Article |
Address correspondence to Mitsunobu Hoshino, Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. Tel.: 81-42-346-1722. Fax: 81-42-346-1752. email: hoshinom{at}ncnp.go.jp
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Abstract |
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Key Words: calcium; MAPK; Ras; Rho
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Introduction |
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The newly discovered GTPase, Rin, and its Drosophila homologue Ric have been classified into the Ras superfamily (Lee et al., 1996; Wes et al., 1996; Shao et al., 1999; Reuther and Der, 2000). Rin binds GTP in vitro and exhibits an intrinsic GTPase activity (Lee et al., 1996; Shao et al., 1999). Rin shares a high sequence identity with the Ras and has a highly conserved but distinct G2 effector region (HDPTIEDAY) in which histidine is substituted for tyrosine at position 32, and alanine is substituted for serine at position 39 (Lee et al., 1996). Moreover, Rin displays unique characteristics that are not observed in other members of the Ras superfamily, such as CaM-binding activity and the lack of a typical COOH-terminal prenylation motif (CAAX motif) required for membrane association (Lee et al., 1996). Rin is expressed only in neurons and binds CaM in a calcium-dependent manner (Lee et al., 1996). The expression of Rin is more prominent in the adult brain than in the brain at earlier stages (Lee et al., 1996). We demonstrated previously that Rin is activated by growth factor stimulation using a Rin pull-down assay system (Hoshino and Nakamura, 2002). These studies raised the possibility that Rin plays important roles in the control of the calcium/CaM-mediated signaling pathways in the nervous system. To date, however, few of the intracellular functions and signal transduction pathways of Rin have been characterized.
Rat pheochromocytoma cell line PC12 cells are a useful model system for examining mechanisms of neuronal differentiation and signal transduction (Greene and Tischler, 1976). They differentiate in response to NGF with growth arrest and show neurite outgrowth resembling that of sympathetic neurons (Greene and Tischler, 1976). After NGF stimulation, a number of signal transduction pathways in PC12 cells are activated, including the Trk receptor tyrosine kinaseRas-MAPK cascade (Kaplan and Stephens, 1994). It has been shown that sustained MAPK activation is necessary for NGF-induced neurite outgrowth in PC12 cells (Marshall, 1995).
The Rho GTPase family proteins, which consist of the closely related GTPases Rho, Rac, and Cdc42, participate in the actin cytoskeleton dynamics, reactive oxygen generation, and tumorigenesis (Lim et al., 1996; Bishop and Hall, 2000). In Swiss 3T3 fibroblasts, Rho regulates growth factorstimulated stress fiber formation, whereas Rac and Cdc42 regulate growth factorstimulated membrane ruffling and filopodium formation, respectively (Lim et al., 1996; Bishop and Hall, 2000). To date, many Rho family effector proteins have been identified. For example, the GTP-bound Rho can bind to the Rho-associated coiled-coilforming kinases, whereas the GTP-bound Rac and Cdc42 can bind to the p21-activated kinases (PAKs) through their Cdc42/Rac interactive binding (CRIB) domain, and these kinases control the regulation of the actin cytoskeleton system (Lim et al., 1996; Bishop and Hall, 2000).
CaM is a ubiquitous, highly conserved protein and is recognized as a major calcium sensor for diverse intracellular enzymes (Rhoads and Friedberg, 1997). When cells are responding to an increase in the intracellular calcium concentration, CaM undergoes a conformational change, binds to its target proteins, and evokes many cellular processes, including cell cycle regulation, cytoskeletal organization, and ion channel regulation (Rhoads and Friedberg, 1997).
Here, we examined the role of Rin in neuronal signaling, especially focusing on the formation and morphology of neurite processes in calcium/CaM-mediated signaling pathway. Our results showed that the expression of Rin induced neurite outgrowth without NGF stimulation or MAPK activation, and this phenomenon was markedly inhibited by coexpression with the dominant negative Rac/Cdc42 protein or treatment with CaM inhibitor. We also found that Rin proteins activated endogenous Rac/Cdc42 proteins and that Rin-induced neurite outgrowth required Rin association with CaM. Moreover, we show that endogenous Rin proteins are involved in calcium-mediated neurite outgrowth and propose that they play pivotal roles in regulating neuronal signaling pathways.
