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Identification and Characterization of a General Nuclear Translocation Signal in Signaling Proteins
Molecular Cell
Article Identification and Characterization of a General Nuclear Translocation Signal in Signaling Proteins Dana Chuderland,1 Alexander Konson,1 and Rony Seger1,* 1Department of Biological Regulation, The Weizmann Institute of Science, 76100 Rehovot, Israel *Correspondence: [email protected] DOI 10.1016/j.molcel.2008.08.007
SUMMARY
Upon stimulation, many proteins translocate into the nucleus in order to regulate a variety of cellular processes. The mechanism underlying the translocation is not clear since many of these proteins lack a canonical nuclear localization signal (NLS). We searched for an alternative mechanism in extracellular signal-regulated kinase (ERK)-2 and identified a 3 amino acid domain (SPS) that is phosphorylated upon stimulation to induce nuclear translocation of ERK2. A 19 amino acid stretch containing this phosphorylated domain inserts nondiffusible proteins to the nucleus autonomously. The phosphorylated SPS acts by binding to importin7 and the release from nuclear pore proteins. This allows its functioning both in passive and active ERK transports. A similar domain appears in many cytonuclear shuttling proteins, and we found that phosphorylation of similar sequences in SMAD3 or MEK1 also induces their nuclear accumulation. Therefore, our findings show that this phosphorylated domain acts as a general nuclear translocation signal (NTS). INTRODUCTION To ensure accurate cellular functioning, the spatial distribution of proteins needs to be delicately regulated and coordinated. This is particularly apparent in many signaling proteins that dynamically and rapidly change their localization upon extracellular stimulation. In order to maintain such regulation, the nucleus is separated from the cytoplasm by a double membrane envelope that allows a selective entrance of proteins through specialized nuclear pore complexes (NPC; Nigg, 1997). The selectivity of nuclear localization is primarily mediated by a nuclear localization signal (NLS) harbored within the sequence of the nuclear protein (Schlenstedt, 1996). The NLS is recognized by special carrier proteins, the importins, which, upon binding, facilitate the transfer of proteins across the NPC (Tran and Wente, 2006). However, not all cytonuclear shuttling proteins contain the canonical NLS and, therefore, use other, NLS-independent mechanisms for their passage through the NPC. Some of the characterized
NLS-independent mechanisms include passive diffusion of small proteins (<30–40 kDa), distinct nuclear-directing motifs (Christophe et al., 2000), interaction with NLS-containing proteins, or alternatively, a direct interaction with the nuclear pore proteins (NUPs; Xu and Massague, 2004). However, these mechanisms do not always allow timely regulation of nuclear processes and, therefore, the molecular mechanism(s) that allows the rapid and reversible NLS-independent translocation of signaling proteins upon stimulation is still obscure. Examples for signaling proteins that translocate into the nucleus upon stimulation in an NLS-independent manner include ERKs, MEKs (Yoon and Seger, 2006), and SMADs (Massague et al., 2005). The importance of these proteins in regulation of proliferation and differentiation led to many studies on the nuclear translocation of ERKs (Chen et al., 1992; Chuderland et al., 2008; Lorenzen et al., 2001; Matsubayashi et al., 2001; Yazicioglu et al., 2007), MEKs (Fukuda et al., 1996; Jaaro et al., 1997), and SMAD3 (Chen et al., 2005; Liu et al., 1997; Xiao et al., 2000a; Xiao et al., 2000b; Xu et al., 2003) (for more information see the Supplemental Data available online). Although a possible involvement of the canonical NLS machinery was initially identified in some systems, it was recently suggested that these proteins may translocate into the nucleus by a direct interaction with NUPs (Xu and Massague, 2004). We identify here a sequence (Ser-Pro-Ser, SPS) in ERKs that is phosphorylated upon stimulation to mediate their nuclear translocation. The phosphorylated sequence within a 19 amino acid stretch acts autonomously by binding to the nuclear translocating protein importin7 (Imp7) and by promoting release from NUPs in active and possibly also passive transports. Phosphorylation of the same or similar (Thr-Pro-Thr, TPT) sequence is responsible for nuclear translocation of other proteins such as SMAD3 and MEKs, which is also mediated by Imp7. We propose that phosphorylated S/T-P-S/T acts as a general nuclear translocation signal (NTS) for NLS-lacking proteins that shuttle to the nucleus upon stimulation.
RESULTS The SPS Domain Plays a Role in the Nuclear Accumulation of Overexpressed ERK2 Although the regulation and underlying mechanism of ERKs nuclear translocation remains unclear, previous studies suggest a role for the kinase insert domain (KID; Lee et al., 2004). A
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Figure 1. Identification of SPS as Important Domain for ERK2’s Nuclear Translocation (A) Schematic representation of ERK2, including the TEY and the SPS domains. (B) Role of SPS in nonregulated accumulation of ERK2 in the nucleus. CHO or COS7 cells were grown on cover slips and transfected with either WT-GFP-ERK2 (WT-ERK2) or the mutants 244-6A and D244-6. Twenty-four hours after transfection, the cells were serum starved (0.1% FBS, 16 hr), fixed, stained with DAPI, and visualized using a fluorescent microscope. (C) Role of SPS in the stimulated nuclear translocation of ERK2. CHO cells were cotransfected with HA-MEK1 together with WTERK2, 244-6A, or D244-6. Twenty-four hours after transfection, the cells were serum starved and treated for 15 min with either 250 or 10 nM TPA. After treatments, the cells were fixed and stained with either aHA Ab to detect MEK1 or DAPI for nuclear staining. ERK was detected by its GFP fluorescence. (D) Equilibration of TEY phosphorylation with different TPA concentrations. The TEY phosphorylation of GFP-ERK2 and its mutants was detected by a direct WB using apTEY and a-general ERK Abs (agERK). (E) 244-6A-ERK2 binds to 6His-MEK1 and is released upon phosphorylation. Serum-starved CHO cells expressing WT-ERK2 (WT) or 244-6A-ERK2 were treated with TPA (250 nM, 15 min) followed by IP and stringently washed. The IPed constructs, as well as beads alone, were incubated with 0.5 mg recombinant 6HisMEK1 (30 min, 30 C). Thereafter, the beads were mildly washed and subjected to WB using aMEK and agERK Abs.
as WT-GFP-ERK2, were transfected into CHO cells. Unlike the reported nuclear accumulation of GFPERK2 in these cells (Rubinfeld et al., 1999), both mutants were found primarily in the cytoplasm (Figure 1B). These results were validated using COS7 cells, which express higher amount of ectopic proteins, clearly implicating the SPS domain in determination of ERK2 localization in quiescent cells.
thorough mutagenesis of ERK2’s KID revealed a 3 amino acids sequence, composed of residues Ser244, Pro245, and Ser 246 (SPS), which participates in the regulation of ERK2 localization. To examine the function of this region, we either replaced the three residues to Ala or deleted them (244-6A and D244-6; Figure 1A; Figure S1). The mutated ERKs were then fused to a green fluorescent protein (GFP) and these constructs, as well
The SPS Domain Plays a Role in the Nuclear Translocation of ERK2 upon Stimulation Since overexpressed ERKs translocate to the nucleus mainly by passive diffusion while much of the translocation of endogenous ERKs is mediated by an activated transport (Adachi et al., 1999), it was important to examine the role of the SPS domain in a stimulusdependent translocation. In order to do so, we cotransfected the ERK constructs together with MEK1, which acts as an anchoring protein, to secure cytoplasmic localization of ERKs in resting cells (Fukuda et al., 1997; Rubinfeld et al., 1999). As expected (Rubinfeld et al., 1999), the cotransfected ERK2 was localized in the cytoplasm of the resting CHO cells and translocated into the nucleus upon TPA stimulation (250 nM). However, the two SPS mutants remained localized in the cytoplasm irrespective of TPA stimulation (Figure 1C). A possible reason for this lack of translocation could have been a reduced phosphorylation of the Thr and Tyr residues within the TEY motif of the mutants. Indeed, the rate of phosphorylation of the mutants detected by anti-doubly phosphorylated TEY-ERK (apTEY)
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antibody (Ab) was lower than that of WT-ERK2 (Figure 1D), indicating that the SPS domain is involved in the phosphorylation of ERKs by MEKs. To determine whether the cytoplasmic retention of the SPS mutants is a result of reduced TEY phosphorylation, we equilibrated the phosphorylation levels by using different concentrations of TPA (10 nM for WT-ERK2 and 250 nM for the mutants Figure 1C). Also with equivalent phosphorylation levels, WT-GFP-ERK2 was localized to the nucleus, whereas the SPS mutants remained restricted to the cytoplasm. Furthermore, 244-6A-ERK2 interacted with MEK1 and detached from it upon stimulation, similar to WT-ERK2 (Figure 1E). This indicates that the change by SPS mutation affects only the phosphorylation by MEKs, but not ERKs’ binding or detachment, confirming that no major conformational changes are induced by the mutations. Therefore, the lack of nuclear accumulation of the SPSmutated ERK2 is probably due to other, inherent effect of this region. Taken together, these data implicate the SPS domain in ERKs nuclear translocation, both in resting (mostly passive transport of overexpressed ERKs) and stimulated cells. The Ser Residues in the SPS Domain Are Phosphorylated upon Cellular Stimulation The abrogation of stimulation-dependent nuclear trafficking of SPS-mutated ERK2 and the presence of Ser residues within this domain may unveil a potential phosphorylation site. Therefore, we examined the possibility that the Ser residues within the SPS domain undergo an additional phosphorylation concomitantly with nuclear translocation. Mass spectroscopic (MS) analysis (Maldi-Tof) was performed on immunoprecipitated (IPed) GFP-ERK2 from either stimulated or nonstimulated COS7 cells. In addition to a trypsin-digested peptide containing the pTEY (Yoon and Seger, 2006), we found in proteins derived from stimulated cells also a peptide corresponding to the SPS domain containing one and possibly two incorporated phosphate(s). Since the only phosphoacceptors in this peptide were the two Ser residues in the SPS, it is likely that this higher mass peptide(s) represents phosphorylation of one or possibly two Ser residues within this domain. Characterization of SPS Phosphorylation by a Specific Ab To further study the occurrence and role of the SPS phosphorylation, we raised polyclonal Ab directed against a doubly-phosphorylated SPS peptide. In a western blot (WB), the Ab weakly recognized a 72 kDa band, corresponding to GFP-ERK2 IPed from serum-starved, transfected COS7 cells (Figure 2A). This recognition of GFP-ERK2 was significantly increased when the GFP-ERK2 was IPed from VOOH, TPA, or EGF stimulated cells, confirming that the SPS is phosphorylated upon stimulation. Indeed, time course of EGF stimulation revealed a transient increase in SPS phosphorylation with a peak at 15 min (Figure 2B), which was slower than TEY phosphorylation that peaked at 5 min after stimulation. This rate of SPS phosphorylation correlated better with translocation (data not shown), suggesting sequential events in the activation of ERKs, and their subsequent SPS phosphorylation and translocation to the nucleus. Similar phosphorylation patterns detected by the Ab were also found
with endogenous ERKs from EGF- or VOOH-stimulated cells (Figure 2C). In order to confirm that the Ab is indeed specific to the pSPS, we first used a competition with the antigenic peptide and found that it completely abolishes the Ab recognition (Figure 2D). Moreover, treatment of phosphorylated ERK2 with alkaline phosphatase demonstrated a significant reduction of the immunoreactive pSPS (Figure 2E), as well as the control pTEY (Figure 2F). We then examined whether the Ab recognizes just one of the phosphorylated residues or both of them. To do so, we mutated either Ser244 (AS) or Ser246 (SA) to Ala. These constructs, as well as the WT ERK2 and the ERK2-244-6A mutant, were transfected to COS7 cells, which were either stimulated or left nontreated. We found that the apSPS Ab reacts with the WT and the monophosphorylatable ERK2 mutants, but not with the nonphosphorylatable mutant, only from stimulated cells (Figure 2G). Thus, these results confirm the specificity of the apSPS Ab to the phosphorylated form of SPS and its ability to recognize either the monophosphorylated or the doubly-phosphorylated SPS. These results also show that, in agreement with the MS data, stimulation can cause phosphorylation of either both Ser244 and Ser246 together or just one of them alone. Finally, we found that the nonphosphorylatable TEY mutant of ERK2 (TEY-AAA) can still be recognized by the apSPS Ab upon stimulation, albeit to a slightly lower extent (Figure 2H). This indicates that the Ab does not recognize the phosphorylated TEY and shows again that the SPS phosphorylation occurs independently from that of TEY. Thus, our results identify an important stimulationdependent phosphorylation of ERKs on their SPS domain. SPS Phosphorylation Induces Nuclear Translocation of ERK2 Both the phosphorylation on SPS domain and the abrogation of nuclear translocation by SPS mutants suggest that this phosphorylation may be required for the nuclear translocation of ERKs. To further study this possibility, we replaced the two Ser residues in the SPS domain with Glu that acts as a phosphomimetic residue. GFP-EPE-ERK2 showed a pronounced nuclear localization, while the APA mutant exhibited a cytoplasmic distribution similar to that of the 244-6A construct (Figure 3A; Figure S1). These differences in distribution were confirmed by subcellular fractionation showing that about 75% of WT-ERK2, 95% of EPE-ERK2, and only 5% of APA-ERK2 are localized in the nucleus 48 hr after transfection (Figure 3B). The difference between WT- and EPE-ERK2 was even more pronounced shortly after transfection (8 hrs when the GFP protein is just visible; Figure 3C), although their expression levels were similar, indicating that the translocation of overexpressed EPE-ERK2 into the nucleus is faster than that of WT-ERK2. Similar results were obtained upon treatment with leptomycine B (LMB) that prevents nuclear export (Figure S2), indicating that SPS mutation modulates the nuclear import and not export of ERKs. To further verify and characterize the importance of the SPS domain in mediating the nuclear translocation of ERK2, we used several distinct methods. First, we found that SPS mutants affected the translocation of ERK2 in an in vitro system consisting of recombinant proteins, cytosolic extract, and fixed digitonin-permeabilized cells (Chuderland et al., 2008; Matsubayashi et al.,
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Figure 2. Characterization of SPS Phosphorylation Using apSPS Ab (A) Elevation of pSPS detection upon stimulation. COS7 cells transfected with WT-GFP-ERK2 (GFP-ERK2) were serum-starved and treated with VOOH (V, 100 mM Na3VO4 and 200 mM H2O2, 15 min), TPA (T, 250 nM, 15 min), EGF (E, 50 ng/ml, 15 min), or left untreated (NT). The GFP-ERK2 was IPed with aGFP Ab, extensively washed, and subjected to WB using apSPS and agERK Abs. (B) Time course of SPS phosphorylation. Serumstarved COS7 cells overexpressing GFP-ERK2 were treated with EGF (50 ng/ml) for the indicated times. The GFP-ERK2 was IPed with aGFP Ab, extensively washed, and subjected to WB with apSPS, apTEY and agERK Abs. (C) Elevation of apSPS Ab immunoreactivity upon stimulation of endogenous ERKs. Serum-starved COS7 cells were treated with EGF (50 ng/ml) or VOOH for the indicated times. SPS phosphorylation was detected using the apSPS Ab, and this was compared to the amount of general ERKs detected by agERK. (D) Specificity of the apSPS Ab. Serum-starved COS7 cells overexpressing either GFP-ERK2 or 244-6A were treated with EGF (50 ng/ml, 15 min) following WB using apSPS and agERK Abs. (E and F) Further characterization of the apSPS Ab’s specificity. GFP-ERK2 was IPed from VOOH stimulated (Act) or nonstimulated (NT), transfected COS7 cells. The IPed protein was then incubated with shrimp’s alkaline phosphatase (SAP, 15 units, 30 min, 30 C) and subjected to WB with apSPS (E), apTEY (F), or agERK Abs. (G) Recognition of monophosphorylated SPS by the apSPS Ab. Serum-starved COS7 cells overexpressing the following mutants GFP-ERK2 (WT), GFP-APS-ERK2 (AS), GFP-SPA-ERK2 (SA), and GFP-APA-ERK2 (APA) were stimulated with EGF (50 ng/ml, 10 min) or left untreated (NT). The cells were then extracted and the lysates were subjected to WB using apSPS and agERK Abs. (H) TEY phosphorylation is not required for SPS phosphorylation. COS7 cells overexpressing WT-ERK2 or TEY-AAA were treated and analyzed as described in (D).
