Ran-Binding Protein 3 Phosphorylation Links the Ras and PI3-Kinase Pathways to Nucleocytoplasmic Transport

Molecular Cell

Article Ran-Binding Protein 3 Phosphorylation Links the Ras and PI3-Kinase Pathways to Nucleocytoplasmic Transport Sang-Oh Yoon,1 Sejeong Shin,1 Yuzhen Liu,1 Bryan A. Ballif,2 Michele S. Woo,3 Steven P. Gygi,1 and John Blenis1,* 1Department

of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA of Biology, University of Vermont, 109 Carrigan Drive, Burlington, VT 05405, USA 3Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2007.12.024 2Department

SUMMARY

The major participants of the Ras/ERK and PI3kinase (PI3K) pathways are well characterized. The cellular response to activation of these pathways, however, can vary dramatically. How differences in signal strength, timing, spatial location, and cellular context promote specific cell-fate decisions remains unclear. Nuclear transport processes can have a major impact on the determination of cell fate; however, little is known regarding how nuclear transport is regulated by or regulates these pathways. Here we show that RSK and Akt, which are activated downstream of Ras/ERK and PI3K, respectively, modulate the Ran gradient and nuclear transport by interacting with, phosphorylating, and regulating Ran-binding protein 3 (RanBP3) function. Our findings highlight an important link between two major cell-fate determinants: nuclear transport and the Ras/ERK/RSK and PI3K/ Akt signaling pathways. INTRODUCTION The Ras/ERK and PI3-Kinase (PI3K)/Akt pathways regulate a variety of biological processes (Shaw and Cantley, 2006). Extracellular stimuli such as growth factors transiently activate Ras proteins through the activation of cell-surface receptors. Activated Ras proteins, in turn, regulate multiple downstream effectors that modulate cell proliferation, survival, and differentiation. The best-characterized Ras-regulated pathway is initiated when activated Ras binds to the Raf family of serine/threonine kinases. Raf then activates MAPK/ERK-activated kinases (MEK1/2), which directly phosphorylate and activate ERK1/2 (Murphy and Blenis, 2006). Activated ERKs phosphorylate and activate downstream targets such as the family of 90 kDa ribosomal S6 kinases (RSKs), which consist of RSK1–RSK4 (Roux and Blenis, 2004). RSK isoforms are activated by virtually all extracellular signaling molecules that stimulate the Ras-ERK pathway, i.e., growth factors, cytokines, peptide hormones, and neurotransmitters. RSKs are involved in multiple processes including transcriptional regulation, cell-cycle control, and protein synthesis

(Roux and Blenis, 2004). In addition to the Ras/ERK pathway, the PI3K/Akt pathway is also an essential pathway for determining cell fate. The PI3K/Akt pathway is activated by growth factors and inhibited by negative regulators like the PTEN tumor suppressor (Hennessy et al., 2005). This pathway controls many cellular processes such as cell size/growth, proliferation, survival, and glucose metabolism (Shaw and Cantley, 2006). Even though Ras/ERK/RSK and PI3K/Akt pathways are distinct from each other, their final roles are very similar; cell-cycle control, translational control, protein transport control, and antiapoptosis. RSK and Akt belong to the PKA, PKG, and PKC (AGC) superfamily, and they possess similar substrate specificity, with a preference for a RXRXXS/T motif. As a result, RSK and Akt have some targets that are in common. For example, RSK and Akt can phosphorylate GSK-3b, tRNA methylase METTL1, and p27Kip1 at the same position (Cartlidge et al., 2005; Doble and Woodgett, 2003; Fujita et al., 2003). Interestingly, there are other critical, common targets of RSK and Akt that are phosphorylated at distinct sites but that have similar functions such as BAD (Bonni et al., 1999; Shimamura et al., 2000) and TSC2 (Roux et al., 2004). The nucleus is the defining feature of the eukaryotic cell. The physical separation of the nucleoplasm and cytoplasm provides an added level of spatial regulation of protein activity (Kau et al., 2004). Movement of macromolecules between the nucleus and the cytoplasm occurs via aqueous channels that are formed by the nuclear pore complexes (NPCs) in the nuclear envelope. Nucleocytoplasmic transport of proteins is regulated by the Ras-related small nuclear GTPase, Ran (Quimby and Dasso, 2003; Sazer, 2005). Ran is predominantly located in the nucleus, but due to nucleocytoplasmic cycling, small amounts are also present in the cytoplasm. The Ran GTPase cycle is modulated by various interacting proteins. The guanine nucleotide exchange factor for Ran, RCC1, is restricted to the nucleus, while the GTPase-activating protein for Ran, RanGAP, is concentrated in the cytoplasm (Quimby and Dasso, 2003). Due to the compartmentalization of Ran regulators, the GTP-bound form of Ran is predominantly localized in the nucleus (Kuersten et al., 2001; Sazer, 2005), and the resulting Ran gradient contributes to the import and export of many proteins and some RNAs through the nuclear pore by the importins and exportins (e.g., Crm1), respectively (Kau et al., 2004; Macara, 2001). The control of nuclear-cytoplasmic transport contributes to the spatial and temporal control of transcription, translation, cell cycle, and other

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Figure 1. RanBP3 Is a RSK-Binding Partner (A) Schematic representation of RSK structure. (B) The partial RanBP3 clones (8, 32, and 41) obtained from the two-hybrid mouse library screen are schematically aligned with the two full-length human clones (isoforms RanBP3a and RanBP3b). (C) Clone 8 obtained from two-hybrid screening was transformed into yeast with plasmids bearing the indicated GAL4 DNA-binding domain fusions and grown on minimal media lacking uracil, leucine, and adenine to select for two-hybrid interaction. Clone SKAR, which is known to interact with p70S6K1, was used as a positive control. (D) HEK293 cells were transfected with either HARSK or control vector with FLAG-RanBP3 for 24 hr and then incubated in serum-free media for an additional 20 hr. Cells were treated with 50 ng/ml PMA for 15 min and lysed with either CHAPS or Triton X-100 lysis buffer in the presence or absence of DSP, a crosslinking reagent. Immunoprecipitation and immunoblot analyses were performed. The arrowhead indicates the HA-RSK coimmunoprecipitating FLAG-tagged RanBP3.