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Results |
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Rin-mediated neurite outgrowth is MAPK pathway independent
It has been well established that the MAPK cascade is necessary for neuronal differentiation of PC12 cells (Kaplan and Stephens, 1994). We examined the activity of MAPK in Rin-expressing cells by Western blot analysis using antiphospho-MAPK antibody. As shown in Fig. 2 A, Rin-transfected cells did not show obvious MAPK activation. Moreover, pretreatment with 7.5 µM U0126 (a MAPK kinase inhibitor), which concentration is sufficient for the inhibition of MAPK activity after 50 ng/ml NGF stimulation (Fig. 2 B) and of NGF-induced neurite outgrowth (Fig. 2 C), did not affect the Rin-mediated neurite outgrowth in PC12 cells (Fig. 2 C). These data suggest that the MAPK pathway is not likely to be responsible for the Rin-mediated neurite outgrowth.
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To further confirm this observation, we constructed other CaM-binding defective mutants, which contained point rather than deletion mutation. These point mutants, named RinC-7 and RinC-4, in which all seven basic amino acid residues and four basic residues were replaced with neutral ones in the COOH-terminal of the Rin protein that was deleted in the Rin18 mutant, respectively (Fig. 5 C), also showed no binding activity to CaM (Fig. 5 D). As expected, they activated Rac/Cdc42 (Fig. 4, lanes 5 and 6), but failed to extend neurites in PC12 cells (Fig. 5, A and E). Together, these data strongly suggest that Rin-mediated neurite outgrowth requires both downstream Rac/Cdc42 activation and CaM association.
Endogenous RhoA is activated in Rin-expressed cells
To examine whether Rho is also activated by Rin, we measured the endogenous Rho activity by Rho pull-down assay. This assay system makes use of the Rho-binding domain of mDia1 as a GST fusion protein, which specifically binds to and isolates the GTP-bound Rho (Kimura et al., 2000). As shown in Fig. 6 A, an increase of the amounts of GTP-bound Rho was detected in both the wild-type Rin-transfected (lane 2) and RinQ78L-transfected cell lysates (lane 3). The level of GTP-bound Rho was similar to the lysophosphatidic acid (LPA)-treated cells (Fig. 6 A, lane 7).
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Endogenous Rin protein is also involved in calcium-mediated neurite outgrowth
To investigate the function of the endogenous Rin protein, we examined whether expression of the dominant negative Rin protein altered the function of the endogenous Rin in PC12 cells. Rin mRNA was not detected in nonneuronal Cos-7 cells but certainly existed in PC12 cells, though its mRNA level is rather lower than that of primary culture of neuronal cells (Fig. 7 A). Next, we constructed the dominant negative Rin, RinS34N, in which Ser34 was replaced with Asn. The cognate S17N mutant of the Ras, which is regarded as a dominant negative Ras, cannot bind to the effectors but binds to its GEFs and sequesters them, with the result that it cannot activate its downstream signaling cascade (Vojtek and Der, 1998). However, RinS34N has quite a low expression efficiency compared with the wild-type protein (unpublished data). Therefore, we needed to construct another Rin mutant that act in a dominant negative manner. RinG29V-C-7, in which the CaM-binding motif of the constitutively active RinG29V protein was mutated in a manner similar to that for the RinC-7 mutant, was constructed. As shown in Fig. 7 B, Myc-tagged RinG29V-C-7 could not induce the neurite outgrowth in PC12 cells because of the lack of CaM-binding ability, and it also suppressed the wild-type Rin-mediated neurite outgrowth. These data suggest that RinG29V-C-7 can act as a dominant negative Rin.