2001). In this system, the APA mutation prevented the translocation of recombinant ERK2, while the EPE mutation facilitated it, confirming the importance of the SPS phosphorylation (Figure S3). We then used monophosphomimetic ERK2 mutants to follow the importance of each of the phosphorylation sites and found that each of them is required for the nuclear translocation, although the phospho-Ser246 is somewhat more important (Figure S4). Similar effects of the SPS mutants were obtained when we used MEK1 retention to study stimulated ERK2 translocation (Figure S5). All SPS mutants were retained in the cytoplasm upon overexpression of MEK1, and TPA stimulation allowed nuclear translocation of either doubly- or monophosphomimetic mutants, but not of the APA mutant. Thus, in agreement with the in vitro binding in Figure 1E, these results indicate that the SPS domain or its phopshorylation do not play a role in MEK binding, but the phosphorylation of both or each one of its Sers can induce passive and stimulated nuclear translocation of ERKs. The lack of correlation between SPS phosphorylation and MEK binding or TEY phosphorylation corresponds to previous findings that the nuclear accumulation of ERK2 does not require
TEY phosphorylation (Wolf et al., 2001). Indeed, we found that mutating the TEY motif to Ala does not change the nuclear translocation of ERK2, but this effect is still seen when Ser244 and Ser246 are mutated to Glu (Figure S6). These results are consistent with a model in which the phosphorylation of TEY is important for the detachment from cytoplasmic anchors, while the subsequent SPS phosphorylation is responsible for the actual translocation. Importantly, the SPS-induced translocation seems to be important for the downstream activity of ERKs, as phosphodeficient SPS mutants prevented serum-induced cell proliferation, while the phosphomimetic EPE-ERK2 slightly enhanced this process (Figure S7). Use of b-Galactosidase to Study the Role of Phosphorylated SPS To validate the general importance of the SPS domain and examine whether it can act autonomously, we used a construct of b-Galactosidase fused to GFP (b-Gal, Figure 4A). This 145 kDa construct (Figure 4B), which is too big to freely diffuse to the nucleus, was indeed localized exclusively in the cytoplasm
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Figure 3. SPS Phosphorylation Is Required for the Nuclear Translocation of ERK2 (A) Replacing the Ser residues in the SPS with Glu induces the nuclear accumulation of ERK2. Serum-starved CHO cells overexpressing WT-GFP-ERK2 (WT), APA-GFP-ERK2 (APA), or EPE-GFP-ERK2 (EPE), were fixed, stained with DAPI, and visualized (as in Figure 1). (B) Subcellular fractionation confirms the fluorescence results. Serum-starved COS7 cells overexpressing the ERK2 constructs (as in [A]) were grown in 10 cm plates and then subjected to cellular fractionation. WB of aliquots of the fractions was performed with aGFP Ab to detect ERKs, and with atubulin or ac-Jun Abs as markers for cytoplasmic and nuclear fractions respectively. (C) EPE-ERK2 translocates to the nucleus faster than WTERK2. CHO cells were transfected with either WT-ERK2 or EPE-ERK2 constructs. Eight or 12 hr later the cells were either stained with DAPI and visualized or lysed and subjected to WB with aGFP Ab to confirm equal expression.
Since the SPS-containing region mimics some of the functions of the SPS region in ERKs, we undertook to examine whether its overexpression competes with the translocation machinery and, therefore, acts as a dominant-negative construct of endogenous ERK shuttling. Indeed, overexpression of SPS containing 19 amino acid stretch conjugated to a 2GFP chimera (2GFP-SPS) and more so 2GFPAPA inhibited TPA-induced translocation of endogenous ERKs, while the control 2GFP and the nuclear 2GFP-EPE did not affect it at all (Figure S8). Notably, the mutants had no effect on the localization of ERKs in resting cells, indicating that the SPS domain competes with the translocation of endogenous ERKs mainly upon stimulation. The reason for the lack of inhibition by the EPE mutant could be its rapid translocation to the nucleus, which prevents its interaction with the translocation machinery localized in the cytoplasm and is better accessible to the other two, mostly cytoplasmic. Thus, our results suggest that the phospho-SPS (pSPS) serves as a unique and general nuclear translocation signal (NTS) also in endogenous ERKs.
when expressed in CHO or COS7 cells (Figures 4C and 4D). However, fusion of the SPS domain, within the 19 amino acid stretch surrounding it, to the b-Gal induced some nuclear translocation of the construct, which was prevented by the mutation of the SPS to APA and accelerated by the EPE mutation. Moreover, the fusion of the SPS region made the construct sensitive to stimulation, as TPA facilitated the nuclear translocation of the bGal-SPS (Figure 4E), and this was prevented by the APA mutation. These results clearly demonstrate that the SPS domain within the 19 amino acid stretch functions autonomously.
Imp7 Mediates the Stimulation-Dependent Nuclear Translocation of ERKs The nuclear translocation of ERKs (Chen et al., 1992) can occur either by a passive diffusion or by an active transport (Adachi et al., 1999), which are governed by various mechanisms (Yazicioglu et al., 2007). In Drosophila, it was suggested that D-Imp7 (DIM-7) is responsible for the accumulation of ERKs in the nucleus during development upon activation (Lorenzen et al., 2001). Therefore, we examined whether the mammalian analog of this protein and some other importins play a role in ERK translocation in mammals as well. Transfection of SiRNA of Imp7 into HeLa cells dramatically inhibited EGF-induced translocation of ERK2 (Figure 5A). On the other hand, SiRNAs of importin5 (Imp5), importin-b (Imp-b), and transportin1
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Figure 4. The SPS domain, within a 19 Amino Acid Stretch, Is an Autonomous NTS (A) Schematic representation of the b-Gal constructs. (B) Equal expression of the b-Gal constructs. The constructs were overexpressed in COS7 cells, which were grown for 24 hr, serum starved, extracted, and subjected to WB with aGFP Ab. (C) SPS, APA, and EPE peptides fused to the b-Gal construct modify its subcellular localization. Serum-starved CHO and COS7 cells overexpressing the b-Gal constructs were fixed, stained with DAPI, and visualized. (D) Subcellular fractionation confirms the fluorescence results. Subcellular fractionation was performed on COS7 cells transfected with the b-Gal constructs. WB of aliquots of the fractions was performed with aGFP Ab to detect the b-Gal constructs, and with atubulin and a-histone-H1 Abs as cytoplasmic and nuclear markers respectively. (E) TPA stimulation enhances nuclear localization of the b-Gal-SPS. Serum-starved CHO and COS7 cells, overexpressing b-Gal-GFP, b-GalSPS, and b-Gal-APA were stimulated with TPA (250 nM, 15 min) or left untreated, and then fixed, stained with DAPI and visualized.
the activation-induced, but not as much nonstimulated, passive, nuclear translocation of ERKs.
(Trn1) did not significantly influence the nuclear translocation of ERK2 (Figure S9). Interestingly, the SiRNA of Imp7 had only a partial inhibitory effect on the nonstimulated (passive) mechanism of ERK translocation (Adachi et al., 2000), as detected in either LMB-treated (Figure 5B) or WT-ERK2 overexpressing (Figure 5C) cells. In all these experiments, the cells transfected with SiRNA to Imp7 lost considerable amount of Imp7, but not other proteins, such as ERKs, that served as a control (Figure 5D). Moreover, we found that Imp7 can interact with both overexpressed ERK2 (Figure 5E) and endogenous ERKs (Figure 5F), and this interaction was significantly increased upon stimulation with EGF. These results indicate that Imp7 is an important regulator of
Phosphorylated SPS Domain Is Required for ERK2 Interaction with Imp7 and Release from Nuclear Pore Proteins The finding that Imp7 is involved in ERKs nuclear translocation prompted us to examine whether the phosphorylation of the SPS is required for ERK-Imp7 association. We also examined the possible involvement of Imp-b, which seems to cooperate with Imp7 in some nuclear transport systems (Fu et al., 2006). Thus, we first examined the interaction of the various SPS mutants with Imp7 and Imp-b. Co-IP experiments revealed that WT-ERK2 interaction with Imp7 is significantly increased upon TPA stimulation (Figure 6A). Prevention of SPS phosphorylation using APA mutation reduced the interaction, mainly upon stimulation, while the use of the phosphomimetic EPE mutant dramatically increased the interaction either with or without stimulation. These results were confirmed by in vitro binding assay, in which soluble GST-ERK2, GST-APA-ERK2, GST-EPE-ERK2, or GST proteins were incubated with IPed Imp7 (Figure 6B). Indeed, the phosphomimetic mutant interacted with the Imp7 much stronger than WT-ERK2, while the nonphosphorylatable mutant did not associate with Imp7 at all. Similar results were obtained using the 2GFP constructs (Figure 6C), as the SPS conjugated 2GFP did bind Imp7, the APA mutation reduced this interaction,
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Figure 5. The Nuclear Translocation of ERKs Is Mediated by Imp7 (A) EGF-induced ERKs translocation is prevented by SiRNA of Imp7. HeLa cells were transfected with SiRNA of Imp7 or scrambled control SiRNA using oligofectamin. Seventy-two hours after transfection, the cells were serum starved and then either treated with EGF (50 ng/ml, 15 min) or left untreated. Then, the cells were washed, fixed, and stained with agERK Ab and DAPI. (B) LMB-induced ERKs translocation is partially prevented by SiRNA of Imp7. HeLa cells were transfected with control or Imp7 SiRNAs as above. After serum starvation, the cells were either left untreated (basal) or were treated with LMB (5 ng/ml, 30 min), washed, fixed, and stained with agERK and DAPI. (C) Nuclear accumulation of WT-GFP-ERK2 is partially prevented by SiRNA of Imp7. HeLa cells overexpressing the SiRNA of Imp7 or the control SiRNA (72 hr after transfection) were further cotransfected with WT-GFP-ERK2 (GFP-ERK2). Forty-eight hours after this transfection the cells were serum-starved and then fixed and stained with DAPI. (D) SiRNA abolishes Imp7 expression. HeLa cells overexpressing the SiRNA of Imp7 or control SiRNA were harvested and subjected to WB using aImp7 and agERK Abs. (E) GFP-ERK interaction with Imp7 is stimulation dependent. Serum-starved HeLa cells overexpressing GFP-ERK2 or GFP control were incubated with or without EGF (50 ng/ml, 15 min). This was followed by Co-IP with aGFP Ab and mild washes. The amount of Co-IPed Imp7 and the amount of IPed GFP or GFP-ERK2 were determined by WB using aImp7 and aGFP Abs. (F) Interaction of endogenous Imp7 and ERK2 is elevated by EGF. Serum-starved HeLa cells were treated with EGF (50 ng/ml) for 0, 5, and 15 min and then subjected to Co-IP with agERK2 Ab or beads alone. The amount of Co-IPed Imp7, the amount of the IPed ERKs, and a loading marker (L) were detected by WB using aImp7 and agERK Abs.