To date, there is little evidence for growth-factor-mediated modulation of the Ran pathway. We show that RanBP3 is a target of signal integration between the Ras and PI3K signaling pathways and that RSK and Akt, respectively, provide this connection by binding and phosphorylating RanBP3 in vitro and in vivo. More importantly, our analysis shows that phosphorylated RanBP3 contributes to the formation of the Ran gradient and nuclear transport, providing a new link between this process and two important cellular signaling systems. RESULTS

parameters that are important for cell fate (Sazer, 2005). Therefore, nuclear transport needs to be tightly regulated. For this, many Ran-interacting proteins exist, and they coordinately regulate nuclear import and export with Ran. Ran-binding protein 3 (RanBP3) is a Ran-interacting protein. It has been reported that RanBP3 binds to the Crm1 transport receptor and inhibits the binding of unloaded Crm1 to the NPC (Macara, 2001). In addition, RanBP3 increases the affinity of Crm1 for both Ran-GTP and nuclear export sequences (Englmeier et al., 2001; Lindsay et al., 2001). In this manner, RanBP3 coordinates efficient cargo loading, RanGTP binding, and nuclear export (Macara, 2001).

RanBP3 Is a Binding Partner of RSK To identify new targets of RSK, we performed two-hybrid screening of a mouse embryonic library using full-length RSK, RSK D10 , and RSK D2 as bait (Figure 1A). Three clones (clones 8, 32, and 41) encoding RanBP3 (Figure 1B) interacted strongly with RSK D2 but did not interact with S6K1, full-length RSK, and RSK D10 (Figure 1C). The failure of the RanBP3 clones to interact with full-length RSK may be the result of a weak interaction of full-length RSK with RanBP3 and/or the failure of fulllength RSK to fold properly when expressed as bait in yeast. Mueller et al. (1998) first described the expression of two RanBP3 isoforms, RanBP3a and RanBP3b; however, other groups have found only RanBP3b in their systems (Welch et al., 1999). We examined expression of RanBP3 in HeLa and HEK293 cells using RT-PCR and immunoblot analysis and found that these cells express only RanBP3b (data not shown). We next attempted to

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provide evidence that RanBP3 and RSK interact in mammalian cells. We were unable to confirm an interaction by simple coimmunoprecipitation experiments but were able to coimmunoprecipitate RSK and RanBP3 from phorbol ester (PMA)-stimulated HEK293 cells lysed in the presence of DSP, a crosslinking agent (Figure 1D). These data are consistent with the possibility that the interaction of RanBP3 with full-length RSK is relatively weak. RSK Phosphorylates Serine 58 of RanBP3 In Vitro and In Vivo Another approach that we have used to provide support for an interaction is to determine if RanBP3 is a substrate for RSK. If this is the case, RSK must interact with RanBP3 for the phosphotransferase reaction to occur. We first set out to identify possible RSK phosphorylation sites in RanBP3. RSK often phosphorylates serine or threonine residues in the sequence RXRXXS/T, and RanBP3b has such a consensus sequence: 53RERTSS58 (Figure 2A). To examine the possibility that serine 58 is a RSK phosphorylation site, tryptic peptides of immunopurified RanBP3b from PMA-stimulated HEK293 cells were subjected to analysis by liquid chromatography-tandem mass spectrometry (LC-MS/ MS). The low-energy fragmentation spectrum of the phosphopeptide shown in Figure 2B was confidently identified many times in our analysis with phosphorylation at the indicated serine (serine 58) as being the top SEQUEST match in almost all cases. However, there were insufficient diagnostic fragment ions to confidently distinguish between phosphorylation at threonine 56, serine 57, and serine 58. To evaluate the possibility that RSK can phosphorylate RanBP3 at serine 58, wild-type (WT) RSK and kinase-dead (KD) RSK expression constructs were transfected into HEK293 cells. After immunoprecipitation, a RSK phosphotransferase assay was performed using purified recombinant RanBP3 as substrate. WT RSK, but not KD RSK, isolated from PMA-stimulated cells, was able to phosphorylate WT RanBP3 and S57A RanBP3, but not S58A RanBP3 in vitro (Figure 2C). This indicated that serine 58 in RanBP3 is the likely RSK phosphorylation site. Further studies were performed to determine if serine 58 is the only site for RSK phosphorylation. Although RXRXXS/T is a good RSK phosphorylation site, some proteins such as DAPK can be phosphorylated by RSK at serine or threonine residues in a RXXS/T motif (Anjum et al., 2005). Since RanBP3 has such a motif, 150RSPS153, we generated a S153A RanBP3 mutant to determine whether RSK can also phosphorylate this serine. Activated RSK, immunopurified from PMA-

