RanBP3 inhibits Smad2/3-mediated transcriptional responses in mammalian cells
In an attempt to identify novel intracellular factors controlling TGF-β signaling, we discovered that RanBP3 was a strong candidate for mediating nuclear export of Smad2/3. As a first step in evaluating a possible role of RanBP3 in TGF-β signaling, we examined the effects of RanBP3 on Smad2/3-mediated transcriptional activation using various Smad-dependent gene reporters. Increased expression of human or mouse RanBP3 caused a decrease in TGF-β-dependent transcription from the Smad-binding element (SBE)-Luc reporter (; data not shown). RanBP3 also inhibited TGF-β-induced activation of the natural p21 promoter in HaCaT cells (), as well as the p15 promoter and the plasminogen activator inhibitor type 1 (PAI-1) promoter in HepG2 cells (data not shown). However, RanBP3-wv, a mutant that loses its Ran-binding activity (Englmeier et al., 2001
), failed to block TGF-β-induced promoter activation, although the mutant was expressed at a similar level to that of RanBP3 (). In sharp contrast to its ability to regulate TGF-β signaling, RanBP3 did not interfere with BMP-induced activation of the Id1 promoter in C2C12 cells () or HepG2 cells (Figure S3
), suggesting that RanBP3 specifically acts on TGF-β, but not BMP, signaling.
RanBP3 inhibits TGF-β-induced transcriptional responses in mammalian cells
Consistent with the ability of RanBP3 to inhibit activation of TGF-β-responsive promoters, HaCaT cells stably expressing RanBP3 (, blot a) diminished TGF-β-mediated induction of endogenous p15, p21 and PAI-1 mRNAs (). In addition, the protein level of p21 was also reduced (, blot b). Overexpression of RanBP3, however, did not disrupt the TGF-β-induced C-terminal phosphorylation of Smad2/3 (, blot c and d), nor did it affect the total level of Smad2/3 in the same stable cells (, blot e). These data suggest that RanBP3 acts downstream of Smad2/3 activation.
RanBP3 suppresses activin but not BMP signaling in Xenopus embryos
During early vertebrate development, activin/nodal and BMP signals are known to control embryonic patterning and cell fate determination (Chang et al., 2002
). Activation of TGF-β signaling induces mesoderm- and endoderm-specific gene expression in a dose-dependent fashion in early Xenopus
embryos. To examine whether RanBP3 can regulate TGF-β signaling during Xenopus
embryogenesis, we examined the effects of RanBP3 on activin- or BMP-induced endogenous gene expression in Xenopus
ectodermal explants (animal caps). As shown in , activin induced a whole range of mesendodermal markers, including the endodermal marker Sox17α, the dorsal mesodermal marker chordin, the ventrolateral mesodermal marker XWnt8, and the pan-mesodermal marker XBra at gastrula stages (lane 2), whereas BMP4 induced the expression of Sox17α, XWnt8 and Xbra (lane 5). Co-expression of wildtype RanBP3, but not RanBP3-wv, completely suppressed activin-induced expression of Sox17α and chordin, and moderately reduced the expression of Xbra and XWnt8 (lanes 3 and 4). In contrast, RanBP3 or RanBP3-wv failed to block the expression of the genes activated by BMP4, including Sox17α, Xwnt8 and XBra (lane 6). These results further support the notion that RanBP3 preferentially blocks activin but not BMP signaling in a Ran-binding dependent manner.
Knockdown of RanBP3 enhances TGF-β growth inhibitory and transcriptional responses
We next investigated whether RanBP3 depletion enhances TGF-β responses. We first established HaCaT cell lines that stably express distinct shRNAs against separate target sequences of RanBP3, i.e. shRNA494 (designated RanBP3-KD1) or shRNA-676 (designated RanBP3-KD2 and KD3)(). It is clear that depletion of RanBP3 enhanced TGF-β-induced expression of both the SBE-Luc reporter () and the natural genes including those encoding PAI-1, and CDK inhibitors p15 and p21 (). Consistent with the increased mRNA levels of p15 and p21, depletion of RanBP3 rendered cells more sensitive to TGF-β-induced growth inhibition (). Collectively, these data established a novel role of RanBP3 as negative modulator in TGF-β-mediated transcriptional responses.