and the EPE mutation markedly elevated it. On the other hand, no interaction with Imp-b was detected, indicating that this protein is not directly involved in the translocation and supporting the specificity of the reaction. These results strongly support the involvement of Imp7 in the stimulated pSPS-dependent nuclear translocation of ERKs. Another mechanism that was suggested to play a role in the nuclear translocation of ERKs is their direct interaction with NUPs (Lee et al., 2004; Matsubayashi et al., 2001). Examining this interaction revealed that ERK2 indeed interacts with Nup153, and the interaction is slightly increased upon stimulation (Figure 6D). Surprisingly, the interaction with Nup153 was increased, not decreased, with the APA mutant, while the interaction with EPE mutant was reduced. This result may indicate that the interaction with Nup153 is mediated mostly by other residues of ERK2, and the pSPS is required for release of ERKs from NUPs, which consequently facilitates the nuclear translocation of ERKs. Therefore, the phosphorylation of the SPS domain
seems to play a dual role in the translocation: enhancing interaction with Imp7, mainly after stimulation, and expediting the release from NUPs in both passive and stimulated translocations. Phosphorylated SPS Domain Plays a Role in the Nuclear Translocation of SMAD3 The pSPS-dependent translocation of the inert b-Gal construct prompted us to further study the generality of this domain in the nuclear translocation of signaling molecules. A sequence homology search revealed that SMAD3, a signaling protein that translocates into the nucleus upon TGF-b stimulation (Massague et al., 2000), contains an SPS domain in the MH2 region (Figure S10), which has been implicated in its nuclear translocation (Xu et al., 2003). We found that the TGF-b-induced nuclear translocation is mediated by Imp7, as knockdown of this protein prevented the translocation of the endogenous SMAD3 protein (Figure S11). Using apSPS Ab, we also found that WT-FlagSMAD3 is phosphorylated on its SPS domain upon VOOH,
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TPA, and TGF-b stimulation (Figure 7A). Therefore, it became likely that the SPS of SMAD3 operates similarly to that of ERK2. To examine it, we compared nonphosphorylatable FlagSMAD3 in which the SPS domain was replaced with three Ala residues (AAA-SMAD3) to the WT-Flag-SMAD3. Similar to ERK2, overexpression of WT-Flag-SMAD3 resulted in its nuclear localization, while the mutant was found primarily cytoplasmic (Figure 7B). For additional characterization of the SPS domain of SMAD3, we used a truncated form of the protein containing all the elements required for the proper subcellular localization of the protein (MH2; Xu et al., 2003) and, importantly, also the SPS domain. This MH2 region was fused to GFP (WT-MH2), and the SPS domain was replaced with either three Ala (AAAMH2) or with the phosphomimetic Asp-Pro-Asp (DPD-MH2). Overexpression of WT-MH2 in CHO cells resulted in a nuclear distribution, while AAA-MH2 and DPD-MH2 were localized in the cytoplasm or the nucleus respectively, as detected by fluorescence microscopy (Figure 7C). This result was supported by cellular fractionation experiment in which 75% of WT-MH2, 7% of AAA-MH2, and 88% of DPD-MH2 were localized in the nucleus (Figure 7D). Thus, SMAD3, like ERK2, uses pSPS to induce its nuclear translocation.
Figure 6. pSPS Interacts with Imp7 and Releases Nup153c Binding without Involvement of Imp-b (A) Interaction of ERK2 with Imp7, but not Imp-b, is enhanced by EGF stimulation and EPE mutation. Serum-starved HeLa cells overexpressing WT-ERK (WT), ERK-APA (APA), ERK-EPE (EPE), or GFP were treated with EGF (50 ng/ml, 10 min). Their extracts were then subjected to Co-IP with a-GFP Ab and mild washes. The Co-IPed Imp7 and Imp-b, as well as their loading amounts, were determined by WB using aImp7 and aImp-b Abs. The equal amount of IPed ERKs was determined with aGFP Ab. (B) The interaction of ERK2 and Imp7 in vitro is affected by the SPS domain. Reduced glutathione-released GST-ERK2 or GST constructs were incubated with IPed Imp7. This was followed by a mild wash, boiling, and WB using agERK and aImp7 Abs. (C) Interaction of 2GFP-SPS and its mutants with Imp7. HeLa cells overexpressing 2GFP, 2GFP-SPS, 2GFP-APA, and 2GFP-EPE constructs were subjected to Co-IP as described in (A) (without EGF stimulation). (D) Interaction with Nup153c is elevated in APA-ERK2 but reduced in EPEERK2. IPed and stringently washed WT-ERK2 (WT), APA-ERK2 (APA), or EPE-ERK2 from COS7 cells pretreated with or without TPA (250 nM, 15 min)
Phosphorylated TPT Domain Plays a Role in the Nuclear Translocation of MEK1 Other proteins that translocate into the nucleus upon stimulation are MEKs (Jaaro et al., 1997). Interestingly, we found that similarly to ERK2 and SMAD3, MEKs’ translocation into the nucleus is also mediated, at least in part, by Imp7 (Figure S11). Although MEKs do not contain an SPS domain, they do have a phosphorylatable ThrPro-Thr (TPT) sequence (Figure S10) next to a region that participates in the determination of their subcellular localization (Cha et al., 2001). This TPT sequence was shown to be phosphorylated by ERKs upon cellular stimulation (Matsuda et al., 1993) and to be involved in protein-protein interaction that can determine subcellular localization (Nantel et al., 1998). It was therefore interesting to examine whether the cytonuclear shuttling of MEKs requires the phosphorylation of this putative NTS. Thus, the two Thr residues within the TPT domain of MEK1-GFP were replaced with either Ala or Glu residues and their subcellular localization was followed upon overexpression in CHO cells. As expected from the presence of a nuclear export signal (NES) in MEKs (Fukuda et al., 1996; Jaaro et al., 1997), WT-MEK1-GFP and the mutants were localized primarily in the cytoplasm of nontreated cells (Figure 7E). The addition of the exportin inhibitor LMB (Yao et al., 2001) induced a moderate nuclear accumulation of WT-MEK1, which was increased in EPEMEK1 and abrogated in AAA-MEK1. Stimulation of the cells with TPA in the presence of LMB increased the nuclear WT-MEK1GFP, but not EPE-MEK1 that was localized in the nucleus even without stimulation. Importantly, AAA-MEK1 was localized in the cytoplasm, confirming that in similarity to ERK2, the TPT domain plays a role in stimulated translocation and not only in nonregulated
were incubated with 0.5 mg recombinant His-Nup153c. This was followed by mild washes, and the beads were then subjected to WB using aHis and aGFP Abs.
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Figure 7. The NTS Motif Mediates Nuclear Translocation of SMAD3 and MEK1 (A) Elevation of pSPS detection upon stimulation by SMAD3. Serum-starved COS7 cells overexpressing Flag-SMAD3 were stimulated with VOOH, TPA (T, 250 nM, 15 min), TGF-b (TG, 1 ng/ml, 15 min), or left untreated (NT). Flag-SMAD3 was IPed with aFlag Ab, extensively washed, and subjected to WB with apSPS or aFlag Abs. (B) Replacement of SPS with alanines prevents the nuclear translocation of full-length SMAD3. CHO cells overexpressing either Flag-SMAD3 or its AAA mutant were fixed and stained with both aFlag Ab (Flag) and DAPI. (C) Mutants of the SPS domain modulate the subcellular localization of the MH2 region of SMAD3. CHO cells overexpressing GFP-MH2 domain of SMAD3 (WT-MH2) or the AAA and DPD mutants were fixed and visualized. (D) Subcellular fractionation confirms the fluorescence results. Subcellular fractionation was performed on COS7 cells transfected with the MH2 constructs. WB of aliquots of the fractions was performed with aGFP Ab to detect the SMAD3 constructs and with atubulin or ac-Jun Abs as cytoplasmic and nuclear markers respectively. (E) The NTS motif regulates MEK1 shuttling into the nucleus. Serum-starved CHO cells overexpresing WT-GFP-MEK1, AAA-GFP-MEK1, and EPE-GFP-MEK1 were treated with LMB (5 ng/ml, 1 hr), LMB + TPA (5 ng/ml, 45 min and then 250 nM TPA for an additional 15 min), or left untreated (NT). Subsequently the cells were fixed, stained with DAPI, and visualized.