treated cells, phosphorylated WT and S153A RanBP3 but did not phosphorylate S58A RanBP3 (Figure 2D). U0126, a MEK/ ERK pathway inhibitor, blocked the ability of PMA to activate RSK. Under these conditions, immunoprecipitated RSK was not able to phosphorylate RanBP3. Taken together, these results suggest that RSK and RanBP3 interact and that serine 58 is the only phosphorylation site regulated by RSK in vitro. For further studies, we developed anti-phospho-serine 58RanBP3 antisera. To test specificity of this antibody, WT and mutants of RanBP3 were expressed and subjected to immunoblot analysis. As shown in Figure 2E, this antibody detected endogenous and overexpressed WT RanBP3, and PMA increased the intensity of immunostaining. Importantly, the antibody did not detect the ectopically expressed RanBP3 S58A or S58D proteins. These results indicate that this antibody is specific for phosphoS58-RanBP3. Using this antibody, we provided additional evidence that RSK regulates serine58 phosphorylation of RanBP3 in cells. Coexpression of RSK1 or RSK2 with RanBP3 resulted in increased phosphorylation of both overexpressed and endogenous RanBP3 (Figure 2F). Similarly, knockdown of RSK1/2 blocked PMA-induced RanBP3 phosphorylation (Figure 2G). Akt Regulates RanBP3 Phosphorylation In Vitro and In Vivo RSK and Akt can recognize and phosphorylate the same basophilic sequence (RXRXXS/T). Therefore, we tested the possibility that Akt might also regulate RanBP3 phosphorylation. For this, an Akt immunocomplex kinase assay was performed using purified RanBP3 as described above for RSK. Insulin-stimulated Akt increased phosphorylation of WT RanBP3, S57A RanBP3, and S153A RanBP3 but did not affect S58A RanBP3 phosphorylation (Figure 3A). KD Akt did not generate RanBP3 phosphorylation under any condition. To test whether phosphorylation of RanBP3 by insulin-stimulated Akt was PI3K dependent, cells were treated with LY294002, an inhibitor of PI3K, and an Akt phosphotransferase assay was performed. As shown in Figure 3B, insulin-stimulated Akt increased phosphorylation of WT RanBP3 and S153A RanBP3, but this effect was blocked by LY294002 treatment. The data from Figures 3A and 3B suggest that Akt is an in vitro serine 58 kinase for RanBP3. To determine if Akt contributed to RanBP3 phosphorylation in vivo, we first tested the interaction between Akt and RanBP3. These proteins were expressed in HEK293 cells, and immunoprecipitation was performed in the presence of a crosslinking

Figure 2. RSK Phosphorylates RanBP3 at Serine 58 Residue In Vitro and In Vivo (A) Schematic representation of RanBP3. RanBP3 has a nuclear localization signal (NLS), a Ran-binding domain (RBD), and possible RSK phosphorylation sites. (B) Low-energy collision-induced dissociation spectrum of the RanBP3 pS58 phosphopeptide. (C) HEK293 cells were transfected with either HA-WT-RSK1 or HA-KD-RSK1 (D/D RSK1) for 24 hr and then incubated in serum-free media for an additional 20 hr. Cells were left untreated or treated with 50 ng/ml PMA for 15 min, and immunoprecipitation was performed. RSK phosphotransferase assays were performed with purified GST-WT RanBP3, GST-S57A RanBP3, and GST-S58A RanBP3. (D) HEK293 cells were transfected with HA-WT-RSK1 for 24 hr and then incubated in serum-free media for an additional 20 hr. During the last 1 hr, cells were left untreated or treated with 10 mM U0126. After treatment with 50 ng/ml PMA, immunoprecipitation and RSK phosphotransferase assays were performed. (E) HEK293 cells were transfected with control plasmid, WT RanBP3, S58A RanBP3, or S58D RanBP3 for 24 hr and incubated in serum-free media for an additional 20 hr. After treatment with 50 ng/ml PMA for 15 min, cells were lysed, and immunoblot analysis was performed. (F) HA-RanBP3 plasmid was cotransfected with either HA-RSK1 or HA-RSK2 into HEK293 cells for 24 hr, and then cells were incubated in serum-free medium for an additional 20 hr. Cells were lysed, and immunoblot analysis was performed. (G) RSK knockdown HEK293 cells were generated using RSK1 and RSK2 shRNA. Cells were incubated in serum-free media for 20 hr. After treatment with 50 ng/ ml PMA for 15 min, cells were lysed, and immunoblot analysis was performed.

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Figure 3. Akt Phosphorylates RanBP3 at Serine 58 Residue In Vitro and In Vivo (A) HEK293 cells were transfected with either HA-WT Akt or HA-KD Akt for 24 hr and then incubated in serum-free media for an additional 20 hr. Cells were left untreated or treated with 100 nM insulin for 15 min, and immunoprecipitation was performed. Akt phosphotransferase assays were performed using purified GSTWT RanBP3, GST-S57A RanBP3, GST-S58A RanBP3, and GST-S153A RanBP3 as substrate.

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reagent. As shown in Figure 3C, RanBP3 coimmunoprecipitated with Akt. We next asked whether Akt can phosphorylate RanBP3 in vivo. Ectopic expression of Akt increased RanBP3 phosphorylation at serine 58 (Figure 3D). Further studies were performed using Akt1/2 shRNA and an Akt inhibitor. As shown in Figures 3E and 3F, knockdown of Akt1/2 or use of an Akt inhibitor decreased insulin-induced RanBP3 phosphorylation. Taken together, these results suggest that Akt also regulates RanBP3 phosphorylation in vitro and in vivo. Given that RSK can also phosphorylate serine or threonine residues in the RXRXXS/T motif, we examined the effect of S6K1 and S6K2 on RanBP3 phosphorylation. For this, we used WT and rapamycin-insensitive, constitutively active (CA) S6K1 and S6K2. As shown in Figure S1, available online, ectopic expression of WT S6K1, CA S6K1, or CA S6K2 increased phosphorylation of a known substrate, ribosomal protein S6 (rpS6). Rapamycin effectively inhibited rpS6 phosphorylation by WT S6K1 or WT S6K2 but did not affect rpS6 phosphorylation in cells expressing the activated, rapamycin-resistant forms of S6K1 or S6K2. Importantly, neither S6K1 nor 2 affected RanBP3 phosphorylation. These data indicate that, although RSK, Akt, and S6K have the potential to phosphorylate similar basophilic sites, only RSK and Akt can regulate RanBP3 phosphorylation. Growth Factors Regulate RanBP3 Phosphorylation via PI3K/Akt- and/or ERK/RSK-Dependent Pathways Our data suggested that RanBP3 might be a critical target for the convergence of signaling downstream of the Ras and PI3K pathways. We investigated this possibility further by investigating the contribution of various stimuli and effects of different signaling inhibitors on RanBP3 phosphorylation in cells. HEK293 cells were serum starved and then stimulated with PMA, insulin, or EGF in the presence or absence of selective signaling inhibitors (Figure 4A). All agonists robustly increased phospho-RanBP3. The PMA-stimulated increase in RanBP3 phosphorylation was blocked by U0126, an inhibitor of MEK/ERK signaling. EGF activates both PI3K/Akt and ERK/RSK pathways, which accounts for the reason that both U0126 and LY294002 were necessary to inhibit EGF-stimulated RanBP3 phosphorylation (Figure 4B). A small amount of insulin-stimulated RanBP3 phosphorylation was detected in the presence of LY294002 even though phospho-Akt was not detectable (Figure 4A). With longer exposure times, we could detect a small increase in phospho-ERK and -RSK by insulin, and this was enhanced in the presence of LY294002 (Figure 4C). This observation is consistent with the