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It was reported previously that elevation of extracellular potassium evokes membrane depolarization, and that depolarization-induced calcium entry through voltage-dependent calcium channels sustains neurites and cell survival after NGF withdrawal in PC12 cells (Teng and Greene, 1993). Moreover, Mark et al. (1995) have reported that intracellular cAMP and KCl depolarization synergistically stimulate neurite outgrowth in PC12 cells. Therefore, we investigated whether Rin is involved in calcium entry-mediated neurite outgrowth after KCl-evoked membrane depolarization in PC12 cells.
As shown in Fig. 7 E, 30% of 50 µM forskolin-treated GFP-expressed cells showed neurite outgrowth. After addition of 50 mM KCl, neurite outgrowth of forskolin-stimulated cells was potentiated by KCl-evoked membrane-depolarization and subsequent calcium entry (Fig. 7 E). As demonstrated previously (Mark et al., 1995), depolarization by KCl alone showed no significant induction of neurite outgrowth (not depicted), and forskolin and KCl depolarization-induced neurite outgrowth was calcium entry-dependent because it was inhibited by the L-type calcium channel blockers nitrendipine and diltiazem (Fig. 7 E). Dominant negative RinG29V-C-7 could not inhibit the forskolin-induced neurite outgrowth, but could inhibit the neurite outgrowth stimulated by forskolin and KCl at the level of the stimulation of forskolin alone (Fig. 7 E). After preincubation with the L-type calcium channel blockers nitrendipine or diltiazem, RinG29V-C-7 was unable to further inhibit the neurite outgrowth stimulated by forskolin and KCl, as compared with the forskolin and KCl-stimulated blocker-pretreated cells (Fig. 7 E).
To confirm these findings, we examined whether Rin is certainly involved in the calcium entry-mediated neurite outgrowth by using RNA interference method. As shown in Fig. 7 F, siRNA of Rin eliminated the expression of Rin protein, but did not eliminate the expression of another protein, such as Ras. Control siRNA did not eliminate the expression of Rin protein (Fig. 7 F). These results indicate that siRNA of Rin specifically disrupts Rin protein expression. The neurite outgrowth stimulated by forskolin and KCl is inhibited at the level of the stimulation of forskolin alone after siRNA of Rin introduction (Fig. 7 G). Control siRNA did not show this inhibitory phenomenon (Fig. 7 G). These data suggested that dominant negative Rin or siRNA of Rin inhibit calcium-mediated neurite outgrowth and that Rin may be involved in the calcium-mediated signaling pathways leading to neurite outgrowth.
In addition, we examined the effect of inhibiting Rac/Cdc42 in this calcium-mediated neurite outgrowth. As shown in Fig. 7 H, dominant negative Rac or Cdc42 considerably inhibited neurite outgrowth after forskolin stimulation. Dominant negative Cdc42-expressed cells showed the potentiation of neurite outgrowth after KCl addition, and siRNA of Rin did not inhibit this potentiation further (Fig. 7 H). These data suggest that Rin requires Cdc42 signaling pathway to mediate calcium-induced neurite outgrowth. However, dominant negative Rac-expressed cells did not show the potentiation after KCl addition (Fig. 7 H). Thus, it is uncertain whether Rac is specifically required for Rin-mediated calcium-induced neurite outgrowth.
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Discussion |
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We found that wild-type Rin, as well as constitutively active RinQ78L, induced neurite outgrowth. We (Hoshino and Nakamura, 2002) and Spencer et al. (2002) observed previously that basal levels of GTP-bound wild-type Rin remains quite high and that the amount of wild-type Rin precipitated from the Cos-7 cell lysates is almost the same as that of constitutively active Rin using a Rin pull-down assay system (unpublished data). Considering that the guanine nucleotide dissociation rate of Rin is faster than that of most Ras family member (Shao et al., 1999) and that the intracellular concentration of GTP is much higher than that of GDP (McCormick, 1989), it is expected that most of Rin proteins may be remained active GTP-bound state in the cells. There may be another possibility that it might be based on low activity of RinGAP or that a specific GEF of Rin might be exist and might exhibit marked affinity and catalytic efficiency for wild-type Rin protein.