shuttling. Thus, in similarity to the SPS domains of ERK2 and SMAD3, TPT domain mediates nuclear translocation of MEK1. DISCUSSION Nuclear translocation of signaling molecules is crucial for their proper stimulus-dependent functioning in the regulation of cellular processes such as proliferation and differentiation. Interestingly, many of these proteins, including ERKs, do not contain the canonical NLS, and their mechanism of nuclear translocation is not fully understood. Based on previous publications and the results obtained here, we propose a mechanism for the regulation of ERKs’ subcellular localization (see Movie S1 for a model). Thus, in resting cells, ERKs are associated with several cytoplasmic anchors through several docking domains (Chuderland and Seger, 2005). Phosphorylation of their TEY domain by MEKs induces a conformational change, which activates the proteins
and induces their detachment from most of the anchors (Wolf et al., 2001). The detached proteins are then phosphorylated on their SPS domain, possibly by different protein kinases operating in different conditions. This phosphorylation then allows association of the active ERKs with Imp7 (Figures 5 and 6) that carries the ERKs via the nuclear pores into the nucleus. In this case, the Imp7-dependent translocation does not require the aid of Imp-b (Figure 6), which seems to be required for Imp7 in some, but not all, systems (Gorlich et al., 1997; Jakel and Gorlich, 1998). The actual penetration through the pore requires SPS phosphorylation as well because it seems to alleviate the strong interaction with NUPs (Figure 6) and to allow a proper pore sliding. Our results indicate that Imp7- and NTS-dependent processes are also involved in the nuclear tranlocation of SMAD3 and MEK1 (Figure 7). The scenario above describes the active nuclear translocation of ERKs upon stimulation. However, it was shown that ERKs can
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also translocate into the nucleus via a coexisting passive mechanism as well (Adachi et al., 1999). Our results indicate that the SPS phosphorylation is required for this type of translocation as well, because the overexpressed (Figure 3) or LMB-induced ERKs (Figure 5 and Figure S3) were translocated to nuclei of resting cells in an SPS-dependent manner (Figure 3 and Figure S1). Interestingly, despite the extensive dependence on SPS phosphorylation, the passive diffusion of overexpressed ERK2, or of endogenous ERKs late after LMB treatment, were only partially inhibited by Imp7 knockdown (Figure 5). The limited dependence of passive translocation on Imp7 may indicate that under such conditions, some of the ERK molecules can reach NUPs without the aid of the transport carrier, although a small fraction of the ERK may still use the Imp7 due to a limited amount of SPS phosphorylation in resting or LMB-treated cells. On the other hand, the dependence of passive transport on pSPS could be explained by the necessity of phosphate incorporation to the SPS for the proper sliding through the NUPs as mentioned above. Additionally, our results indicate that dimerization (Khokhlatchev et al., 1998) is not required for the translocation, although autophsophorylation of the SPS domain may participate in the stimulated translocation. This result fits our observation obtained with mutations in ERKs dimerization residues (Wolf et al., 2001). To our surprise, we found that unlike the interaction with importins, SPS phopshorylation decreases rather than increases the binding of ERKs to Nup153 (Figure 6C). This result may suggest that NUPs’ binding is mediated by other residues, adjacent to the SPS domain, which might be complementary residues in the SPS-dependent NTS. The phosphorylation of the SPS either reduces this binding affinity to the interacting residues or elevates its Koff, a process that results in a faster release from the NUPs, and allows a smooth sliding through the nuclear pores. Such a binding might have been mediated by hydrophobic regions, which were previously shown important in the binding of NUPs to various proteins (Bayliss et al., 2002). Thus, hydrophobic regions in the vicinity of the SPS domain have been implicated in the nuclear translocation of SMAD3 (Xu et al., 2003) and ERK2 (Lee et al., 2004), and we noticed that a proximal hydrophobic region exists also in MEK1 (Figure S10). However, our results indicate that such residues do not participate in the translocation (data not shown), and therefore, the interrelationships between the SPS domain and hydrophobic or other NUPbinding regions need further examination. A sequence analysis showed that the NTS is present in the sequences of regulatory nuclear shuttling signaling proteins and might similarly induce their nuclear translocation. One such protein is Cyclin-B, in which the region required for translocation contains SPS domain in a Ser-rich region (Walsh et al., 2003). ERK phosphorylation of Ser residues in this region was shown to be important for the mechanism of translocation and, therefore, is likely to operate via phosphorylated SPS and support the NTS mechanism identified here. Other nuclear shuttling NTS-containing proteins are SMAD2 and SMAD4, JNK2, AKT, STAT4, BRCA1, APC, p53, and more signaling proteins whose mechanism of translocation is not yet clear and could require phosphorylation of S/T-P-S/T. Notably, the requirement for pSPS could be alleviated when one of the amino acids next to
a Pro is acidic (S/T-P-D/E, E/D-P-S/T). Such sequences (DPS) exist in the translocating JNK1 (Mizukami et al., 1997) and b-catenin (Henderson and Fagotto, 2002). Two acidic residues next to the Pro may induce permanent nuclear accumulation, and this may be the case in p38MAPK (Ben-Levy et al., 1998). However, the regulation of translocation of such proteins is probably less stringent and should be further studied. In summary, we identified a unique, general NTS that participates in nuclear translocation of signaling proteins upon extracellular stimulation. Phosphorylation of the NTS is required for the dynamic and reversible shuttling. The generality and autonomous function of the NTS was validated by overexpression of b-Gal-GFP chimera fused to a sequence of 19 amino acids containing the NTS. The mechanism of stimulated translocation involves phosphorylation-dependent interaction with Imp7 and release from NUPs. The passive translocation requires SPS phosphorylation as well, but is not likely to involve Imp7 binding. The NTS-dependent nuclear translocation was identified and characterized for ERK2, SMAD3, and MEK1, but similar sequences in other signaling proteins suggest that the identified NTS may play a general role in the stimulation-dependent translocation of signaling proteins. EXPERIMENTAL PROCEDURES DNA Constructs and Mutations GFP-ERK2 was prepared in pEGFP-C1 (Clontech, Mountain View, CA) as described (Rubinfeld et al., 1999). GST-ERK2 was prepared in pGEX vector as previously described (Chuderland et al., 2008). All mutations described were verified by sequencing. GFP-MEK1 was in pEGFP-N1 (Clontech; Yao et al., 2001), and HA-MEK1 was prepared in pCMV-HA. To construct chimera proteins bearing the SPS domain, 19 amino acid stretch containing the SPS from ERK2 (LDQLNHILGILGSPSQEDL) was fused after a starting Met to the N terminus of fusion protein 2GFP (gift from A. Gross, Weizmann Institute of Science [WIS], Israel) or b-Gal-GFP. Point mutations were performed by site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed by sequencing. The GST-ERK2 constructs were prepared using pGEX-2T vector (AP Biotech, USA). The His-Nup153c was a gift from Dr. Michael Elbaum (WIS, Israel). 6His-MEK construct was produced as described (Jaaro et al., 1997). GFP-MH2-SMAD3 and FLAG-SMAD3 were provided by Dr. Joan Massague` (Mermorial Sloan-Kettering Center, NY). All SiRNAs were from Dharmacon (Lafayette, CO). Cell Culture and Transfection COS7 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and were transfected using the DEAE-dextran method as described (Jaaro et al., 1997) and polyethylenimine (PEI) as described (Shaul and Seger, 2006), respectively. CHO cells were grown in F12-DMEM and transfected using Lipofectamine (Invitrogen, CA). After transfection, the cells were washed and grown under their corresponding conditions. SiRNAs were transfected using oligofectamin (Dharmacon) according to the manufacturer’s instructions. Immunofluorescence Microscopy Cells were fixed (30 min in 3% paraformaldehyde in PBS), followed by 5 min permeabilization with 0.2% Triton X-100 in PBS (23 C). The fixed cells were sequentially incubated with an Ab (45 min), followed by rhodamine-conjugated secondary Ab (45 min) and DAPI. Slides were visualized by a fluorescence microscope (Nikon, Japan 4003 magnification). Western Blotting Cell extracts were collected and separated by 10% SDS-PAGE, which was followed by transfer to nitrocellulose and WB with the appropriate Abs. The blots
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were developed with alkaline phosphatase or horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat Fab Abs. Additional Experimental Procedures can be found in the Supplemental Data.
SUPPLEMENTAL DATA The Supplemental Data include Supplemental Introduction, Supplemental Experimental Procedures, Supplemental References, 11 figures, and one movie and can be found with this article online at http://www.molecule.org/ cgi/content/full/31/6/850/DC1/.
ACKNOWLEDGMENTS We would like to thank Reut Hemo and the MS unit of WIS for their help in the project. This work was supported by grants from the Israel Academy of Sciences and Humanities, MINERVA foundation, and from the European Community’s Sixth Framework Program project IST-2004-027265-SIMAP. R.S. is an incumbent of the Yale Lewine and Ella Miller Lewine professorial chair for cancer research. Received: January 19, 2007 Revised: April 4, 2008 Accepted: August 4, 2008 Published online: August 28, 2008
Fukuda, M., Gotoh, Y., and Nishida, E. (1997). Interaction of MAPK with MAPKK: its possible role in the control of nucleocytoplasmic transport of MAP kinase. EMBO J. 16, 1901–1908. Gorlich, D., Dabrowski, M., Bischoff, F.R., Kutay, U., Bork, P., Hartmann, E., Prehn, S., and Izaurralde, E. (1997). A novel class of RanGTP binding proteins. J. Cell Biol. 138, 65–80. Henderson, B.R., and Fagotto, F. (2002). The ins and outs of APC and betacatenin nuclear transport. EMBO Rep. 3, 834–839. Jaaro, H., Rubinfeld, H., Hanoch, T., and Seger, R. (1997). Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation. Proc. Natl. Acad. Sci. USA 94, 3742–3747. Jakel, S., and Gorlich, D. (1998). Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells. EMBO J. 17, 4491–4502. Khokhlatchev, A.V., Canagarajah, B., Wilsbacher, J., Robinson, M., Atkinson, M., Goldsmith, E., and Cobb, M.H. (1998). Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell 93, 605– 615. Lee, T., Hoofnagle, A.N., Kabuyama, Y., Stroud, J., Min, X., Goldsmith, E.J., Chen, L., Resing, K.A., and Ahn, N.G. (2004). Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry. Mol. Cell 14, 43–55.
REFERENCES
Liu, X., Sun, Y., Constantinescu, S.N., Karam, E., Weinberg, R.A., and Lodish, H.F. (1997). Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc. Natl. Acad. Sci. USA 94, 10669–10674.
Adachi, M., Fukuda, M., and Nishida, E. (1999). Two co-existing mechanisms for nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer. EMBO J. 18, 5347–5358.
Lorenzen, J.A., Baker, S.E., Denhez, F., Melnick, M.B., Brower, D.L., and Perkins, L.A. (2001). Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin. Development 128, 1403–1414.
Adachi, M., Fukuda, M., and Nishida, E. (2000). Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism. J. Cell Biol. 148, 849–856. Bayliss, R., Littlewood, T., Strawn, L.A., Wente, S.R., and Stewart, M. (2002). GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta. J. Biol. Chem. 277, 50597–50606. Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H.F., and Marshall, C.J. (1998). Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr. Biol. 8, 1049–1057. Cha, H., Lee, E.K., and Shapiro, P. (2001). Identification of a C-terminal region that regulates mitogen-activated protein kinase kinase-1 cytoplasmic localization and ERK activation. J. Biol. Chem. 276, 48494–48501. Chen, H.B., Rud, J.G., Lin, K., and Xu, L. (2005). Nuclear targeting of transforming growth factor-beta-activated Smad complexes. J. Biol. Chem. 280, 21329–21336.
Massague, J., Blain, S.W., and Lo, R.S. (2000). TGFbeta signaling in growth control, cancer, and heritable disorders. Cell 103, 295–309. Massague, J., Seoane, J., and Wotton, D. (2005). Smad transcription factors. Genes Dev. 19, 2783–2810. Matsubayashi, Y., Fukuda, M., and Nishida, E. (2001). Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells. J. Biol. Chem. 276, 41755–41760. Matsuda, S., Gotoh, Y., and Nishida, E. (1993). Phosphorylation of Xenopus mitogen-activated protein (MAP) kinase kinase by MAP kinase kinase kinase and MAP kinase. J. Biol. Chem. 268, 3277–3281. Mizukami, Y., Yoshioka, K., Morimoto, S., and Yoshida, K. (1997). A novel mechanism of JNK1 activation. Nuclear translocation and activation of JNK1 during ischemia and reperfusion. J. Biol. Chem. 272, 16657–16662.
Chen, R.H., Sarnecki, C., and Blenis, J. (1992). Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol. Cell. Biol. 12, 915–927.
Nantel, A., Mohammad-Ali, K., Sherk, J., Posner, B.I., and Thomas, D.Y. (1998). Interaction of the Grb10 adapter protein with the Raf1 and MEK1 kinases. J. Biol. Chem. 273, 10475–10484.
Christophe, D., Christophe-Hobertus, C., and Pichon, B. (2000). Nuclear targeting of proteins: how many different signals? Cell. Signal. 12, 337–341.
Nigg, E.A. (1997). Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386, 779–787.
Chuderland, D., and Seger, R. (2005). Protein-protein interactions in the regulation of the extracellular signal-regulated kinase. Mol. Biotechnol. 29, 57–74.
Rubinfeld, H., Hanoch, T., and Seger, R. (1999). Identification of a cytoplasmicretention sequence in ERK2. J. Biol. Chem. 274, 30349–30352.
Chuderland, D., Marmor, G., Shainskaya, A., and Seger, R. (2008). Calciummediated interactions regulate the subcellular localization of extracellular signal-regulated kinases (ERKs). J. Biol. Chem. 283, 11176–11188.
Shaul, Y.D., and Seger, R. (2006). ERK1c regulates Golgi fragmentation during mitosis. J. Cell Biol. 172, 885–897.
Fu, X., Choi, Y.K., Qu, D., Yu, Y., Cheung, N.S., and Qi, R.Z. (2006). Identification of nuclear import mechanisms for the neuronal Cdk5 activator. J. Biol. Chem. 281, 39014–39021. Fukuda, M., Gotoh, I., Gotoh, Y., and Nishida, E. (1996). Cytoplasmic localization of mitogen-activated protein kinase kinase directed by its NH2-terminal, leucine-rich short amino acid sequence, which acts as a nuclear export signal. J. Biol. Chem. 271, 20024–20028.
Schlenstedt, G. (1996). Protein import into the nucleus. FEBS Lett. 389, 75–79.
Tran, E.J., and Wente, S.R. (2006). Dynamic nuclear pore complexes: life on the edge. Cell 125, 1041–1053. Walsh, S., Margolis, S.S., and Kornbluth, S. (2003). Phosphorylation of the cyclin b1 cytoplasmic retention sequence by mitogen-activated protein kinase and Plx. Mol. Cancer Res. 1, 280–289. Wolf, I., Rubinfeld, H., Yoon, S., Marmor, G., Hanoch, T., and Seger, R. (2001). Involvement of the activation loop of ERK in the detachment from cytosolic anchoring. J. Biol. Chem. 276, 24490–24497.
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Molecular Cell Importin7-Dependent Nuclear Translocation Signal
Xiao, Z., Liu, X., Henis, Y.I., and Lodish, H.F. (2000a). A distinct nuclear localization signal in the N terminus of Smad 3 determines its ligand-induced nuclear translocation. Proc. Natl. Acad. Sci. USA 97, 7853–7858. Xiao, Z., Liu, X., and Lodish, H.F. (2000b). Importin beta mediates nuclear translocation of Smad 3. J. Biol. Chem. 275, 23425–23428. Xu, L., Alarcon, C., Col, S., and Massague, J. (2003). Distinct domain utilization by Smad3 and Smad4 for nucleoporin interaction and nuclear import. J. Biol. Chem. 278, 42569–42577. Xu, L., and Massague, J. (2004). Nucleocytoplasmic shuttling of signal transducers. Nat. Rev. Mol. Cell Biol. 5, 209–219.
Yao, Z., Flash, I., Raviv, Z., Yung, Y., Asscher, Y., Pleban, S., and Seger, R. (2001). Non-regulated and stimulated mechanisms cooperate in the nuclear accumulation of MEK1. Oncogene 20, 7588–7596. Yazicioglu, M.N., Goad, D.L., Ranganathan, A., Whitehurst, A.W., Goldsmith, E.J., and Cobb, M.H. (2007). Mutations in ERK2 binding sites affect nuclear entry. J. Biol. Chem. 282, 28759–28767. Yoon, S., and Seger, R. (2006). The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24, 21–44.
