; CAT, the catalytic domain of Rho-kinase; Y-27632, (R)(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; ERM proteins, ezrin, radixin, and moesin. * Corresponding author. Fax: +81-52-744-2083. E-mail address: [email protected] (K. Kaibuchi).
cal hormones are involved in the regulation of NKA. Among them, dopamine inhibits the NKA activity by causing endocytosis via its phosphorylation by protein kinase C [2,3]. Thus, the distribution of NKA is thought to be crucial for its function. Members of the Rho family GTPases have been implicated in the regulation of cytoskeletons [4–6]. They have both GTP-bound active and GDP-bound inactive forms. Rho plays pivotal roles in the regulation of various cellular functions such as stress fiber formation [7], smooth muscle contraction [8], cytokinesis [9], neurite retraction [10], microvilli-like structure formation [11], and endocytosis [12]. The downstream effectors of Rho are thought to cause these events. Among them, Rhokinase/ROCK/ROK plays pivotal roles in the regulation of cellular functions by phosphorylation of its effector proteins [13,14]. In this study, we investigated the role of Rho family GTPases in the regulation of NKA distribution and found that the activity of Rho affects the distribution of NKA.
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Materials and methods Materials and chemicals. NRK-52E cells were purchased from ATCC (American Type Culture Collection, Manassas, VA). The expression plasmid of C3 was provided from Dr. A. Hall (University College London, London, UK). GST-RhoAVal14 , GST-Rac1Val12 , GST-Cdc42Val12 , and GST-C3 were produced and purified from Escherichia coli. For microinjection, GST-tagged proteins were cleaved by thrombin, purified to remove the GST, and concentrated [15]. MBP and MBP-RB/PH (TT) were also produced and purified from E. coli. GST-Rho-kinase-CAT was produced by baculovirus-infected Spodoptera frugiperda cells and purified [16]. Anti-NKA and anti-E-cadherin monoclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY) and BD Biosciences (San Jose, CA), respectively. A polyclonal antibody against moesin phosphorylated at 558th threonine (anti-pT558), which also recognizes other ERM proteins phosphorylated at 567th threonine (ezrin) and at 564th threonine (radixin), was raised [17]. Y-27632 was provided by Mitsubishi Pharma. Co. (Osaka, Japan). Other materials were obtained from commercial sources. Cell culture, microinjection, and immunostaining. NRK-52E cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma Chemical Co., St. Louis, MO) with 5% calf serum and 1% nonessential amino acids. NRK-52E cells were seeded at 5 103 cells onto 13-mm poly-D lysine-coated coverslips and cultured for 3 days. Recombinant GTPases were microinjected at 0.5 mg/ml with a marker protein (rabbit IgG at 1.5 mg/ml) into the cytoplasm of cells. MBP or MBP-RB/PH (TT) was injected at 5.0 mg/ml in combination with RhoAVal14 . Injected cells were cultured at 37°C for 2 h. For treatment with Y-27632, cells were pretreated with 10 lM Y-27632 for 30 min before the microinjection, and then incubated in the presence of the compound. For immunostaining of NKA, cells were fixed with ice-cold methanol on ice for 10 min and permeabilized with 0.3% Triton X-100. The cells were incubated with anti-NKA antibody for 12 h at 4°C and then with Cy3conjugated anti-mouse Ig antibody for 1 h at room temperature. For immunostaining of E-cadherin, cells were fixed in 3.7% formaldehyde for 10 min and permeabilized with 0.2% Triton X-100 for 10 min. The other procedures were the same as those for NKA. The injected cells were identified with immunostaining with flourescein isothiocyanate (FITC)-conjugated anti-rabbit mouse Ig antibody or by marking the locations on the coverslips with a grid. The observation was performed with a laser scanning confocal microscope LSM510 (Carl Zeiss, Oberkochen, Germany).