fact that the PI3K pathway can act as a negative regulator of the ERK pathway (Zimmermann and Moelling, 1999). Thus, both ERK and PI3K pathways must be inhibited to block RanBP3 phosphorylation (Figure 4D). Like EGF (Figure 4B), serum also increased phospho-Akt, phospho-ERK, and phospho-RanBP3 levels (Figure 4E). Only cotreatment with LY294002 and U0126 inhibited this increase. Taken together, these results indicate that both PI3K/Akt and ERK/RSK pathways are responsible for RanBP3 phosphorylation, and for many agonists, the extent of RanBP3 phosphorylation will depend on the level of integration by both pathways. Finally, although all agonists studied here are known to activate S6K1/2, rapamycin, a potent inhibitor of S6K1/2 and rpS6 phosphorylation, had no effect on agonist-induced RanBP3 phosphorylation at Ser58 in HEK293 cells (Figure 4F). Thus, combined with the S6K1/2 overexpression data provided in Figure S1, these results suggest that S6 kinases have little, if any, activity toward RanBP3 phosphorylation under the conditions studied.

RanBP3 Phosphorylation Increases Its Affinity toward Ran To support the potential importance of RanBP3 phosphorylation by RSK and Akt, we determined if this was a conserved site in various mammals. The RSK and Akt phosphorylation sequence RERTSS, as well as the adjacent nuclear localization sequence, is strictly conserved (Figure 5A). This indicates that the RanBP3 phosphorylation site and nuclear localization sequences may have important functions in mammals. To investigate the function of phospho-RanBP3, we first examined the localization of RanBP3. Since the nuclear localization signal (NLS) in RanBP3 is just N terminal to the phosphorylation site (Figure 5A), we postulated that phosphorylation may affect RanBP3 localization. However, all forms of RanBP3 (endogenous, WT, and S58A) were predominantly localized to the nucleus (Figure 5B). Based on the fact that RanBP3 interacts with Crm1 in vitro (Englmeier et al., 2001; Lindsay et al., 2001), we next examined the in vivo interaction between RanBP3 and Crm1. As shown in Figure 5C, the interaction between overexpressed RanBP3 and endogenous Crm1 was detected in CHAPS lysis buffer, but not in Triton X-100 and NP-40 lysis buffer. However, this interaction pattern was not changed by serum addition. We further examined this interaction using WT RanBP3 and the S58A mutant RanBP3 in the CHAPS lysis buffer. Neither serum nor mutation of RanBP3 changed this interaction (Figure 5D).

(B) HEK293 cells were transfected with HA-WT Akt for 24 hr and then incubated in serum-free medium for an additional 20 hr. During the last 1 hr, cells were left untreated or treated with 20 mM LY294002. After treatment with 100 nM insulin, immunoprecipitation and Akt phosphotransferase assays were performed using GST-WT RanBP3, GST-S58A RanBP3, and GST-S153A RanBP3 as substrates. (C) HEK293 cells were transfected with either HA-Akt or control vector with FLAG-RanBP3 for 24 hr and then incubated in serum-free media for an additional 20 hr. Cells were treated with 100 nM insulin for 15 min and lysed with either CHAPS- or Triton X-100-containing buffer in the presence or absence of DSP, a crosslinking reagent. Immunoprecipitation and immunoblot analysis were performed. The arrowheads indicate the HA-Akt coimmunoprecipitating FLAG-tagged RanBP3. (D) HEK293 cells were transfected with HA-Akt for 24 hr and then incubated in serum-free medium for an additional 20 hr. Cells were lysed, and immunoblot analysis was performed. (E) Akt knockdown HEK293 cells were generated using Akt1 and Akt2 shRNA. Cells were incubated in serum-free media for 20 hr. After treatment with 100 nM insulin for 15 min, cells were lysed, and immunoblot analysis was performed. (F) HEK293 cells were incubated in serum-free media for 20 hr. During the last 1 hr, cells were treated with pleckstrin homology domain-dependent Akt inhibitor. After treatment with 100 nM insulin for 15 min, cells were lysed, and immunoblot analysis was performed.

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Figure 4. Growth Factors Regulate RanBP3 Phosphorylation in PI3K/Akt- and ERK/RSK-Dependent Pathways (A–F) HEK293 cells were incubated in serum-free media for 20 hr. During the last 1 hr, cells were treated with inhibitors (10 mM U0126, 20 mM LY294002, or 20 ng/ ml rapamycin). After treatment with indicated agonists (50 ng/ml PMA, 100 nM insulin, or 50 ng/ml EGF) for 15 min, cells were lysed, and immunoblot analysis was performed.