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Article Identification and Characterization of a General Nuclear Translocation Signal in Signaling Proteins Dana Chuderland,1 Alexander Konson,1 and Rony Seger1,* 1Department of Biological Regulation, The Weizmann Institute of Science, 76100 Rehovot, Israel *Correspondence: [email protected] DOI 10.1016/j.molcel.2008.08.007
SUMMARY
Upon stimulation, many proteins translocate into the nucleus in order to regulate a variety of cellular processes. The mechanism underlying the translocation is not clear since many of these proteins lack a canonical nuclear localization signal (NLS). We searched for an alternative mechanism in extracellular signal-regulated kinase (ERK)-2 and identified a 3 amino acid domain (SPS) that is phosphorylated upon stimulation to induce nuclear translocation of ERK2. A 19 amino acid stretch containing this phosphorylated domain inserts nondiffusible proteins to the nucleus autonomously. The phosphorylated SPS acts by binding to importin7 and the release from nuclear pore proteins. This allows its functioning both in passive and active ERK transports. A similar domain appears in many cytonuclear shuttling proteins, and we found that phosphorylation of similar sequences in SMAD3 or MEK1 also induces their nuclear accumulation. Therefore, our findings show that this phosphorylated domain acts as a general nuclear translocation signal (NTS). INTRODUCTION To ensure accurate cellular functioning, the spatial distribution of proteins needs to be delicately regulated and coordinated. This is particularly apparent in many signaling proteins that dynamically and rapidly change their localization upon extracellular stimulation. In order to maintain such regulation, the nucleus is separated from the cytoplasm by a double membrane envelope that allows a selective entrance of proteins through specialized nuclear pore complexes (NPC; Nigg, 1997). The selectivity of nuclear localization is primarily mediated by a nuclear localization signal (NLS) harbored within the sequence of the nuclear protein (Schlenstedt, 1996). The NLS is recognized by special carrier proteins, the importins, which, upon binding, facilitate the transfer of proteins across the NPC (Tran and Wente, 2006). However, not all cytonuclear shuttling proteins contain the canonical NLS and, therefore, use other, NLS-independent mechanisms for their passage through the NPC. Some of the characterized
NLS-independent mechanisms include passive diffusion of small proteins (<30–40 kDa), distinct nuclear-directing motifs (Christophe et al., 2000), interaction with NLS-containing proteins, or alternatively, a direct interaction with the nuclear pore proteins (NUPs; Xu and Massague, 2004). However, these mechanisms do not always allow timely regulation of nuclear processes and, therefore, the molecular mechanism(s) that allows the rapid and reversible NLS-independent translocation of signaling proteins upon stimulation is still obscure. Examples for signaling proteins that translocate into the nucleus upon stimulation in an NLS-independent manner include ERKs, MEKs (Yoon and Seger, 2006), and SMADs (Massague et al., 2005). The importance of these proteins in regulation of proliferation and differentiation led to many studies on the nuclear translocation of ERKs (Chen et al., 1992; Chuderland et al., 2008; Lorenzen et al., 2001; Matsubayashi et al., 2001; Yazicioglu et al., 2007), MEKs (Fukuda et al., 1996; Jaaro et al., 1997), and SMAD3 (Chen et al., 2005; Liu et al., 1997; Xiao et al., 2000a; Xiao et al., 2000b; Xu et al., 2003) (for more information see the Supplemental Data available online). Although a possible involvement of the canonical NLS machinery was initially identified in some systems, it was recently suggested that these proteins may translocate into the nucleus by a direct interaction with NUPs (Xu and Massague, 2004). We identify here a sequence (Ser-Pro-Ser, SPS) in ERKs that is phosphorylated upon stimulation to mediate their nuclear translocation. The phosphorylated sequence within a 19 amino acid stretch acts autonomously by binding to the nuclear translocating protein importin7 (Imp7) and by promoting release from NUPs in active and possibly also passive transports. Phosphorylation of the same or similar (Thr-Pro-Thr, TPT) sequence is responsible for nuclear translocation of other proteins such as SMAD3 and MEKs, which is also mediated by Imp7. We propose that phosphorylated S/T-P-S/T acts as a general nuclear translocation signal (NTS) for NLS-lacking proteins that shuttle to the nucleus upon stimulation.
RESULTS The SPS Domain Plays a Role in the Nuclear Accumulation of Overexpressed ERK2 Although the regulation and underlying mechanism of ERKs nuclear translocation remains unclear, previous studies suggest a role for the kinase insert domain (KID; Lee et al., 2004). A
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Figure 1. Identification of SPS as Important Domain for ERK2’s Nuclear Translocation (A) Schematic representation of ERK2, including the TEY and the SPS domains. (B) Role of SPS in nonregulated accumulation of ERK2 in the nucleus. CHO or COS7 cells were grown on cover slips and transfected with either WT-GFP-ERK2 (WT-ERK2) or the mutants 244-6A and D244-6. Twenty-four hours after transfection, the cells were serum starved (0.1% FBS, 16 hr), fixed, stained with DAPI, and visualized using a fluorescent microscope. (C) Role of SPS in the stimulated nuclear translocation of ERK2. CHO cells were cotransfected with HA-MEK1 together with WTERK2, 244-6A, or D244-6. Twenty-four hours after transfection, the cells were serum starved and treated for 15 min with either 250 or 10 nM TPA. After treatments, the cells were fixed and stained with either aHA Ab to detect MEK1 or DAPI for nuclear staining. ERK was detected by its GFP fluorescence. (D) Equilibration of TEY phosphorylation with different TPA concentrations. The TEY phosphorylation of GFP-ERK2 and its mutants was detected by a direct WB using apTEY and a-general ERK Abs (agERK). (E) 244-6A-ERK2 binds to 6His-MEK1 and is released upon phosphorylation. Serum-starved CHO cells expressing WT-ERK2 (WT) or 244-6A-ERK2 were treated with TPA (250 nM, 15 min) followed by IP and stringently washed. The IPed constructs, as well as beads alone, were incubated with 0.5 mg recombinant 6HisMEK1 (30 min, 30 C). Thereafter, the beads were mildly washed and subjected to WB using aMEK and agERK Abs.
as WT-GFP-ERK2, were transfected into CHO cells. Unlike the reported nuclear accumulation of GFPERK2 in these cells (Rubinfeld et al., 1999), both mutants were found primarily in the cytoplasm (Figure 1B). These results were validated using COS7 cells, which express higher amount of ectopic proteins, clearly implicating the SPS domain in determination of ERK2 localization in quiescent cells.
thorough mutagenesis of ERK2’s KID revealed a 3 amino acids sequence, composed of residues Ser244, Pro245, and Ser 246 (SPS), which participates in the regulation of ERK2 localization. To examine the function of this region, we either replaced the three residues to Ala or deleted them (244-6A and D244-6; Figure 1A; Figure S1). The mutated ERKs were then fused to a green fluorescent protein (GFP) and these constructs, as well
The SPS Domain Plays a Role in the Nuclear Translocation of ERK2 upon Stimulation Since overexpressed ERKs translocate to the nucleus mainly by passive diffusion while much of the translocation of endogenous ERKs is mediated by an activated transport (Adachi et al., 1999), it was important to examine the role of the SPS domain in a stimulusdependent translocation. In order to do so, we cotransfected the ERK constructs together with MEK1, which acts as an anchoring protein, to secure cytoplasmic localization of ERKs in resting cells (Fukuda et al., 1997; Rubinfeld et al., 1999). As expected (Rubinfeld et al., 1999), the cotransfected ERK2 was localized in the cytoplasm of the resting CHO cells and translocated into the nucleus upon TPA stimulation (250 nM). However, the two SPS mutants remained localized in the cytoplasm irrespective of TPA stimulation (Figure 1C). A possible reason for this lack of translocation could have been a reduced phosphorylation of the Thr and Tyr residues within the TEY motif of the mutants. Indeed, the rate of phosphorylation of the mutants detected by anti-doubly phosphorylated TEY-ERK (apTEY)
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antibody (Ab) was lower than that of WT-ERK2 (Figure 1D), indicating that the SPS domain is involved in the phosphorylation of ERKs by MEKs. To determine whether the cytoplasmic retention of the SPS mutants is a result of reduced TEY phosphorylation, we equilibrated the phosphorylation levels by using different concentrations of TPA (10 nM for WT-ERK2 and 250 nM for the mutants Figure 1C). Also with equivalent phosphorylation levels, WT-GFP-ERK2 was localized to the nucleus, whereas the SPS mutants remained restricted to the cytoplasm. Furthermore, 244-6A-ERK2 interacted with MEK1 and detached from it upon stimulation, similar to WT-ERK2 (Figure 1E). This indicates that the change by SPS mutation affects only the phosphorylation by MEKs, but not ERKs’ binding or detachment, confirming that no major conformational changes are induced by the mutations. Therefore, the lack of nuclear accumulation of the SPSmutated ERK2 is probably due to other, inherent effect of this region. Taken together, these data implicate the SPS domain in ERKs nuclear translocation, both in resting (mostly passive transport of overexpressed ERKs) and stimulated cells. The Ser Residues in the SPS Domain Are Phosphorylated upon Cellular Stimulation The abrogation of stimulation-dependent nuclear trafficking of SPS-mutated ERK2 and the presence of Ser residues within this domain may unveil a potential phosphorylation site. Therefore, we examined the possibility that the Ser residues within the SPS domain undergo an additional phosphorylation concomitantly with nuclear translocation. Mass spectroscopic (MS) analysis (Maldi-Tof) was performed on immunoprecipitated (IPed) GFP-ERK2 from either stimulated or nonstimulated COS7 cells. In addition to a trypsin-digested peptide containing the pTEY (Yoon and Seger, 2006), we found in proteins derived from stimulated cells also a peptide corresponding to the SPS domain containing one and possibly two incorporated phosphate(s). Since the only phosphoacceptors in this peptide were the two Ser residues in the SPS, it is likely that this higher mass peptide(s) represents phosphorylation of one or possibly two Ser residues within this domain. Characterization of SPS Phosphorylation by a Specific Ab To further study the occurrence and role of the SPS phosphorylation, we raised polyclonal Ab directed against a doubly-phosphorylated SPS peptide. In a western blot (WB), the Ab weakly recognized a 72 kDa band, corresponding to GFP-ERK2 IPed from serum-starved, transfected COS7 cells (Figure 2A). This recognition of GFP-ERK2 was significantly increased when the GFP-ERK2 was IPed from VOOH, TPA, or EGF stimulated cells, confirming that the SPS is phosphorylated upon stimulation. Indeed, time course of EGF stimulation revealed a transient increase in SPS phosphorylation with a peak at 15 min (Figure 2B), which was slower than TEY phosphorylation that peaked at 5 min after stimulation. This rate of SPS phosphorylation correlated better with translocation (data not shown), suggesting sequential events in the activation of ERKs, and their subsequent SPS phosphorylation and translocation to the nucleus. Similar phosphorylation patterns detected by the Ab were also found
with endogenous ERKs from EGF- or VOOH-stimulated cells (Figure 2C). In order to confirm that the Ab is indeed specific to the pSPS, we first used a competition with the antigenic peptide and found that it completely abolishes the Ab recognition (Figure 2D). Moreover, treatment of phosphorylated ERK2 with alkaline phosphatase demonstrated a significant reduction of the immunoreactive pSPS (Figure 2E), as well as the control pTEY (Figure 2F). We then examined whether the Ab recognizes just one of the phosphorylated residues or both of them. To do so, we mutated either Ser244 (AS) or Ser246 (SA) to Ala. These constructs, as well as the WT ERK2 and the ERK2-244-6A mutant, were transfected to COS7 cells, which were either stimulated or left nontreated. We found that the apSPS Ab reacts with the WT and the monophosphorylatable ERK2 mutants, but not with the nonphosphorylatable mutant, only from stimulated cells (Figure 2G). Thus, these results confirm the specificity of the apSPS Ab to the phosphorylated form of SPS and its ability to recognize either the monophosphorylated or the doubly-phosphorylated SPS. These results also show that, in agreement with the MS data, stimulation can cause phosphorylation of either both Ser244 and Ser246 together or just one of them alone. Finally, we found that the nonphosphorylatable TEY mutant of ERK2 (TEY-AAA) can still be recognized by the apSPS Ab upon stimulation, albeit to a slightly lower extent (Figure 2H). This indicates that the Ab does not recognize the phosphorylated TEY and shows again that the SPS phosphorylation occurs independently from that of TEY. Thus, our results identify an important stimulationdependent phosphorylation of ERKs on their SPS domain. SPS Phosphorylation Induces Nuclear Translocation of ERK2 Both the phosphorylation on SPS domain and the abrogation of nuclear translocation by SPS mutants suggest that this phosphorylation may be required for the nuclear translocation of ERKs. To further study this possibility, we replaced the two Ser residues in the SPS domain with Glu that acts as a phosphomimetic residue. GFP-EPE-ERK2 showed a pronounced nuclear localization, while the APA mutant exhibited a cytoplasmic distribution similar to that of the 244-6A construct (Figure 3A; Figure S1). These differences in distribution were confirmed by subcellular fractionation showing that about 75% of WT-ERK2, 95% of EPE-ERK2, and only 5% of APA-ERK2 are localized in the nucleus 48 hr after transfection (Figure 3B). The difference between WT- and EPE-ERK2 was even more pronounced shortly after transfection (8 hrs when the GFP protein is just visible; Figure 3C), although their expression levels were similar, indicating that the translocation of overexpressed EPE-ERK2 into the nucleus is faster than that of WT-ERK2. Similar results were obtained upon treatment with leptomycine B (LMB) that prevents nuclear export (Figure S2), indicating that SPS mutation modulates the nuclear import and not export of ERKs. To further verify and characterize the importance of the SPS domain in mediating the nuclear translocation of ERK2, we used several distinct methods. First, we found that SPS mutants affected the translocation of ERK2 in an in vitro system consisting of recombinant proteins, cytosolic extract, and fixed digitonin-permeabilized cells (Chuderland et al., 2008; Matsubayashi et al.,
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Figure 2. Characterization of SPS Phosphorylation Using apSPS Ab (A) Elevation of pSPS detection upon stimulation. COS7 cells transfected with WT-GFP-ERK2 (GFP-ERK2) were serum-starved and treated with VOOH (V, 100 mM Na3VO4 and 200 mM H2O2, 15 min), TPA (T, 250 nM, 15 min), EGF (E, 50 ng/ml, 15 min), or left untreated (NT). The GFP-ERK2 was IPed with aGFP Ab, extensively washed, and subjected to WB using apSPS and agERK Abs. (B) Time course of SPS phosphorylation. Serumstarved COS7 cells overexpressing GFP-ERK2 were treated with EGF (50 ng/ml) for the indicated times. The GFP-ERK2 was IPed with aGFP Ab, extensively washed, and subjected to WB with apSPS, apTEY and agERK Abs. (C) Elevation of apSPS Ab immunoreactivity upon stimulation of endogenous ERKs. Serum-starved COS7 cells were treated with EGF (50 ng/ml) or VOOH for the indicated times. SPS phosphorylation was detected using the apSPS Ab, and this was compared to the amount of general ERKs detected by agERK. (D) Specificity of the apSPS Ab. Serum-starved COS7 cells overexpressing either GFP-ERK2 or 244-6A were treated with EGF (50 ng/ml, 15 min) following WB using apSPS and agERK Abs. (E and F) Further characterization of the apSPS Ab’s specificity. GFP-ERK2 was IPed from VOOH stimulated (Act) or nonstimulated (NT), transfected COS7 cells. The IPed protein was then incubated with shrimp’s alkaline phosphatase (SAP, 15 units, 30 min, 30 C) and subjected to WB with apSPS (E), apTEY (F), or agERK Abs. (G) Recognition of monophosphorylated SPS by the apSPS Ab. Serum-starved COS7 cells overexpressing the following mutants GFP-ERK2 (WT), GFP-APS-ERK2 (AS), GFP-SPA-ERK2 (SA), and GFP-APA-ERK2 (APA) were stimulated with EGF (50 ng/ml, 10 min) or left untreated (NT). The cells were then extracted and the lysates were subjected to WB using apSPS and agERK Abs. (H) TEY phosphorylation is not required for SPS phosphorylation. COS7 cells overexpressing WT-ERK2 or TEY-AAA were treated and analyzed as described in (D).