Results Constitutively active mutant of RhoA induces translocation of NKA To determine if Rho influences the distribution of NKA, microinjection of the constitutively active mutant of RhoA (RhoAVal14 ), which is supposed to stay in GTPbound active form in cells [18], in combination with rabbit IgG as a marker into NRK-52E cells was performed. In control cells, NKA was localized mainly at cell–cell contact sites (Fig. 1A). By microinjection of RhoAVal14 into NKR-52E cell, NKA was translocated to the fine spike-like protrusions over the cell surfaces (Fig. 1B), which may be consistent with the structures observed in Swiss 3T3 cells [19]. The formation of stress fibers in cells microinjected with RhoAVal14 was confirmed by staining of F-actin with phalloidin (data not shown). On the other hand, in cells microinjected with
constitutively active mutants of other GTPases of Rho family, Rac1Val12 and Cdc42Val12 , NKA was mainly localized at cell–cell contact sites (Figs. 1C and D). Compared with cells microinjected with buffer only, Rac1Val12 and Cdc42Val12 did not cause significant changes in the distribution of NKA. C3 is known to specifically ADP-ribosylate Rho and inactivate it [20]. We examined the distribution of NKA in NRK-52E cells microinjected with C3 and RhoAVal14 . Coinjection of C3 with RhoAVal14 partially inhibited the accumulation of NKA at the cell surface, compared with RhoAVal14 alone (data not shown). E-cadherin is also localized at the basolateral membrane and is a calcium-dependent cell– cell adhesion molecule that forms a complex with catenins [21]. Microinjection of RhoAVal14 did not show significant changes in the distribution of E-cadherin (Fig. 1F), compared with that of buffer alone (Fig. 1E). This is consistent with the observation of MDCK cells stably expressing constitutively active RhoA [22]. Z-axis scanning revealed that NKA was localized in spike-like protrusions at the apical membrane (Fig. 2A) in the RhoAVal14 -injected cells. In the basal membrane, NKA was detected aroud cell periphery (Fig. 2B). The vertical image confirmed that NKA was mainly localized in spike-like protrusions over the apical surfaces, which seemed to be microvilli-like structures (Fig. 2C). Involvement of Rho-kinase in the translocation of NKA induced by RhoA We next examined the involvement of Rho-kinase/ ROCK/ROK, the effector of Rho, in the translocation of NKA induced by RhoAVal14 . RB/PH (TT) is the carboxyl terminal portion of Rho-kinase and it serves as a dominant negative mutant of Rho-kinase [23]. Coinjection of MBP with RhoAVal14 induced the translocation of NKA in about 60% of total injected cells (Figs. 3A and E), whereas that of MBP-RB/PH (TT) effectively reduced the ratio of the translocation to about 30% of cells (Figs. 3B and E). Y-27632 is a compound that specifically inhibits the activity of Rho-kinase [24]. When the cells were pretreated with 10 lM Y-27632 for 30 min and incubated in the presence of the compound after injection, the RhoAVal14 -induced translocation of NKA was inhibited (Figs. 3C and E). These results indicate the involvement of Rho-kinase as a downstream molecule of RhoA in the translocation of NKA. We next examined the effect of GST-Rho-kinase-CAT (CAT), a constitutively active mutant of Rho-kinase [25]. When purified CAT was microinjected into NRK-52E cells at a concentration up to 10 mg/ml, the typical translocation of NKA was not induced. NKA seemed to be aggregated in large dot-like structures (Fig. 3D) in which actin filaments accumulated. The nature of the large dot-like structures was unclear. By the staining of F-actin, stress fiber formation in cells microinjected with CAT was confirmed.
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Fig. 1. The immunostaining images of NKA in cells microinjected with buffer (A), or constitutively active mutants of Rho family GTPases at 0.5 mg/ ml, RhoAVal14 (B), Rac1Val12 (C), and Cdc42Val12 (D). The localization of NKA in cells microinjected with buffer was not changed from that of noninjected cells (A). NKA was localized in spike-like protrusions in the cells microinjected with RhoAVal14 (B). In cells microinjected with Rac1Val12 (C) and Cdc42Val12 (D), NKA was mainly localized at cell–cell contact sites. The distribution of E-cadherin in cells injected with RhoAVal14 (F) was not changed from that in cells injected with buffer (E). The arrows indicate microinjected cells. The arrowheads denote the localization of E-cadherin at cell–cell contact sites. Bar, 10 lm.