Knockdown of RanBP3 enhances TGF-β growth inhibitory and transcriptional responses
RanBP3 enhances nuclear export of Smad2/3
Because RanBP3 has been implicated in nuclear export by its ability to bind Ran, nuclear pore components and CRM1 (Mueller et al., 1998
; Englmeier et al., 2001
; Lindsay et al., 2001
), we sought to evaluate if RanBP3 has a direct role in regulating Smad2/3 nuclear export. The ability of RanBP3 to affect TGF-β-induced nuclear accumulation of endogenous Smad3 was examined in HaCaT cells. As seen in , indirect immunostaining of Smad3 revealed that overexpression of RanBP3 decreased the intensity of nuclear Smad3 by 55.3%(+/-4.2%). Similar results were observed with mouse RanBP3 (data not shown). To further confirm this observation, TGF-β-induced nuclear accumulation of endogenous Smad2 was examined in parental HaCaT cells, RanBP3-expressing stable cells (RanBP3-OE), and RanBP3 knockdown cells (RanBP3-KD1). The results demonstrate that the level of nuclear Smad2/3 is inversely proportional to that of RanBP3 in the nucleus. As shown in , TGF-β stimulation rapidly induced Smad2 accumulation in the nucleus in parent HaCaT cells. Increased expression of RanBP3 reduced the level of nuclear Smad2 in RanBP3-OE cells, whilst knockdown of endogenous RanBP3 expression rendered more endogenous Smad2 accumulated in the nucleus of RanBP3-KD1 cells.
RanBP3 enhances nuclear export of Smad2/3
To specifically evaluate Smad2/3 export without interference of continuous import, we next examined the TGF-β-induced nuclear accumulation of Smad2/3 in the presence of TβRI inhibitor SB431542, which blocks Smad2/3 phosphorylation in the cytoplasm and nuclear accumulation thereafter (Laping et al., 2002
; Inman et al., 2002a
). In parental HaCaT cells, Smad2 and Smad4 were accumulated in the nucleus after 2 h of TGF-β stimulation, but redistributed throughout the whole cells after removal of TGF-β and simultaneous addition of SB431542 for 2 h. Treatment with CRM1 inhibitor LMB blocked Smad4 but not Smad2 redistribution (, top panels). In RanBP3-KD1 cells, sustained Smad2 nuclear localization was observed in the absence or presence of LMB (, second row panels from the bottom). On the contrary, knockdown of RanBP3 did not affect the Smad4 cellular redistribution (, bottommost panels).
We next sought to specifically examine how much Smad2/3 are exported into the cytoplasm. To this end, cells were similarly treated as in and harvested at different time point during 4 h export process. Smad2/3 levels in the cytoplasmic vs. nuclear fractions at each time point were analyzed by Western blotting. The quality of cytoplasmic/nuclear fractionation was determined by the presence of cytoplasmic GADPH and nuclear Lamin A/C in the fractions. At time point 0 (immediately after 60 min TGF-β treatment), a very low level of Smad2/3 in the cytoplasmic fractions of both wildtype and knockdown cell lines was detected, indicating most of the Smad2/3 resided in the nucleus. SB431542 treatment promoted the cycling back of Smad2/3 in the cytoplasm in parental HaCaT cells, as indicated by the gradual increase in the level of cytoplasmic Smad2/3 and the corresponding decrease in the nuclear Smad2/3 (). In contrast, knockdown of RanBP3 slowed down the cytoplasmic-to-nuclear shift of Smad2/3 in RanBP3-KD1 cells (). Taken together, the data in support the notion that RanBP3 positively regulates the nuclear export of Smad2/3.