2001). In this system, the APA mutation prevented the translocation of recombinant ERK2, while the EPE mutation facilitated it, confirming the importance of the SPS phosphorylation (Figure S3). We then used monophosphomimetic ERK2 mutants to follow the importance of each of the phosphorylation sites and found that each of them is required for the nuclear translocation, although the phospho-Ser246 is somewhat more important (Figure S4). Similar effects of the SPS mutants were obtained when we used MEK1 retention to study stimulated ERK2 translocation (Figure S5). All SPS mutants were retained in the cytoplasm upon overexpression of MEK1, and TPA stimulation allowed nuclear translocation of either doubly- or monophosphomimetic mutants, but not of the APA mutant. Thus, in agreement with the in vitro binding in Figure 1E, these results indicate that the SPS domain or its phopshorylation do not play a role in MEK binding, but the phosphorylation of both or each one of its Sers can induce passive and stimulated nuclear translocation of ERKs. The lack of correlation between SPS phosphorylation and MEK binding or TEY phosphorylation corresponds to previous findings that the nuclear accumulation of ERK2 does not require
TEY phosphorylation (Wolf et al., 2001). Indeed, we found that mutating the TEY motif to Ala does not change the nuclear translocation of ERK2, but this effect is still seen when Ser244 and Ser246 are mutated to Glu (Figure S6). These results are consistent with a model in which the phosphorylation of TEY is important for the detachment from cytoplasmic anchors, while the subsequent SPS phosphorylation is responsible for the actual translocation. Importantly, the SPS-induced translocation seems to be important for the downstream activity of ERKs, as phosphodeficient SPS mutants prevented serum-induced cell proliferation, while the phosphomimetic EPE-ERK2 slightly enhanced this process (Figure S7). Use of b-Galactosidase to Study the Role of Phosphorylated SPS To validate the general importance of the SPS domain and examine whether it can act autonomously, we used a construct of b-Galactosidase fused to GFP (b-Gal, Figure 4A). This 145 kDa construct (Figure 4B), which is too big to freely diffuse to the nucleus, was indeed localized exclusively in the cytoplasm
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Figure 3. SPS Phosphorylation Is Required for the Nuclear Translocation of ERK2 (A) Replacing the Ser residues in the SPS with Glu induces the nuclear accumulation of ERK2. Serum-starved CHO cells overexpressing WT-GFP-ERK2 (WT), APA-GFP-ERK2 (APA), or EPE-GFP-ERK2 (EPE), were fixed, stained with DAPI, and visualized (as in Figure 1). (B) Subcellular fractionation confirms the fluorescence results. Serum-starved COS7 cells overexpressing the ERK2 constructs (as in [A]) were grown in 10 cm plates and then subjected to cellular fractionation. WB of aliquots of the fractions was performed with aGFP Ab to detect ERKs, and with atubulin or ac-Jun Abs as markers for cytoplasmic and nuclear fractions respectively. (C) EPE-ERK2 translocates to the nucleus faster than WTERK2. CHO cells were transfected with either WT-ERK2 or EPE-ERK2 constructs. Eight or 12 hr later the cells were either stained with DAPI and visualized or lysed and subjected to WB with aGFP Ab to confirm equal expression.
Since the SPS-containing region mimics some of the functions of the SPS region in ERKs, we undertook to examine whether its overexpression competes with the translocation machinery and, therefore, acts as a dominant-negative construct of endogenous ERK shuttling. Indeed, overexpression of SPS containing 19 amino acid stretch conjugated to a 2GFP chimera (2GFP-SPS) and more so 2GFPAPA inhibited TPA-induced translocation of endogenous ERKs, while the control 2GFP and the nuclear 2GFP-EPE did not affect it at all (Figure S8). Notably, the mutants had no effect on the localization of ERKs in resting cells, indicating that the SPS domain competes with the translocation of endogenous ERKs mainly upon stimulation. The reason for the lack of inhibition by the EPE mutant could be its rapid translocation to the nucleus, which prevents its interaction with the translocation machinery localized in the cytoplasm and is better accessible to the other two, mostly cytoplasmic. Thus, our results suggest that the phospho-SPS (pSPS) serves as a unique and general nuclear translocation signal (NTS) also in endogenous ERKs.
when expressed in CHO or COS7 cells (Figures 4C and 4D). However, fusion of the SPS domain, within the 19 amino acid stretch surrounding it, to the b-Gal induced some nuclear translocation of the construct, which was prevented by the mutation of the SPS to APA and accelerated by the EPE mutation. Moreover, the fusion of the SPS region made the construct sensitive to stimulation, as TPA facilitated the nuclear translocation of the bGal-SPS (Figure 4E), and this was prevented by the APA mutation. These results clearly demonstrate that the SPS domain within the 19 amino acid stretch functions autonomously.
Imp7 Mediates the Stimulation-Dependent Nuclear Translocation of ERKs The nuclear translocation of ERKs (Chen et al., 1992) can occur either by a passive diffusion or by an active transport (Adachi et al., 1999), which are governed by various mechanisms (Yazicioglu et al., 2007). In Drosophila, it was suggested that D-Imp7 (DIM-7) is responsible for the accumulation of ERKs in the nucleus during development upon activation (Lorenzen et al., 2001). Therefore, we examined whether the mammalian analog of this protein and some other importins play a role in ERK translocation in mammals as well. Transfection of SiRNA of Imp7 into HeLa cells dramatically inhibited EGF-induced translocation of ERK2 (Figure 5A). On the other hand, SiRNAs of importin5 (Imp5), importin-b (Imp-b), and transportin1
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Figure 4. The SPS domain, within a 19 Amino Acid Stretch, Is an Autonomous NTS (A) Schematic representation of the b-Gal constructs. (B) Equal expression of the b-Gal constructs. The constructs were overexpressed in COS7 cells, which were grown for 24 hr, serum starved, extracted, and subjected to WB with aGFP Ab. (C) SPS, APA, and EPE peptides fused to the b-Gal construct modify its subcellular localization. Serum-starved CHO and COS7 cells overexpressing the b-Gal constructs were fixed, stained with DAPI, and visualized. (D) Subcellular fractionation confirms the fluorescence results. Subcellular fractionation was performed on COS7 cells transfected with the b-Gal constructs. WB of aliquots of the fractions was performed with aGFP Ab to detect the b-Gal constructs, and with atubulin and a-histone-H1 Abs as cytoplasmic and nuclear markers respectively. (E) TPA stimulation enhances nuclear localization of the b-Gal-SPS. Serum-starved CHO and COS7 cells, overexpressing b-Gal-GFP, b-GalSPS, and b-Gal-APA were stimulated with TPA (250 nM, 15 min) or left untreated, and then fixed, stained with DAPI and visualized.
the activation-induced, but not as much nonstimulated, passive, nuclear translocation of ERKs.
(Trn1) did not significantly influence the nuclear translocation of ERK2 (Figure S9). Interestingly, the SiRNA of Imp7 had only a partial inhibitory effect on the nonstimulated (passive) mechanism of ERK translocation (Adachi et al., 2000), as detected in either LMB-treated (Figure 5B) or WT-ERK2 overexpressing (Figure 5C) cells. In all these experiments, the cells transfected with SiRNA to Imp7 lost considerable amount of Imp7, but not other proteins, such as ERKs, that served as a control (Figure 5D). Moreover, we found that Imp7 can interact with both overexpressed ERK2 (Figure 5E) and endogenous ERKs (Figure 5F), and this interaction was significantly increased upon stimulation with EGF. These results indicate that Imp7 is an important regulator of
Phosphorylated SPS Domain Is Required for ERK2 Interaction with Imp7 and Release from Nuclear Pore Proteins The finding that Imp7 is involved in ERKs nuclear translocation prompted us to examine whether the phosphorylation of the SPS is required for ERK-Imp7 association. We also examined the possible involvement of Imp-b, which seems to cooperate with Imp7 in some nuclear transport systems (Fu et al., 2006). Thus, we first examined the interaction of the various SPS mutants with Imp7 and Imp-b. Co-IP experiments revealed that WT-ERK2 interaction with Imp7 is significantly increased upon TPA stimulation (Figure 6A). Prevention of SPS phosphorylation using APA mutation reduced the interaction, mainly upon stimulation, while the use of the phosphomimetic EPE mutant dramatically increased the interaction either with or without stimulation. These results were confirmed by in vitro binding assay, in which soluble GST-ERK2, GST-APA-ERK2, GST-EPE-ERK2, or GST proteins were incubated with IPed Imp7 (Figure 6B). Indeed, the phosphomimetic mutant interacted with the Imp7 much stronger than WT-ERK2, while the nonphosphorylatable mutant did not associate with Imp7 at all. Similar results were obtained using the 2GFP constructs (Figure 6C), as the SPS conjugated 2GFP did bind Imp7, the APA mutation reduced this interaction,
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Figure 5. The Nuclear Translocation of ERKs Is Mediated by Imp7 (A) EGF-induced ERKs translocation is prevented by SiRNA of Imp7. HeLa cells were transfected with SiRNA of Imp7 or scrambled control SiRNA using oligofectamin. Seventy-two hours after transfection, the cells were serum starved and then either treated with EGF (50 ng/ml, 15 min) or left untreated. Then, the cells were washed, fixed, and stained with agERK Ab and DAPI. (B) LMB-induced ERKs translocation is partially prevented by SiRNA of Imp7. HeLa cells were transfected with control or Imp7 SiRNAs as above. After serum starvation, the cells were either left untreated (basal) or were treated with LMB (5 ng/ml, 30 min), washed, fixed, and stained with agERK and DAPI. (C) Nuclear accumulation of WT-GFP-ERK2 is partially prevented by SiRNA of Imp7. HeLa cells overexpressing the SiRNA of Imp7 or the control SiRNA (72 hr after transfection) were further cotransfected with WT-GFP-ERK2 (GFP-ERK2). Forty-eight hours after this transfection the cells were serum-starved and then fixed and stained with DAPI. (D) SiRNA abolishes Imp7 expression. HeLa cells overexpressing the SiRNA of Imp7 or control SiRNA were harvested and subjected to WB using aImp7 and agERK Abs. (E) GFP-ERK interaction with Imp7 is stimulation dependent. Serum-starved HeLa cells overexpressing GFP-ERK2 or GFP control were incubated with or without EGF (50 ng/ml, 15 min). This was followed by Co-IP with aGFP Ab and mild washes. The amount of Co-IPed Imp7 and the amount of IPed GFP or GFP-ERK2 were determined by WB using aImp7 and aGFP Abs. (F) Interaction of endogenous Imp7 and ERK2 is elevated by EGF. Serum-starved HeLa cells were treated with EGF (50 ng/ml) for 0, 5, and 15 min and then subjected to Co-IP with agERK2 Ab or beads alone. The amount of Co-IPed Imp7, the amount of the IPed ERKs, and a loading marker (L) were detected by WB using aImp7 and agERK Abs.