Fig. 2. Z-axis scanning of NRK-52E cells microinjected with RhoAVal14 . All the cells displayed here were microinjected with RhoAVal14 at 0.5 mg/ml. NKA was localized in spike-like protrusions at the apical membrane (A). In the basal membrane, NKA was detected aroud cell periphery (B). The vertical image confirmed that NKA was mainly localized in spike-like protrusions over the apical surfaces, which seemed to be microvilli-like structures (C). Bar, 10 lm.
NKA is localized at microvilli-like structures in cells injected with RhoAVal14 The spike-like protrusions in which NKA was localized in cells microinjected with RhoAVal14 seemed to be
microvilli-like structures. Our previous study indicated that the Rho/Rho-kinase pathway is involved in the formation of microvilli-like structures through phosphorylation of moesin at 558th threonine [17,26]. We next performed double immunostaining of NRK-52E
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at microvilli-like structures in cells microinjected with RhoAVal14 (Fig. 4E). The merged image revealed that NKA and phosphorylated ERM proteins seemed to be colocalized at microvilli-like structures (Fig. 4F). The pretreatment of RhoAVal14 -microinjected cells with Y27632 canceled the accumulation of phosphorylated ERM proteins in microvill-like structures (Fig. 4H). In the cells pretreated with Y-27632, the colocalization of NKA and phosphorylated ERM proteins was weakly observed at cell–cell contact sites but not at microvillilike structures (Fig. 4I).
Discussion
Fig. 3. Involvement of Rho-kinase in the RhoAVal14 -induced translocation of NKA. Coinjection of MBP (5.0 mg/ml) with RhoAVal14 (A) had no effects on the translocation of NKA induced by RhoAVal14 alone. Coinjection of MBP-RB/PH (TT) (5.0 mg/ml) with RhoAVal14 suppressed the translocation of NKA (B). RhoAVal14 -injected cells pretreated with and incubated with 10 lM Y-27632 (C) did not exhibit typical changes of NKA distribution. Microinjection of CAT (D) could not induce the translocation of NKA observed in cells microinjected with RhoAVal14 . The arrows and arrowhead indicate microinjected cells and large dot-like structure, respectively. Bar, 10 lm. (E) The ratio of the cells displaying NKA translocation into microvilli-like structures. Bars represent means SEM for at least three indepedent experiments.
cells with anti-NKA and anti-phosphorylated ERM antibodies. In control cells, phosphorylated ERM proteins were localized at cell–cell contact sites and diffusely in the cytoplasm (Fig. 4B). The apparent colocalization of NKA and phosphorylated ERM proteins was partly observed at cell–cell contact sites in the merged image (Fig. 4C). Phosphorylated ERM proteins accumulated
The restricted distribution of NKA in the basolateral membrane is important for its function in sodium homeostasis. In this paper, we found that RhoAVal14 induced translocation of NKA into microvilli-like structures. Neither Rac1Val12 nor Cdc42Val12 induced this translocation. The inhibition of Rho-kinase canceled the effects of RhoAVal14 . These results indicate that Rho pathway is supposed to participate in the regulation of sodium and water homeostasis through the translocation of NKA. In the microvilli-like structures, NKA was colocalized with phosphorylated ERM proteins. In this study, because of technical restriction by the microinjection we did not observe the ratio of phosphorylation in total ERM proteins by the biochemical methods. Previous data demonstrated that RhoAVal14 induced phosphorylation of ERM proteins in COS7 cells [17] or NIH3T3 cells [11]. We speculate that ERM proteins should be phosphorylated through Rho pathway rather than phosphorylated ERM proteins were relocated to the microvilli-like structures. Although the precise interaction between the translocation of NKA and the phosphorylation of ERM proteins remains to be clarified, phosphorylation of ERM proteins might be involved in this translocation. The following possibilities may account for this translocation. (i) ERM proteins recruit NKA to microvilli-like structures in an activated Rho-dependent manner. In gastric parietal cells, proton secretion is mediated by the translocation of Hþ ; Kþ -ATPase (HKA) to apical membrane in microvilli [27]. Ezrin is one of the candidates to connect HKA with the actin cytoskeleton [28]. HKA was co-precipitated with ezrin by anti-ezrin antibody from the lysate of gastric parietal cells [29]. Because both HKA and NKA belong to Ptype ATPase and share a 65% identity in their amino acid sequences, it is possible that NKA also interacts with ERM proteins. Our previous data indicate that phosphorylation of moesin by Rho-kinase is crucial for the formation of microvilli-like structures in the signaling pathway from RhoA [17,26]. The role of phosphorylation of ERM proteins in the translocation of