RanBP3 enhances Smad2 nuclear export in a Ran-binding dependent manner
Since RanBP3 is a Ran-binding protein, we sought to examine whether RanBP3 requires Ran to export Smad2/3 by using in vitro
export assay. In these assays, HaCaT cells stably expressing GFP-Smad2 were first treated with TGF-β for 2 h to induce Smad2 nuclear accumulation, and then pulsed with digitonin for 5 min to permeabilize cell membrane. After washing, the remaining level of GFP-Smad2, which represented nuclear Smad2 that was not exported, was determined by Western blotting analysis. Several dosages of digitonin were tested to produce substantial loss of cytoplasmic proteins yet retain the intact nuclear envelope (Figure S1
). To avoid over-permeabilization, we chose 30 ng/ml of digitonin in the subsequent assays. Proper cell membrane permeabilization was confirmed by lack of GAPDH in the total lysates of permeabilized cells (). In addition, functional integrity of the nuclear envelope was assessed by similar levels of GFP-Smad2 in digitonin-permeabilized and non-permeabilized cells at time point 0 (normalized against that of Lamin A/C) as well as the disappearance of cytoplasmic GADPH after 5 min of digitonin treatment (, lane 4). In vitro
export of GFP-Smad2 was progressive over time, as indicated by a reduced level of GFP-Smad2 protein (, lanes 4-6) or diminished GFP fluorescence (data not shown) in the permeabilized cells. Retention of GFP-Smad2 in the nucleus appeared to partially, if not completely, depend on Ran activity and energy because it decreased after addition of recombinant Ran proteins to the cells (lanes 9 and 10) and increased when incubated on ice (lanes 7 and 8).
RanBP3 mediates Smad2 export dependent of its Ran-binding ability
We then analyzed the effect of the Ran-binding deficiency of RanBP3-wv on Smad2 export. Albeit at low efficiency, recombinant RanBP3 and RanBP3-wv proteins could similarly enter the nucleus of permeabilized cells (Figure S2
). It is apparent that RanBP3 was capable of exporting Smad2 (, lanes 4 and 5). In sharp contrast, RanBP3-wv had no effect on Smad2 export in vitro
(lanes 6 and 7). To further compare the effect of RanBP3 or RanBP3-wv mutant, we also used a highly sensitive quantitative export assay (Cullen, 2004
), which has been previously used to analyze Smad2 nuclear export (Xu et al., 2002
; Lin et al., 2006
). Consistent with our data in vitro
, we found that RanBP3 increased Smad2 export by 5.2-folds, whereas RanBP3-wv failed to produce any effects (). These results strongly suggest that RanBP3 promotes Smad2 nuclear export in a Ran-dependent manner.
RanBP3 specifically interacts with Smad2/3
Having established the role of RanBP3 in mediating nuclear export of Smad2/3, we investigated if RanBP3 could physically associate with Smads. Co-immunoprecipitation (co-IP) assay was used to examine the ability of RanBP3 to interact with Smads in transfected HEK293 cells. We detected the presence of RanBP3 in the immuno-complex of Smad2 (, lanes 2 and 3), but not in that of Smad1 (, lane 4-5). RanBP3 could also bind to Smad3 (, lanes 1 and 2), which is the closest homolog of Smad2 and also functions as TGF-β signal transducer. The interaction of RanBP3 with Smad4, a common Smad for both TGF-β and BMP signaling, was comparatively weaker (, lanes 3 and 4). Furthermore, immunoprecipitation of endogenous RanBP3 retrieved endogenous Smad2 and Smad3 proteins in human keratinocytes HaCaT (, lane 4). Taken together, these data reveal an interaction between RanBP3 and TGF-β-specific Smad2/3 under physiological conditions. Notably, activation of the TGF-β receptor appeared to decrease the Smad2/3-RanBP3 interaction (, lane 5, to be discussed later).