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We next examined the in vivo interaction of RanBP3 and Ran. Although the Ran protein is very abundant in cells, we could not detect any interaction between overexpressed RanBP3 and endogenous Ran in the presence or absence of N-ethylmaleimide (NEM), an alkylating agent that blocks RanGAP activity (data not shown). We next generated RanBP3 and Ran double-knockdown cells and expressed RanBP3 and E46G Ran to nearendogenous level (Figure 5E). This Ran mutant is insensitive to RanGAP but undergoes RCC1-mediated nucleotide exchange; therefore, it is GTP bound in cells (Richards et al., 1997). Given the fact that the RanBP3-binding affinity for Ran-GTP in vitro is very weak compared to the interaction of RanBP1 and Ran (Noguchi et al., 1997), we used the crosslinking reagent, DSP, in the CHAPS lysis buffer. As shown in Figure 5F, serum stimulation increased this interaction. Furthermore, PI3-K and MEK/ERK inhibitors blocked the interaction induced by serum. These results suggest the possibility that phosphorylation of RanBP3 may increase its affinity for Ran. This conclusion is supported by the decrease interaction of S58A RanBP3 with E46G Ran in these cells (Figure S2). RanBP3 Phosphorylation Regulates the Ran Gradient in Part by Controlling RCC1 Activity Previous work has shown that overexpression of either a dominant-active or a dominant-negative form of trafficking-related molecule often inhibits trafficking because it disrupts the trafficking balance. To avoid disrupting this balance, our strategy was to knock down endogenous RanBP3 and to replace it by ectopically expressing endogenous levels of WT RanBP3 or S58A RanBP3. For the knockdown of RanBP3 in cells, two different lentiviral RanBP3 shRNA sequences were used. As shown in Figure 6A, we obtained more than 80% knockdown of RanBP3 with both lentiviral constructs. RanBP3 has been shown to interact with CRM1, RCC1, and Ran in vitro; therefore, we investigated possible changes in complex formation of these proteins in RanBP3 knockdown cells. Unexpectedly, we detected no differences in the levels of CRM1, RCC1, and Ran proteins when RanBP3 expression was dramatically reduced (Figure 6A). We next investigated the localization of these proteins. Control and knockdown cells did not exhibit any difference in CRM1 and RCC1 localization. Interestingly, however, the cellular Ran distribution was altered in RanBP3 knockdown cells (Figure 6B). In control cells, most of the Ran was localized in the nucleus, whereas much more Ran was observed in the cytosol of RanBP3 knockdown cells (Figure 6B and Figure S3). The same results were obtained with a second RanBP3 shRNA lentiviral construct (data not shown). These data indicate that RanBP3 plays an important role in establishing the Ran gradient in cells. To test whether RanBP3 phosphorylation affected the Ran gradient, we stably expressed WT RanBP3 and S58A RanBP3 to endogenous levels in RanBP3 knockdown cells (Figure 6C). Using these cells, we examined the localization of RCC1 and Ran. Importantly, WT RanBP3, but not S58A RanBP3, restored the disrupted Ran gradient observed in RanBP3 knockdown cells (Figure 6D and Figure S4). RCC1 is one of the major factors that establishes the Ran gradient, and RanBP3 has been shown to interact with RCC1 and increase RCC1 activity (Macara, 2001). RanBP3 also forms a trimeric complex with Ran-GTP

and RCC1 (Mueller et al., 1998). Since S58A RanBP3 did not restore the Ran gradient, we hypothesized that RanBP3 phosphorylation may be important for RCC1 activity. We therefore investigated the effect of phosphorylated RanBP3 and S58A RanBP3 on RCC1 activity. As shown in Figure 6E, S58A RanBP3 had an inhibitory effect on RCC1 activity. RanBP3 Regulates Nuclear Transport Given that the Ran gradient has an important function in nucleocytoplasmic transport, especially for the proper nuclear import of proteins, we assessed the possibility that RanBP3 phosphorylation could affect nuclear import. For this purpose, microinjection studies were performed using purified GST-NLS-GFP protein and tetramethylrhodamine-dextran (molecular weight 70 kDa) as an injection marker. Because of the high molecular weight and the absence of a NLS, this fluorescent injection marker cannot move through the nuclear pore. Therefore, when injected into the cytoplasm of control cells, GST-NLS-GFP protein moves into the nucleus while the marker remains in the cytoplasm. We compared nuclear import efficiency in WT RanBP3- or S58A RanBP3expressing cells. As shown in Figure 7A, RanBP3 knockdown cells expressing S58A RanBP3 exhibited an attenuated import efficiency compared to WT RanBP3 rescued cells. These nuclear import data are quantitated in Figure S5A. To determine if RanBP3 phosphorylation regulated the transport of a known target of Ran function, we examined the nuclear import efficiency of ribosomal protein L12 (Plafker and Macara, 2002). As shown in Figure 7B and Figure S5B, nuclear import of this protein at 3 min after microinjection was significantly impaired in RanBP3 knockdown cells rescued with S58A RanBP3 when compared with WT RanBP3. The Ran mutant Ran Q69L has been shown to antagonize nuclear import of proteins (Palacios et al., 1996), and we have shown that RanBP3 phosphorylation can modulate Ran function and the nuclear import efficiency of the ribosomal protein L12. Since alterations in ribosomal protein import can directly affect ribosome assembly, overall protein synthesis, and thus cell growth, we therefore asked if RanBP3 contributes to the regulation of cell proliferation. As shown in Figure 7C, expression of the Ran Q69L mutant inhibited cell proliferation. Similarly, RanBP3 knockdown cells expressing S58A RanBP3 exhibited reduced cell proliferation compared to WT RanBP3 cells (Figure 7D). DISCUSSION The PI3K/Akt and Ras/ERK pathways are two major signaling systems that regulate cell-fate decisions in a variety of cell types in both normal and disease settings. Although the basic backbone of these pathways has been defined, subtle but critical differences in the strength, location, and duration of signaling by these pathways is now known to occur. Exactly how these different aspects of PI3K/Akt and Ras/ERK signaling are regulated and how these differences result in the differential regulation of downstream effectors and cell fates are not well understood. It is clear that signaling in the cytoplasm versus the nucleus will yield very different outcomes; therefore, regulating nucleocytoplasmic transport may provide a means for modifying the cellular response to these ‘‘generic’’ signaling pathways. The data reported