and the EPE mutation markedly elevated it. On the other hand, no interaction with Imp-b was detected, indicating that this protein is not directly involved in the translocation and supporting the specificity of the reaction. These results strongly support the involvement of Imp7 in the stimulated pSPS-dependent nuclear translocation of ERKs. Another mechanism that was suggested to play a role in the nuclear translocation of ERKs is their direct interaction with NUPs (Lee et al., 2004; Matsubayashi et al., 2001). Examining this interaction revealed that ERK2 indeed interacts with Nup153, and the interaction is slightly increased upon stimulation (Figure 6D). Surprisingly, the interaction with Nup153 was increased, not decreased, with the APA mutant, while the interaction with EPE mutant was reduced. This result may indicate that the interaction with Nup153 is mediated mostly by other residues of ERK2, and the pSPS is required for release of ERKs from NUPs, which consequently facilitates the nuclear translocation of ERKs. Therefore, the phosphorylation of the SPS domain
seems to play a dual role in the translocation: enhancing interaction with Imp7, mainly after stimulation, and expediting the release from NUPs in both passive and stimulated translocations. Phosphorylated SPS Domain Plays a Role in the Nuclear Translocation of SMAD3 The pSPS-dependent translocation of the inert b-Gal construct prompted us to further study the generality of this domain in the nuclear translocation of signaling molecules. A sequence homology search revealed that SMAD3, a signaling protein that translocates into the nucleus upon TGF-b stimulation (Massague et al., 2000), contains an SPS domain in the MH2 region (Figure S10), which has been implicated in its nuclear translocation (Xu et al., 2003). We found that the TGF-b-induced nuclear translocation is mediated by Imp7, as knockdown of this protein prevented the translocation of the endogenous SMAD3 protein (Figure S11). Using apSPS Ab, we also found that WT-FlagSMAD3 is phosphorylated on its SPS domain upon VOOH,
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TPA, and TGF-b stimulation (Figure 7A). Therefore, it became likely that the SPS of SMAD3 operates similarly to that of ERK2. To examine it, we compared nonphosphorylatable FlagSMAD3 in which the SPS domain was replaced with three Ala residues (AAA-SMAD3) to the WT-Flag-SMAD3. Similar to ERK2, overexpression of WT-Flag-SMAD3 resulted in its nuclear localization, while the mutant was found primarily cytoplasmic (Figure 7B). For additional characterization of the SPS domain of SMAD3, we used a truncated form of the protein containing all the elements required for the proper subcellular localization of the protein (MH2; Xu et al., 2003) and, importantly, also the SPS domain. This MH2 region was fused to GFP (WT-MH2), and the SPS domain was replaced with either three Ala (AAAMH2) or with the phosphomimetic Asp-Pro-Asp (DPD-MH2). Overexpression of WT-MH2 in CHO cells resulted in a nuclear distribution, while AAA-MH2 and DPD-MH2 were localized in the cytoplasm or the nucleus respectively, as detected by fluorescence microscopy (Figure 7C). This result was supported by cellular fractionation experiment in which 75% of WT-MH2, 7% of AAA-MH2, and 88% of DPD-MH2 were localized in the nucleus (Figure 7D). Thus, SMAD3, like ERK2, uses pSPS to induce its nuclear translocation.
Figure 6. pSPS Interacts with Imp7 and Releases Nup153c Binding without Involvement of Imp-b (A) Interaction of ERK2 with Imp7, but not Imp-b, is enhanced by EGF stimulation and EPE mutation. Serum-starved HeLa cells overexpressing WT-ERK (WT), ERK-APA (APA), ERK-EPE (EPE), or GFP were treated with EGF (50 ng/ml, 10 min). Their extracts were then subjected to Co-IP with a-GFP Ab and mild washes. The Co-IPed Imp7 and Imp-b, as well as their loading amounts, were determined by WB using aImp7 and aImp-b Abs. The equal amount of IPed ERKs was determined with aGFP Ab. (B) The interaction of ERK2 and Imp7 in vitro is affected by the SPS domain. Reduced glutathione-released GST-ERK2 or GST constructs were incubated with IPed Imp7. This was followed by a mild wash, boiling, and WB using agERK and aImp7 Abs. (C) Interaction of 2GFP-SPS and its mutants with Imp7. HeLa cells overexpressing 2GFP, 2GFP-SPS, 2GFP-APA, and 2GFP-EPE constructs were subjected to Co-IP as described in (A) (without EGF stimulation). (D) Interaction with Nup153c is elevated in APA-ERK2 but reduced in EPEERK2. IPed and stringently washed WT-ERK2 (WT), APA-ERK2 (APA), or EPE-ERK2 from COS7 cells pretreated with or without TPA (250 nM, 15 min)
Phosphorylated TPT Domain Plays a Role in the Nuclear Translocation of MEK1 Other proteins that translocate into the nucleus upon stimulation are MEKs (Jaaro et al., 1997). Interestingly, we found that similarly to ERK2 and SMAD3, MEKs’ translocation into the nucleus is also mediated, at least in part, by Imp7 (Figure S11). Although MEKs do not contain an SPS domain, they do have a phosphorylatable ThrPro-Thr (TPT) sequence (Figure S10) next to a region that participates in the determination of their subcellular localization (Cha et al., 2001). This TPT sequence was shown to be phosphorylated by ERKs upon cellular stimulation (Matsuda et al., 1993) and to be involved in protein-protein interaction that can determine subcellular localization (Nantel et al., 1998). It was therefore interesting to examine whether the cytonuclear shuttling of MEKs requires the phosphorylation of this putative NTS. Thus, the two Thr residues within the TPT domain of MEK1-GFP were replaced with either Ala or Glu residues and their subcellular localization was followed upon overexpression in CHO cells. As expected from the presence of a nuclear export signal (NES) in MEKs (Fukuda et al., 1996; Jaaro et al., 1997), WT-MEK1-GFP and the mutants were localized primarily in the cytoplasm of nontreated cells (Figure 7E). The addition of the exportin inhibitor LMB (Yao et al., 2001) induced a moderate nuclear accumulation of WT-MEK1, which was increased in EPEMEK1 and abrogated in AAA-MEK1. Stimulation of the cells with TPA in the presence of LMB increased the nuclear WT-MEK1GFP, but not EPE-MEK1 that was localized in the nucleus even without stimulation. Importantly, AAA-MEK1 was localized in the cytoplasm, confirming that in similarity to ERK2, the TPT domain plays a role in stimulated translocation and not only in nonregulated
were incubated with 0.5 mg recombinant His-Nup153c. This was followed by mild washes, and the beads were then subjected to WB using aHis and aGFP Abs.
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Figure 7. The NTS Motif Mediates Nuclear Translocation of SMAD3 and MEK1 (A) Elevation of pSPS detection upon stimulation by SMAD3. Serum-starved COS7 cells overexpressing Flag-SMAD3 were stimulated with VOOH, TPA (T, 250 nM, 15 min), TGF-b (TG, 1 ng/ml, 15 min), or left untreated (NT). Flag-SMAD3 was IPed with aFlag Ab, extensively washed, and subjected to WB with apSPS or aFlag Abs. (B) Replacement of SPS with alanines prevents the nuclear translocation of full-length SMAD3. CHO cells overexpressing either Flag-SMAD3 or its AAA mutant were fixed and stained with both aFlag Ab (Flag) and DAPI. (C) Mutants of the SPS domain modulate the subcellular localization of the MH2 region of SMAD3. CHO cells overexpressing GFP-MH2 domain of SMAD3 (WT-MH2) or the AAA and DPD mutants were fixed and visualized. (D) Subcellular fractionation confirms the fluorescence results. Subcellular fractionation was performed on COS7 cells transfected with the MH2 constructs. WB of aliquots of the fractions was performed with aGFP Ab to detect the SMAD3 constructs and with atubulin or ac-Jun Abs as cytoplasmic and nuclear markers respectively. (E) The NTS motif regulates MEK1 shuttling into the nucleus. Serum-starved CHO cells overexpresing WT-GFP-MEK1, AAA-GFP-MEK1, and EPE-GFP-MEK1 were treated with LMB (5 ng/ml, 1 hr), LMB + TPA (5 ng/ml, 45 min and then 250 nM TPA for an additional 15 min), or left untreated (NT). Subsequently the cells were fixed, stained with DAPI, and visualized.