RanBP3 interacts with TGF-β specific Smad2 and Smad3
We further examined the RanBP3-Smad3 interaction by mapping their respective interacting domains. As shown in , both the N-terminal MH1 domain and C-terminal MH2 domain could bind to RanBP3 in vitro
. The domains in RanBP3, namely the N-terminal domain (N), intermediate domain domain (F) and C-terminal Ran:GTP-binding domain (R) (Mueller et al., 1998
; Lindsay et al., 2001
), all associated with Smad3 yet weakly than the full-length RanBP3 (). Among all the domains, R apparently exhibited stronger affinity to Smad3 than N or F (, lane 4) and directly interacted with Smad3 in vitro
(, lane 3).
To further identify the structural determinants that define the RanBP3-interacting specificity of Smad3, we generated a series of chimeras between Smad1 (unable to bind to RanBP3) and Smad3 (able to bind to RanBP3). In these chimeras, one or two domains of Smad3 were replaced with the counterpart(s) of Smad1 (, right panel). The ability of these chimeras to bind to RanBP3 was assessed and compared with wildtype Smad 3 in co-IP experiments. As observed earlier, RanBP3 interacted with wildtype Smad3, but not Smad1 (, lanes 2 and 3). RanBP3 also interacted with Smad1-Smad3 chimeras containing the MH1 and/or MH2 domain of Smad3 (, lanes 4, 5 and 7-9). However, RanBP3 did not interact with Smad131 with the linker region only of Smad3 (, lane 6), suggesting the MH1 and MH2 domain mediate the RanBP3-Smad3 interaction. Interestingly, among all the chimeras, Smad313, which contains both the MH1 and MH2 domains of Smad3, exhibited the strongest interaction with RanBP3 that is comparable to that of wildtype Smad3 (, lanes 3 and 8). Taken together, data in suggest that at least two different epitopes in Smad3 are recognized by RanBP3 and cooperatively mediate the Smad3-RanBP3 interaction.
RanBP3 binds to Smad2/3 in their unphosphorylated forms
As shown in , the interaction between RanBP3 and Smad2/3 was apparently weaker in the presence of active TGF-β signaling, either by expression of a constitutively active mutant form of TβRI, i.e. ALK5(T202D) (, lane 3) or TGF-β ligand stimulation (, lane 5). Since RanBP3 resides in the nucleus (Mueller et al., 1998
) (), where Smad2/3 dephosphorylation by nuclear phosphatase PPM1A also occurs (Lin et al., 2006
), we examined the effect of PPM1A on the RanBP3-Smad3 interaction. The level of TGF-β-induced Smad3 phosphorylation was reduced by PPM1A, but was not affected by overexpressed RanBP3 (Figure S4
). Notably, increased expression of PPM1A clearly enhanced the association between RanBP3 and Smad3 (, lanes 3 and 4). These data support the notion that RanBP3 preferentially binds to dephosphorylated Smad2/3.
RanBP3 binds to Smads in its unphosphorylated form in the nucleus
We then carried co-IP experiments to examine the Smad2 SXS motif mutant that either lacks the C-terminal serine phosphorylation (inactive, 2SA) or harbors phosphorylation-mimetic residues (active, 2SD). Analysis of the results revealed different binding affinity of these mutants toward RanBP3. As shown in , RanBP3 pulled down more Smad2(2SA) than Smad2(2SD) mutant (lanes 3 and 4). Since the SXS motif phosphorylation induces Smad2/3-Smad4 interaction, we sought to determine whether TGF-β-induced Smad2-Smad4 interaction contributed to the decreased association between Smad2 and RanBP3. As shown in , overexpression of Smad4 did not affect the interaction between RanBP3 and the Smad2(2SD) mutant (lanes 2 and 3), which readily interact with Smad4 in the absence of TGF-β stimulation (data not shown). Overexpression of RanBP3 also had no affect on the interaction between Smad2(2SD) mutant and Smad4 (). These data suggest that Smad4 and RanBP3 do not interfere with each other for binding to Smad2/3.