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Figure 5. RanBP3 Phosphorylation Does Not Affect RanBP3 Localization and Binding Affinity toward Crm1 (A) Alignment of RanBP3 protein sequences in various mammals. (B) For the detection of endogenous RanBP3 localization in cells, HeLa cells were stained with anti-RanBP3 antibody and subjected to confocal microscopy. For exogenous RanBP3 detection, HeLa cells were transfected with either HA-WT RanBP3 or HA-S58A RanBP3 and stained with anti-HA antibody. The localization of these proteins was visualized by confocal microscopy.

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here highlight a previously unreported role for the PI3K/Akt and Ras/ERK pathways on nuclear transport. We demonstrate that two basophilic protein serine/threonine kinases, RSK and Akt, associate with and phosphorylate a relatively uncharacterized Ran-binding protein, RanBP3, at the same site. This observation immediately raised the possibility that RanBP3 is a site of signal convergence downstream of the Ras and PI3K pathways, respectively. Furthermore, these results suggested the possibility that both pathways might modulate the function of the small GTPase, Ran, in response to growth-factor signaling. Indeed, we have found that RanBP3 is involved in the formation of the Ran gradient, and phosphorylation of RanBP3 by RSK and Akt regulates Ran gradient formation and nuclear transport. The Ran gradient plays a key role in the proper spatial activation of important regulatory proteins by regulating their nuclear transport (Li et al., 2003; Sazer, 2005); thus, the finding that RanBP3 contributes to establishing the Ran gradient in interphase cells is an important discovery. The establishment of the Ran-GTP gradient across the nuclear envelope imposes directionality on nucleocytoplasmic transport by insuring that both import and export cargo bind to and subsequently dissociate from their respective transport receptors in the appropriate cellular compartment. Ran-GTP is thus perfectly positioned to provide the spatial cues essential for insuring that many proteins, particularly those that participate in or regulate the cell cycle, are present and activated in the right place at the right time (Sazer, 2005). Despite the important function of the Ran gradient, the precise mechanisms by which this gradient is established and prevented from collapsing remain largely unknown. The most acceptable theory is that the compartmentalization of RCC1 (nucleus) and RanGAP (cytosol) is responsible for this gradient. In addition, nuclear transport factor 2 (NTF2) appears to be involved in maintenance of the Ran gradient by regulating RanGDP transport (Smith et al., 1998). NTF2 binds to Ran-GDP in the cytosol and facilitates import of Ran-GDP into the nucleus. Here, we show that RanBP3 regulates the Ran gradient without affecting RCC1 protein levels or localization. Our data suggest that RanBP3 may regulate the Ran gradient in part by a direct interaction with Ran and in part by regulating RCC1 activity, a wellknown contributing factor for establishment of the Ran gradient. RanBP3 has a NLS (Welch et al., 1999), and in interphase cells, most RanBP3 is found in the nucleus under a variety of growth conditions such as during serum deprivation; after stimulation of cells with serum, defined growth factors, or tumor-promoting PMA; and in the absence or presence of a variety of signaling inhibitors (S.-O.Y., unpublished data). Although the interaction is relatively weak compared to RanBP1 (Noguchi et al., 1997), RanBP3 has a Ran-binding domain and prefers Ran-GTP to

Ran-GDP for its binding partner (Macara, 2001; Mueller et al., 1998). Through the interaction with Crm1 and cargo molecules, RanBP3 facilitates the export of the Ran-GTP-cargo complex through the nuclear pore (Englmeier et al., 2001; Lindsay et al., 2001). In this regard, the RanBP3 function uncovered in our study is closely related to the function of NTF2. Whereas NTF2 binds to and transports Ran-GDP into the nucleus, which becomes RanGTP by RCC1, RanBP3 interacts with Ran-GTP and contributes to its transport into the cytosol, where Ran-GTP is hydrolyzed to Ran-GDP by RanGAP. Therefore, RanBP3 and NTF2 regulate the formation of the Ran gradient by modulating the cycling of Ran-GDP and Ran-GTP. At this point, however, we cannot exclude the possibility that RanBP3 may sequester free Ran-GTP in the nucleus for later usage. In addition to nucleocytoplasmic transport in interphase cells, the Ran gradient is also essential for kinetochore function, spindle assembly, microtubule dynamics, nuclear envelope reformation, and other mitotic events during mitosis (Clarke, 2005; Sazer, 2005). As observed during interphase, the mitotic Ran gradient is similarly formed by RCC1 (Clarke, 2005; Moore et al., 2002). Moreover, Crm1, a nuclear exporter in interphase cells, was recently shown to be an essential factor for the formation of kinetochore fibers and for faithful chromosome segregation, thereby regulating mitotic spindle assembly during mitosis (Arnaoutov et al., 2005). Considering our data and the previously known function of RanBP3 as a binding partner of Ran, RCC1, and Crm1, it will be of interest to determine if RanBP3 also regulates the Ran gradient during mitosis. It is intriguing that Ras/ERK/RSK and PI3K/Akt pathways can contribute to the regulation of nuclear transport through RanBP3 phosphorylation. There are several ways that nuclear transport can be regulated (Kau et al., 2004). The first is by posttranslational modification of the cargo molecules themselves or their binding partners that mask or increase their transport, which in turn affects their ability to interact with their cognate transporter. A second way that nucleocytoplasmic transport is modulated is through the regulation of the level of transporters. Some importins are differentially expressed in specific tissues and therefore might transport cargoes only during specific stages of development or function in a particular cell type. Finally, the nuclear pore itself can offer an added level of regulation. The number of functional pores varies depending on the growth state of the cell, which in turn affects the overall permeability of the nucleus. Here we identify a previously unreported mechanism for regulating nuclear transport—through growth-factor-regulated posttranslational modification of a transporter cofactor. Considering the growing evidence linking subtle differences in the spatial and temporal regulation of signaling pathways to specific cell-fate