Spencer et al. (2002) reported previously that neither GFP-tagged wild-type Rin nor GFP-tagged RinQ78L induces neurite outgrowth in PC6 cells, which is a subline of PC12 cells. This discrepancy may be due to the difference of the cell lines. The large size of GFP (27 K) could interfere with the distribution and function of the fusion protein. In fact, we verified that neither GFP-tagged wild-type Rin nor GFP-tagged RinQ78L induced neurite outgrowth in PC12 cells (unpublished data). To circumvent this problem, we transfected tiny tag-fused (Myc-tagged or HA-tagged) Rin into the pEF-Bos vector, which is a powerful mammalian expression vector (Mizushima and Nagata, 1990).
We demonstrate that Rac/Cdc42 activity is required for the Rin-induced neurite outgrowth and that Rin activates endogenous Rac/Cdc42 in PC12 cells. To confirm this observation, we cotransfected cells with wild-type Rin and dominant negative PAK1 protein, one of the effector molecules of Rac/Cdc42 proteins (Zhao et al., 1998). Dominant negative PAK1 protein also suppressed the Rin-mediated neurite outgrowth. (There were 76.45 ± 2.00% of neurite-bearing cells for wild-type Rin-expressed cells, compared with 59.82 ± 1.14 of neurite-bearing cells for dominant negative PAK1 and wild-type Rin-coexpressed cells; P < 0.05). To examine whether Rin directly associates with Rac/Cdc42, we performed the coimmunoprecipitation assay. As a result, we failed to detect the direct interaction between Rin and Rac/Cdc42 (unpublished data). Probably, there must be a linking molecule between Rin and Rac/Cdc42, or their GEFs, such as Vav or Cdc24. Because RhoG is shown to be located upstream of Rac/Cdc42 (Kato et al., 2000), we investigated whether RhoG participated in the Rin and Rac/Cdc42 pathway using RNA interference method. As a result, RhoG is not likely to be involved in Rin-mediated neurite outgrowth (unpublished data). Identification of a molecular link between Rin and Rac/Cdc42 should be further clarified.
We showed that CaM association with Rin is necessary to induce neurite outgrowth. We did not analyze the downstream target of CaM, but there is a possibility that calcium/CaM-dependent protein kinases (CaMKs) and/or IQGAP1 are the targets. CaMKI and CaMKII are enriched in neuronal processes and synapses (Curtis and Finkbeiner, 1999), and they can phosphorylate the serine 133 of cAMP response element binding protein, a well-known transcription factor involved in synaptic plasticity (Curtis and Finkbeiner, 1999). IQGAP1 has an affinity for CaM through its IQ motif and an actin-binding protein, whose function is cross-linking the actin filaments into bundles (Hart et al., 1996). IQGAP1 is also a Rac/Cdc42 effector and serves as a direct molecular link between Rac/Cdc42 and actin filaments under the regulation of CaM (Hart et al., 1996; Bashour et al., 1997). Recently, Rac/Cdc42 has been shown to interact with microtubules through IQGAP1 and CLIP-170, a microtubule plus end tracking protein, and forms a complex at the leading edge, resulting in a polarized microtubule array and cell polarization (Fukata et al., 2002). We examined whether IQGAP1 associates RinCaM complex and found that IQGAP1 formed a ternary complex with Rin and CaM by coimmunoprecipitation assay (unpublished data). There is a possibility that CaMK and/or IQGAP1 might contribute to the Rin-mediated neurite outgrowth.
We found that Rho is also activated by Rin. Rho family proteins are central regulators of neuronal morphology including neurite branching (Luo, 2000). We found that Rin-induced neurite has a plenty of branchings. It has been shown that Rac/Cdc42 are positive regulators for dendritic branching and remodeling whereas Rho is a negative regulator for branch formation (Li et al., 2000; Nakayama et al., 2000). It is an interesting possibility that Rin may induce the branchings of neurite by exquisite balancing between the downstream Rac/Cdc42 and Rho signaling.