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Fig. 4. Colocalization of NKA with phosphorylated ERM proteins. In cells injected with buffer, NKA was localized at cell–cell contact sites and cell periphery (A). Phosphorylated ERM proteins were detected at cell–cell contact sites and diffusely in the cytoplasm (B). In the merged image (C), the apparent colocalization of NKA and phosphorylated ERM proteins was partly observed at cell–cell contact sites. Both NKA (D) and phosporylated ERM proteins (E) were localized at microvilli-like structures in RhoAVal14 -microinjected cells. The colocalization of NKA and phosphorylated ERM proteins in microvilli-like structures is observed in the merged image (F). The pretreatment of RhoAVal14 -microinjected cells with Y-27632 canceled accumulation of phosphorylated ERM proteins in microvilli-like structures (H). In the cells pretreated with Y-27632, colocalization of NKA and phosphorylated ERM proteins was weakly observed at cell–cell contact sites (I). The arrows indicate microinjected cells. The arrowheads denote the colocalization of NKA and phosphorylated ERM proteins at cell–cell contact sites. Bar, 10 lm.
NKA remains to be investigated. Among ERM proteins, ezrin and moesin were equally detected in the lysate of NRK-52E cells by immunoblotting with antiERM antibody (date not shown). Perturbation of moesin by antisense phosphorothioate oligonucleotide affected the formation of microvilli in thymoma cells,
whereas that of ezrin or radixin did not [30]. Taken together these results, it is plausible that among ERM proteins moesin should be involved in the translocation of NKA into microvilli-like structures in NRK-52E cells. (ii) Activated RhoA relocates NKA through reorganization of cytoskeletons. It has been shown that
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NKA is delivered to both apical and basolateral membranes equally and is stabilized at the basolateral membrane by the membrane cytoskeleton [31]. NKA is known to interact with ankyrin and spectrin, which are members of the cortical actin meshwork. RhoAVal14 might stabilize the cortical actin cytoskeleton both at the apical and basolateral sites and hence enhance the interaction of NKA with these binding partners. To address the downstream effectors participating in the translocation, we examined the involvement of Rho-kinase by a Rho-kinase inhibitor or the dominant negative mutant of Rho-kinase. The translocation of NKA by RhoAVal14 was canceled by the inhibition of Rho-kinase. On the other hand, microinjection of the constitutively active mutant of Rho-kinase did not induce the translocation. These results indicate that Rho-kinase is considered to be necessary but not sufficient for the translocation of NKA. Effectors of RhoA other than Rho-kinase may be involved. (iii) Activated RhoA alters NKA localization by modulating membrane trafficking. Introduction of the constitutively active mutant of RhoA inhibits transferrin internalization in HeLa cells [12]. In polarized MDCK cells, RhoAVal14 inhibits recycling of transferrin receptors at the basolateral membrane [32]. It has been demonstrated that newly synthesized NKA is delivered both to apical and basolateral membranes. The inhibition of endocytosis of delivered NKA from the apical membrane by RhoAVal14 might be the cause of this translocation. The precise mechanism of the translocation of NKA by Rho is still to be clarified, but these findings will provide insights into the function of Rho in the regulation of vectorial water and sodium transport.
[4] [5] [6]
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Acknowledgments We thank Dr. A. Hall (University College London, UK) for providing pGEX-C3 and Dr. T. Urushidani (University of Tokyo, Japan) for helpful discussion. We are also grateful to Mrs. T. Ishii for secretarial assistance. This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Society for the Promotion of Science Research for the Future Science.
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