(C) HEK293 cells were transfected with either HA-RanBP3 or control plasmid for 24 hr and then incubated in serum-free media for an additional 20 hr. Cells were left untreated or treated with 20% serum for 15 min and lysed with Triton X-100, NP-40, or CHAPS buffer. Immunoprecipitation and immunblot analysis were performed. (D) HEK293 cells were transfected with control plasmid, HA-WT RanBP3, or HA-S58A RanBP3 for 24 hr and then incubated in serum-free media for an additional 20 hr. Cells were left untreated or treated with 20% serum, and immunoprecipitation and immunoblot analysis were performed. (E) RanBP3 and Ran double-knockdown HEK293 cells were generated using RanBP3 and Ran shRNA. Knockdown cells were transfected with RanBP3 and E46G-Ran, and immunoblot analysis was performed. (F) RanBP3 and Ran double-knockdown cells were transfected with either HA-E46G Ran or control vector plus Flga-RanBP3 for 24 hr and then incubated in serum-free media for an additional 20 hr. During the last 1 hr, cells were treated with inhibitors (10 mM U0126 and 20 mM LY294002). After incubation with 20% serum for 15 min, cells were lysed, and immunoprecipitation was performed.

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Figure 6. RanBP3 Modulates the Ran Gradient (A) RanBP3 knockdown HeLa cells were generated as described in the Experimental Procedures. Stable cells were lysed, and immunoblot analysis was performed. (B) RanBP3 knockdown and control HeLa cells were stained with antibodies against RanBP3, CRM1, RCC1, and Ran. Localization of these proteins was visualized by confocal microscopy. (C) Stable WT RanBP3 and S58A RanBP3 HeLa cells were generated from RanBP3 knockdown cells, and immunoblot analysis was performed. (D) Stable WT RanBP3 and S58A RanBP3 HeLa cells were stained with anti-RCC1 and anti-Ran antibodies and subjected to confocal microscopy. (E) RanGEF (RCC1) assay was performed as described in methods. Data represent the mean ± SD of three independent experiments. Results were statistically significant (*p < 0.01) using Student’s t test.

decisions, the finding that Ras/ERK/RSK and PI3K/Akt signaling can modulate the Ran gradient expands our understanding and views of how nucleocytoplasmic transport can be modulated by mitogenic inputs. Since RanBP3 phosphorylation by RSK and Akt enhances nuclear transport rate, the strength of activation of these enzymes, the duration of activation, their cellular localization, and the differential regulation of these pathways will all contribute to their ability to differentially modulate RanBP3 func-

tion and the Ran gradient throughout the cell cycle. This level of regulation will be layered upon the other mechanisms of regulating nucleocytoplasmic transport as discussed above, thus providing another way to fine-tune this critical process at a general or very specific level. Many signaling molecules, transcription factors, cell-cycle regulators, growth-factor receptors, viral proteins, and disease-related proteins are transported in and out of the nucleus (Xu and

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Figure 7. RanBP3 Phosphorylation Regulates Nuclear Transport (A) GST-NLS-GFP and injection marker were injected into the cytoplasm of HeLa cells. After 10 min, cells were fixed, and the injected proteins were visualized by confocal microscopy. (B) GFP-L12 and injection marker were injected into the cytoplasm of HeLa cells. After 3 min, cells were fixed, and the injected proteins were visualized by confocal microscopy. (C) Cells were transfected with control vector or Ran constructs, and cell numbers were counted at the indicated times. Data represent the mean ± SD of three independent experiments. Results were statistically significant (*p < 0.01) using Student’s t test. (D) RanBP3 knockdown cells expressing stable WT RanBP3 and S58A RanBP3 were plated, and cell numbers were counted at indicated times. Data represent the mean ± SD of three independent experiments. Results were statistically significant (*p < 0.01) using Student’s t test.

Massague, 2004). Some proteins shuttle between the nucleus and cytoplasm continuously, and some do so only once. Other proteins do not shuttle at all but localize permanently in or out of the nucleus following translation (Tartakoff et al., 2000). For nucleocytoplasmic transport, many proteins have a NLS and/or nuclear export signal (NES). Importins (nuclear importers such as importin a and b) and exportins (such as the Crm1 nuclear exporter) recognize a cargo’s NLS and NES sequence, respectively, and as a result properly transport them (Macara, 2001; Weis, 2003). However, nuclear import and/or export of some proteins are importin independent and/or exportin independent even

when cargos have NLS and/or NES signals. In addition, although most nucleocytoplasmic protein transport is dependent on the Ran gradient, some proteins move into and/or out of the nucleus independently of Ran. Moreover, transport rates vary for different cargos. These observations imply that there are likely many ways to contribute to the general as well as the specific regulation of nucleocytoplasmic transport. It is not surprising that the regulation of nuclear transport is very complicated and involves the intricate interplay of multiple protein components, since nucleocytoplasmic transport contributes to the proper regulation of transcription, translation, cell-cycle progression, and other