We investigated the function of endogenous Rin using dominant negative Rin. In RT-PCR analysis, the expression of Rin at the mRNA level in PC12 cells was low. For this reason, the endogenous Rin expression in PC12 cells was too low to detect with Western blotting using an anti-Rin antibody (unpublished data). At first, we constructed a RinS34N expression vector, but it could not be efficiently expressed in the cells, probably due to the cell toxicity of RinS34N. This fact indicates that endogenous Rin may play an important role in regulating the cell growth. Then, we made another dominant negative Rin mutant vector encoding RinG29V-C-7, which can be efficiently expressed in the cells. It was expected that RinG29V-C-7 can bind the effector molecules through their effector region but suppress signals to the downstream molecules leading to the neurite outgrowth. We have shown that dominant negative RinG29V-C-7 could not inhibit the NGF-induced neurite outgrowth. Spencer et al. (2002) demonstrated previously that dominant negative RinS34N potently inhibits NGF-induced neurite outgrowth. This may be due to the fact that RinS34N has some toxic effect to the cells. Moreover, we (this paper) and Spencer et al. (2002) observed that neither wild-type nor constitutively active Rin induced MAPK activation. It has been shown that sustained MAPK activation is necessary for NGF-stimulated neurite outgrowth (Marshall, 1995). Thus, we consider that Rin is not likely to be a component of the NGFMAPK pathway leading to neurite outgrowth.
We have demonstrated that dominant negative RinG29V-C-7 or siRNA of Rin inhibits the forskolin plus KCl-induced neurite outgrowth at the level of stimulation of forskolin alone, and that Rin exists in a calcium-mediated signaling pathway using calcium channel blockers. The importance of this calciumcAMP signaling pathway is illustrated by Wong's report. Wong et al. (1999) showed that calcium-stimulated adenylate cyclase activity is essential for long-term memory and late phase long-term potentiation. Intracellular calcium increase through voltage-dependent calcium channels or N-methyl-D-aspartate receptors can activate calcium-sensitive adenylate cyclase, and generated cAMP can activate protein kinase A or cyclic AMP response element binding proteinmediated transcription pathway leading to long-term potentiation and memory formation (Wong et al., 1999; Poser and Storm, 2001). Thus, Rin may play a role in a long term effect on neuronal function as well as neuritogenesis.
In conclusion, we have identified a novel function of Rin protein, i.e., inducing neurite outgrowth in PC12 cells. However, many problems about Rin signaling, for example, whether there is a specific GEF or GAP of Rin, remains to be clarified. Further studies focusing on these problems will shed light on our understanding of the Rin-signaling pathway and its roles in the regulation of neuronal cell morphology and the functions of the nervous system.
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Materials and methods |
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Expression plasmids
Myc-Rin and HA-Rin mammalian expression pEF-Bos vectors were constructed as described previously (Hoshino and Nakamura, 2002). The dominant negative Rac1 vector (pEF-Bos Myc-RacS17N) and Cdc42 vector (pEF-Bos Myc-Cdc42S17N) were provided by Y. Takai (Osaka University, Osaka, Japan) and H. Miki (University of Tokyo, Tokyo, Japan). Constitutively active Rin mutants (RinQ78L and RinG29V), Rin deletion mutant (Rin18), Rin point mutants (RinC-7 and RinC-4), and dominant negative Rin mutants (RinS34N and RinG29V-C-7) were generated by PCR-based site-directed mutagenesis. The dominant negative PAK1 vector was constructed as described previously (Zhao et al., 1998). The coding region of all constructs was confirmed by DNA sequencing.