shuttling. Thus, in similarity to the SPS domains of ERK2 and SMAD3, TPT domain mediates nuclear translocation of MEK1. DISCUSSION Nuclear translocation of signaling molecules is crucial for their proper stimulus-dependent functioning in the regulation of cellular processes such as proliferation and differentiation. Interestingly, many of these proteins, including ERKs, do not contain the canonical NLS, and their mechanism of nuclear translocation is not fully understood. Based on previous publications and the results obtained here, we propose a mechanism for the regulation of ERKs’ subcellular localization (see Movie S1 for a model). Thus, in resting cells, ERKs are associated with several cytoplasmic anchors through several docking domains (Chuderland and Seger, 2005). Phosphorylation of their TEY domain by MEKs induces a conformational change, which activates the proteins
and induces their detachment from most of the anchors (Wolf et al., 2001). The detached proteins are then phosphorylated on their SPS domain, possibly by different protein kinases operating in different conditions. This phosphorylation then allows association of the active ERKs with Imp7 (Figures 5 and 6) that carries the ERKs via the nuclear pores into the nucleus. In this case, the Imp7-dependent translocation does not require the aid of Imp-b (Figure 6), which seems to be required for Imp7 in some, but not all, systems (Gorlich et al., 1997; Jakel and Gorlich, 1998). The actual penetration through the pore requires SPS phosphorylation as well because it seems to alleviate the strong interaction with NUPs (Figure 6) and to allow a proper pore sliding. Our results indicate that Imp7- and NTS-dependent processes are also involved in the nuclear tranlocation of SMAD3 and MEK1 (Figure 7). The scenario above describes the active nuclear translocation of ERKs upon stimulation. However, it was shown that ERKs can
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also translocate into the nucleus via a coexisting passive mechanism as well (Adachi et al., 1999). Our results indicate that the SPS phosphorylation is required for this type of translocation as well, because the overexpressed (Figure 3) or LMB-induced ERKs (Figure 5 and Figure S3) were translocated to nuclei of resting cells in an SPS-dependent manner (Figure 3 and Figure S1). Interestingly, despite the extensive dependence on SPS phosphorylation, the passive diffusion of overexpressed ERK2, or of endogenous ERKs late after LMB treatment, were only partially inhibited by Imp7 knockdown (Figure 5). The limited dependence of passive translocation on Imp7 may indicate that under such conditions, some of the ERK molecules can reach NUPs without the aid of the transport carrier, although a small fraction of the ERK may still use the Imp7 due to a limited amount of SPS phosphorylation in resting or LMB-treated cells. On the other hand, the dependence of passive transport on pSPS could be explained by the necessity of phosphate incorporation to the SPS for the proper sliding through the NUPs as mentioned above. Additionally, our results indicate that dimerization (Khokhlatchev et al., 1998) is not required for the translocation, although autophsophorylation of the SPS domain may participate in the stimulated translocation. This result fits our observation obtained with mutations in ERKs dimerization residues (Wolf et al., 2001). To our surprise, we found that unlike the interaction with importins, SPS phopshorylation decreases rather than increases the binding of ERKs to Nup153 (Figure 6C). This result may suggest that NUPs’ binding is mediated by other residues, adjacent to the SPS domain, which might be complementary residues in the SPS-dependent NTS. The phosphorylation of the SPS either reduces this binding affinity to the interacting residues or elevates its Koff, a process that results in a faster release from the NUPs, and allows a smooth sliding through the nuclear pores. Such a binding might have been mediated by hydrophobic regions, which were previously shown important in the binding of NUPs to various proteins (Bayliss et al., 2002). Thus, hydrophobic regions in the vicinity of the SPS domain have been implicated in the nuclear translocation of SMAD3 (Xu et al., 2003) and ERK2 (Lee et al., 2004), and we noticed that a proximal hydrophobic region exists also in MEK1 (Figure S10). However, our results indicate that such residues do not participate in the translocation (data not shown), and therefore, the interrelationships between the SPS domain and hydrophobic or other NUPbinding regions need further examination. A sequence analysis showed that the NTS is present in the sequences of regulatory nuclear shuttling signaling proteins and might similarly induce their nuclear translocation. One such protein is Cyclin-B, in which the region required for translocation contains SPS domain in a Ser-rich region (Walsh et al., 2003). ERK phosphorylation of Ser residues in this region was shown to be important for the mechanism of translocation and, therefore, is likely to operate via phosphorylated SPS and support the NTS mechanism identified here. Other nuclear shuttling NTS-containing proteins are SMAD2 and SMAD4, JNK2, AKT, STAT4, BRCA1, APC, p53, and more signaling proteins whose mechanism of translocation is not yet clear and could require phosphorylation of S/T-P-S/T. Notably, the requirement for pSPS could be alleviated when one of the amino acids next to
a Pro is acidic (S/T-P-D/E, E/D-P-S/T). Such sequences (DPS) exist in the translocating JNK1 (Mizukami et al., 1997) and b-catenin (Henderson and Fagotto, 2002). Two acidic residues next to the Pro may induce permanent nuclear accumulation, and this may be the case in p38MAPK (Ben-Levy et al., 1998). However, the regulation of translocation of such proteins is probably less stringent and should be further studied. In summary, we identified a unique, general NTS that participates in nuclear translocation of signaling proteins upon extracellular stimulation. Phosphorylation of the NTS is required for the dynamic and reversible shuttling. The generality and autonomous function of the NTS was validated by overexpression of b-Gal-GFP chimera fused to a sequence of 19 amino acids containing the NTS. The mechanism of stimulated translocation involves phosphorylation-dependent interaction with Imp7 and release from NUPs. The passive translocation requires SPS phosphorylation as well, but is not likely to involve Imp7 binding. The NTS-dependent nuclear translocation was identified and characterized for ERK2, SMAD3, and MEK1, but similar sequences in other signaling proteins suggest that the identified NTS may play a general role in the stimulation-dependent translocation of signaling proteins. EXPERIMENTAL PROCEDURES DNA Constructs and Mutations GFP-ERK2 was prepared in pEGFP-C1 (Clontech, Mountain View, CA) as described (Rubinfeld et al., 1999). GST-ERK2 was prepared in pGEX vector as previously described (Chuderland et al., 2008). All mutations described were verified by sequencing. GFP-MEK1 was in pEGFP-N1 (Clontech; Yao et al., 2001), and HA-MEK1 was prepared in pCMV-HA. To construct chimera proteins bearing the SPS domain, 19 amino acid stretch containing the SPS from ERK2 (LDQLNHILGILGSPSQEDL) was fused after a starting Met to the N terminus of fusion protein 2GFP (gift from A. Gross, Weizmann Institute of Science [WIS], Israel) or b-Gal-GFP. Point mutations were performed by site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed by sequencing. The GST-ERK2 constructs were prepared using pGEX-2T vector (AP Biotech, USA). The His-Nup153c was a gift from Dr. Michael Elbaum (WIS, Israel). 6His-MEK construct was produced as described (Jaaro et al., 1997). GFP-MH2-SMAD3 and FLAG-SMAD3 were provided by Dr. Joan Massague` (Mermorial Sloan-Kettering Center, NY). All SiRNAs were from Dharmacon (Lafayette, CO). Cell Culture and Transfection COS7 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and were transfected using the DEAE-dextran method as described (Jaaro et al., 1997) and polyethylenimine (PEI) as described (Shaul and Seger, 2006), respectively. CHO cells were grown in F12-DMEM and transfected using Lipofectamine (Invitrogen, CA). After transfection, the cells were washed and grown under their corresponding conditions. SiRNAs were transfected using oligofectamin (Dharmacon) according to the manufacturer’s instructions. Immunofluorescence Microscopy Cells were fixed (30 min in 3% paraformaldehyde in PBS), followed by 5 min permeabilization with 0.2% Triton X-100 in PBS (23 C). The fixed cells were sequentially incubated with an Ab (45 min), followed by rhodamine-conjugated secondary Ab (45 min) and DAPI. Slides were visualized by a fluorescence microscope (Nikon, Japan 4003 magnification). Western Blotting Cell extracts were collected and separated by 10% SDS-PAGE, which was followed by transfer to nitrocellulose and WB with the appropriate Abs. The blots
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were developed with alkaline phosphatase or horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat Fab Abs. Additional Experimental Procedures can be found in the Supplemental Data.
SUPPLEMENTAL DATA The Supplemental Data include Supplemental Introduction, Supplemental Experimental Procedures, Supplemental References, 11 figures, and one movie and can be found with this article online at http://www.molecule.org/ cgi/content/full/31/6/850/DC1/.
ACKNOWLEDGMENTS We would like to thank Reut Hemo and the MS unit of WIS for their help in the project. This work was supported by grants from the Israel Academy of Sciences and Humanities, MINERVA foundation, and from the European Community’s Sixth Framework Program project IST-2004-027265-SIMAP. R.S. is an incumbent of the Yale Lewine and Ella Miller Lewine professorial chair for cancer research. Received: January 19, 2007 Revised: April 4, 2008 Accepted: August 4, 2008 Published online: August 28, 2008
Fukuda, M., Gotoh, Y., and Nishida, E. (1997). Interaction of MAPK with MAPKK: its possible role in the control of nucleocytoplasmic transport of MAP kinase. EMBO J. 16, 1901–1908. Gorlich, D., Dabrowski, M., Bischoff, F.R., Kutay, U., Bork, P., Hartmann, E., Prehn, S., and Izaurralde, E. (1997). A novel class of RanGTP binding proteins. J. Cell Biol. 138, 65–80. Henderson, B.R., and Fagotto, F. (2002). The ins and outs of APC and betacatenin nuclear transport. EMBO Rep. 3, 834–839. Jaaro, H., Rubinfeld, H., Hanoch, T., and Seger, R. (1997). Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation. Proc. Natl. Acad. Sci. USA 94, 3742–3747. Jakel, S., and Gorlich, D. (1998). Importin beta, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells. EMBO J. 17, 4491–4502. Khokhlatchev, A.V., Canagarajah, B., Wilsbacher, J., Robinson, M., Atkinson, M., Goldsmith, E., and Cobb, M.H. (1998). Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell 93, 605– 615. Lee, T., Hoofnagle, A.N., Kabuyama, Y., Stroud, J., Min, X., Goldsmith, E.J., Chen, L., Resing, K.A., and Ahn, N.G. (2004). Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry. Mol. Cell 14, 43–55.
REFERENCES
Liu, X., Sun, Y., Constantinescu, S.N., Karam, E., Weinberg, R.A., and Lodish, H.F. (1997). Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc. Natl. Acad. Sci. USA 94, 10669–10674.
Adachi, M., Fukuda, M., and Nishida, E. (1999). Two co-existing mechanisms for nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer. EMBO J. 18, 5347–5358.
Lorenzen, J.A., Baker, S.E., Denhez, F., Melnick, M.B., Brower, D.L., and Perkins, L.A. (2001). Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin. Development 128, 1403–1414.
Adachi, M., Fukuda, M., and Nishida, E. (2000). Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism. J. Cell Biol. 148, 849–856. Bayliss, R., Littlewood, T., Strawn, L.A., Wente, S.R., and Stewart, M. (2002). GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta. J. Biol. Chem. 277, 50597–50606. Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H.F., and Marshall, C.J. (1998). Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr. Biol. 8, 1049–1057. Cha, H., Lee, E.K., and Shapiro, P. (2001). Identification of a C-terminal region that regulates mitogen-activated protein kinase kinase-1 cytoplasmic localization and ERK activation. J. Biol. Chem. 276, 48494–48501. Chen, H.B., Rud, J.G., Lin, K., and Xu, L. (2005). Nuclear targeting of transforming growth factor-beta-activated Smad complexes. J. Biol. Chem. 280, 21329–21336.
Massague, J., Blain, S.W., and Lo, R.S. (2000). TGFbeta signaling in growth control, cancer, and heritable disorders. Cell 103, 295–309. Massague, J., Seoane, J., and Wotton, D. (2005). Smad transcription factors. Genes Dev. 19, 2783–2810. Matsubayashi, Y., Fukuda, M., and Nishida, E. (2001). Evidence for existence of a nuclear pore complex-mediated, cytosol-independent pathway of nuclear translocation of ERK MAP kinase in permeabilized cells. J. Biol. Chem. 276, 41755–41760. Matsuda, S., Gotoh, Y., and Nishida, E. (1993). Phosphorylation of Xenopus mitogen-activated protein (MAP) kinase kinase by MAP kinase kinase kinase and MAP kinase. J. Biol. Chem. 268, 3277–3281. Mizukami, Y., Yoshioka, K., Morimoto, S., and Yoshida, K. (1997). A novel mechanism of JNK1 activation. Nuclear translocation and activation of JNK1 during ischemia and reperfusion. J. Biol. Chem. 272, 16657–16662.
Chen, R.H., Sarnecki, C., and Blenis, J. (1992). Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol. Cell. Biol. 12, 915–927.
Nantel, A., Mohammad-Ali, K., Sherk, J., Posner, B.I., and Thomas, D.Y. (1998). Interaction of the Grb10 adapter protein with the Raf1 and MEK1 kinases. J. Biol. Chem. 273, 10475–10484.
Christophe, D., Christophe-Hobertus, C., and Pichon, B. (2000). Nuclear targeting of proteins: how many different signals? Cell. Signal. 12, 337–341.
Nigg, E.A. (1997). Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386, 779–787.
Chuderland, D., and Seger, R. (2005). Protein-protein interactions in the regulation of the extracellular signal-regulated kinase. Mol. Biotechnol. 29, 57–74.
Rubinfeld, H., Hanoch, T., and Seger, R. (1999). Identification of a cytoplasmicretention sequence in ERK2. J. Biol. Chem. 274, 30349–30352.
Chuderland, D., Marmor, G., Shainskaya, A., and Seger, R. (2008). Calciummediated interactions regulate the subcellular localization of extracellular signal-regulated kinases (ERKs). J. Biol. Chem. 283, 11176–11188.
Shaul, Y.D., and Seger, R. (2006). ERK1c regulates Golgi fragmentation during mitosis. J. Cell Biol. 172, 885–897.
Fu, X., Choi, Y.K., Qu, D., Yu, Y., Cheung, N.S., and Qi, R.Z. (2006). Identification of nuclear import mechanisms for the neuronal Cdk5 activator. J. Biol. Chem. 281, 39014–39021. Fukuda, M., Gotoh, I., Gotoh, Y., and Nishida, E. (1996). Cytoplasmic localization of mitogen-activated protein kinase kinase directed by its NH2-terminal, leucine-rich short amino acid sequence, which acts as a nuclear export signal. J. Biol. Chem. 271, 20024–20028.
Schlenstedt, G. (1996). Protein import into the nucleus. FEBS Lett. 389, 75–79.
Tran, E.J., and Wente, S.R. (2006). Dynamic nuclear pore complexes: life on the edge. Cell 125, 1041–1053. Walsh, S., Margolis, S.S., and Kornbluth, S. (2003). Phosphorylation of the cyclin b1 cytoplasmic retention sequence by mitogen-activated protein kinase and Plx. Mol. Cancer Res. 1, 280–289. Wolf, I., Rubinfeld, H., Yoon, S., Marmor, G., Hanoch, T., and Seger, R. (2001). Involvement of the activation loop of ERK in the detachment from cytosolic anchoring. J. Biol. Chem. 276, 24490–24497.
860 Molecular Cell 31, 850–861, September 26, 2008 ª2008 Elsevier Inc.
Molecular Cell Importin7-Dependent Nuclear Translocation Signal
Xiao, Z., Liu, X., Henis, Y.I., and Lodish, H.F. (2000a). A distinct nuclear localization signal in the N terminus of Smad 3 determines its ligand-induced nuclear translocation. Proc. Natl. Acad. Sci. USA 97, 7853–7858. Xiao, Z., Liu, X., and Lodish, H.F. (2000b). Importin beta mediates nuclear translocation of Smad 3. J. Biol. Chem. 275, 23425–23428. Xu, L., Alarcon, C., Col, S., and Massague, J. (2003). Distinct domain utilization by Smad3 and Smad4 for nucleoporin interaction and nuclear import. J. Biol. Chem. 278, 42569–42577. Xu, L., and Massague, J. (2004). Nucleocytoplasmic shuttling of signal transducers. Nat. Rev. Mol. Cell Biol. 5, 209–219.
Yao, Z., Flash, I., Raviv, Z., Yung, Y., Asscher, Y., Pleban, S., and Seger, R. (2001). Non-regulated and stimulated mechanisms cooperate in the nuclear accumulation of MEK1. Oncogene 20, 7588–7596. Yazicioglu, M.N., Goad, D.L., Ranganathan, A., Whitehurst, A.W., Goldsmith, E.J., and Cobb, M.H. (2007). Mutations in ERK2 binding sites affect nuclear entry. J. Biol. Chem. 282, 28759–28767. Yoon, S., and Seger, R. (2006). The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24, 21–44.
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