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cellular processes that determine cell fate: survival, apoptosis, differentiation, etc. (Weis, 2003). For example, we have found that nuclear import of the ribosomal protein L12 is affected by RanBP3. Ribosome biogenesis is a multistep process: ribosomal protein synthesis in the cytoplasm, import into the nucleus, ribosome assembly in the nucleolus, and export to the cytoplasm. For proper functioning of ribosomes, all these steps should be efficiently controlled. These preliminary data indicate that RanBP3 may be involved in a step of ribosome biogenesis, a possibility that will require more experimentation. Further studies are also needed to identify and characterize other targets of RanBP3mediated transport. Despite the important function of Ran and its effectors, little is known about how these effectors are regulated in cells. Recently, it was shown that RCC1 is phosphorylated in mitosis by Cdc2 kinase (Li and Zheng, 2004). This phosphorylation is essential for positioning a high Ran-GTP concentration on mitotic chromosomes and for spindle assembly in mammalian cells. Another Ran modulator that may be regulated is a Ran-GDP dissociation factor for the NTF2-RanGDP complex. As described above, NTF2 binds to Ran-GDP in the cytosol with high affinity and promotes translocation of this complex into the nucleus. In the nucleus, Ran-GDP is released from NTF2 and converted into Ran-GTP by RCC1. Ran-binding proteins within the nucleus sequester free Ran-GTP and further nucleotide exchange by mass action (Smith et al., 1998). However, how Ran-GDP is dissociated from NTF2 is unknown. Recently, it was proposed that there is an unidentified protein that stimulates dissociation of Ran-GDP from NTF2 (Yamada et al., 2004). From the data obtained from their experiments, the authors proposed that this unidentified protein might interact with RCC1 and enhance RCC1 catalytic activity. Interestingly, it was also suggested that the function of this unknown protein could be regulated by phosphorylation. From our data, the localization and function of RanBP3 reflect the properties of this unidentified protein. Since NTF2 and RanBP3 play important roles in Ran gradient formation, it will be of interest to investigate the relationship between these proteins. It is clear that the regulation of nucleocytoplasmic transport is integrated with subtle differences in temporal and spatial regulation of signaling proteins to ensure a proper biological response to extracellular signals. Deregulation of nuclear transport, on the other hand, has been linked to several human diseases (Davis et al., 2007; Fabbro and Henderson, 2003; Kau et al., 2004), likely the result of failed signaling at the right place and/or right time. The diseases linked to improper nuclear transport include acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), familial hypercholesterolemia, cystic fibrosis, schizophrenia, retinitis pigmentosa, nephrogenic diabetes insipidus, and others (Davis et al., 2007). Failure to properly localize certain proteins can also render cells resistant to drugs. In the case of cancers, altered localization of tumor suppressors (p53, INI1/hSNF5, BRCA1), cell-cycle regulators (p21WAF-1, p27Kip1), and transcription factors (FOXO, NF-kB) are often found to be closely linked to tumor progression (Kau et al., 2004). The contribution of inappropriate localization of important regulatory proteins to the development or progression of various diseases has made this process an excellent target for therapeutic intervention. Therefore, further examination of nuclear transport mechanisms

may reveal different ways to treat a growing number of diseases ranging from metabolic disorders to cancer that have been linked to improper regulation of nucleocytoplasmic transport (Davis et al., 2007). In this regard, our studies provide important new insights into how the Ran gradient is established and how the protein kinases RSK and Akt, enzymes linked to many critical biological processes, such as cell growth, proliferation, survival, and migration, can modulate nucleocytoplasmic protein transport. Future work should now be focused toward understanding how an improperly regulated RanBP3 contributes to various human disorders and diseases. EXPERIMENTAL PROCEDURES Crosslinking DSP crosslinking was performed according to the manufacturer’s protocol (Pierce). Briefly, cells were lysed in buffer (40 mM HEPES [pH 7.5], 120 mM NaCl, 1 mM EDTA, 10 mM b-glycerophosphate, 50 mM NaF, 2 mM phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM DTT) containing 0.8 mg/ml DSP and detergent (1% Triton X-100, 1% NP40, or 0.2% CHAPS). After incubation at room temperature for 30 min, the crosslinking reaction was quenched by adding 1 M Tris (pH 7.5) to a final concentration of 20 mM. After additional incubation for 20 min, immunoprecipitation was performed. Supplemental Data Supplemental Data include Supplemental Experimental Procedures, Supplemental References, and five figures and can be found with this article online at http://www.molecule.org/cgi/content/full/29/3/362/DC1/. ACKNOWLEDGMENTS We thank Drs. Ian G. Macara, Philip N. Tsichlis, Yoshihiro Yoneda, Karsten Weis, Scott M. Plafker, Andrew Wilde, William C. Hahn, Mien-Chie Hung, Andrew L. Kung, and David Baltimore for generously providing reagents. We would like to thank all the Blenis lab members for critical reading of the manuscript. We also thank Jennifer Waters, Lara Petrak, and the Nikon Imaging Center at Harvard Medical School for microscopy. This work was supported by National Institutes of Health grants R37 CA46595 (J.B.), RO1 GM51405 (J.B.), and HG3456 (S.P.G.). Received: April 23, 2007 Revised: August 17, 2007 Accepted: December 15, 2007 Published: February 14, 2008 REFERENCES Anjum, R., Roux, P.P., Ballif, B.A., Gygi, S.P., and Blenis, J. (2005). The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling. Curr. Biol. 15, 1762–1767. Arnaoutov, A., Azuma, Y., Ribbeck, K., Joseph, J., Boyarchuk, Y., Karpova, T., McNally, J., and Dasso, M. (2005). Crm1 is a mitotic effector of Ran-GTP in somatic cells. Nat. Cell Biol. 7, 626–632. Bonni, A., Brunet, A., West, A.E., Datta, S.R., Takasu, M.A., and Greenberg, M.E. (1999). Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286, 1358–1362. Cartlidge, R.A., Knebel, A., Peggie, M., Alexandrov, A., Phizicky, E.M., and Cohen, P. (2005). The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells. EMBO J. 24, 1696–1705. Clarke, P.R. (2005). The Crm de la creme of mitosis. Nat. Cell Biol. 7, 551–552. Davis, J.R., Kakar, M., and Lim, C.S. (2007). Controlling protein compartmentalization to overcome disease. Pharm. Res. 24, 17–27.

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