Cell cultivation and transfection
Cos-7 cells and PC12 cells were cultured in DME (Nissui) supplemented with 10% FBS (Intergen), 2 mM L-glutamine and penicillin/streptomycin, and incubated at 37°C in an atmosphere of 5% CO2. Serum-starved cells were obtained by incubation for 24 h in the medium containing 0.5% serum. Semi-confluent cells were transiently transfected with expression vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
CaM-binding assay
The CaM-binding assay was performed as described previously (Hoshino and Nakamura, 2002).
Measurement of endogenous Rac/Cdc42 activity
The CRIB domain of PAK1 was cloned into the pGEX vector and was expressed in Escherichia coli as a fusion protein. Transfected PC12 cells were serum starved for 24 h and lysed with an ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2.5 mM MgCl2, 10% glycerol, 1% NP-40, 10 mM NaF, 1 mM Na3VO4, 0.1 mM PMSF, and 10 µg/ml aprotinin and leupeptin). Cell lysates were centrifuged for 10 min at 13,000 g at 4°C, and each supernatant was incubated with 10 µg of purified GST-PAK CRIB immobilized on glutathione-Sepharose beads at 4°C for 30 min. After the beads were washed twice with a lysis buffer, the bound proteins were suspended in 20 µl Laemmli sample buffer and separated by 14% SDS-PAGE.
Measurement of endogenous Rho activity
Rho pull-down assay was performed similar to the Rac/Cdc42 pull-down assay as described in the previous paragraph, using Rho-binding domain of GST-mDia1 protein instead of GST-PAK CRIB protein. The beads-bound Rho proteins were visualized by Western blotting using anti-RhoA antibody.
Western blotting
Protein samples were separated by SDS-PAGE, transferred to nitrocellulose membrane (Schleicher and Schuell), and probed with a primary antibody in PBS containing 0.1% Tween 20 and 5% nonfat dry milk (Becton Dickinson). The primary antibody was visualized with HRP-conjugated secondary antibody (Amersham Biosciences) and ECL (PerkinElmer).
RT-PCR
Total RNA was extracted from the cells using Isogen reagent (Wako). 5 µg of extracted total RNA was used as a template for oligo-dTprimed first-strand cDNA synthesis using SuperScript reverse transcriptase (Invitrogen), and 2% of the resultant cDNA was used for PCR amplification. PCR was performed by 30 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. The primers used were as follows: for Rin, forward, 5'-CTCTTGCTCGAGACTACAAC-3' and reverse, 5'-CCTTCCTGCGTATTTCTCTC-3' (105 bp); and for GAPDH, forward, 5'-CCTGCACCACCAACTGCTTAGC-3' and reverse, 5'-GCCAGTGAGCTTCCCGTTCAGC-3' (230 bp). The identity of the amplified cDNAs was confirmed by DNA sequencing.
Immunofluorescence and neurite outgrowth analysis
48 h after transfection, PC12 cells were washed with PBS buffer and fixed with 3% PFA-PBS for 20 min. Next, cells were membrane permeabilized in 0.2% Triton X-100 PBS for 5 min. After the residual PFA and Triton X-100 were washed away with PBS buffer, cells were probed with a primary antibody in PBS for 2 h, followed by incubation with secondary antibody for 2 h. Stained cells were examined under appropriate illumination on a fluorescence microscope (Carl Zeiss MicroImaging, Inc.). About 200 cells were counted at each experiment. Cells were counted as positive for neurite outgrowth if one or more neurites exceeded one cell body diameter in length.
RNA interference
Single-stranded RNA was synthesized by standard protocols (Elbashir et al., 2001). Complementary single-stranded RNAs were annealed by heating them to 90°C and cooling to 37°C. Disruption of Rin protein function was performed by transfecting double-stranded RNAs into cells using Lipofectamine 2000 reagent.
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Acknowledgments |
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This research was supported by an Advanced Brain Research grant from the Ministry of Education, Science, Sports, Culture, and Technology, and Health Sciences Research grants from the Organization of Pharmaceutical Safety and Research and Research on Advanced Medical Technology, nano-001.
Submitted: 13 August 2003
Accepted: 8 October 2003
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