To further explore how the phosphorylation affects the RanBP3-Smad2/3 interaction, we carried out in vitro binding assays to examine the RanBP3 interaction with the recombinant Smad2 MH2 (aa 241–467) protein either in its phosphorylated or unphosphorylated form. The results revealed that GST-RanBP3 could bound to unphosphorylated Smad2 MH2 domain (, lane 1), but had no binding affinity to the phosphorylated Smad2 MH2 domain (, lane 2). Collectively, these data establish that RanBP3 preferentially binds to the unphosphorylated and recently dephosphorylated Smad2/3.
RanBP3 interacts with Smad2/3 in the nucleus
To identify the subcellular compartment where RanBP3 interacts with Smad2/3, we used a YFP-based fluorescence complementation system that allows easy visualization of protein interactions with microscopy (Hu et al., 2002
). In this system, YFP fragment YN (aa 1-154) and YC (aa 155-238) were separately fused with each of the interacting protein partners. Protein interaction brings together the two YFP halves to reconstitute a functional YFP that produces a fluorescent signal. In HaCaT cells co-expressing YC-RanBP3 and YN-Smad3, YFP fluorescence was detected in the nucleus in the absence of TGF-β (, set a); similar results were observed when the reverse pair, i.e. YN-RanBP3 and YC-Smad2, were used (, set b). As negative controls, the YC-RanBP3/YN-vector pair and YN-Smad2/YC-vector pair did not yield fluorescence (, set c and d).
We next examined how the RanBP3-Smad2 interaction responds to TGF-β. As shown in , endogenous Smad2 resided in both the cytoplasm and the nucleus in HaCaT cell at the resting state (0.2% FBS treatment overnight). Whilst TGF-β stimulation for 2 h induced nuclear accumulation of Smad2, TβRI inhibitor SB431542 rendered Smad2 mostly accumulated in the cytoplasm. The distribution of transiently expressed YC-Smad2 mimics endogenous Smad2. When YC-Smad2 and YN-RanBP3 were co-expressed, YFP fluorescence resided exclusively in the nucleus under all the conditions examined, strongly suggesting that RanBP3 interacts with Smad2/3 in the nucleus regardless TGF-β signaling.
RanBP3 does not affect the DNA-binding ability of Smad3
As RanBP3 also associates with the MH1 domain of Smad3 (, lane 2), we sought to examine whether RanBP3 would affect the Smad3's DNA-binding ability using DNA pulldown assay. The biotin-labeled SBE oligo comprises of four copies of the SBE sequences and was used to retrieve SBE-containing complex from the lysates of HaCaT control cells and RanBP3-KD1 stable cells. As shown in , TGF-β induced binding of Smad3 and Smad4 to biotinylated SBE (lane 3 and 5). RanBP3 was undetectable in the SBE-containing complex (lane 2 and 3), which is consistent with its position downstream of the Smad complex dissociation and dephosphorylation of Smad3. Moreover, RanBP3 depletion did not affect the level of SBE-bound Smad3 (lane 5), suggesting that RanBP3 would not affect the Smad3's DNA-binding activity.
RanBP3-R dominantly blocks Smad2 export and enhances TGF-β signaling
To evaluate how the RanBP3-Smad interaction modulates Smad2/3 export as well as TGF-β signaling, we explored the use of the Smad-interacting region in RanBP3 as a competitive inhibitor for this interaction. The RanBP3-R resides in the cytoplasm when exogenously expressed (Welch et al., 1999
). Since RanBP3 interacts with Smad2/3 in the nucleus () and partly through the R domain of RanBP3 (), we introduced the nuclear localization signal (NLS) in the N terminus of RanBP3-R (generating NLS-R) in order to force RanBP3-R to be localized in the nucleus. When NLS-R was co-transfected with RanBP3, the association of RanBP3 with Smad2 () and Smad3 () was disrupted in HEK293T cells, clearly demonstrating that RanBP3-R competes with RanBP3 for Smad2/3 binding. We then examined the effect of NLS-R in Smad2 export and TGF-β-induced transcriptional responses. Quantitative Smad2 export assay revealed NLS-R attenuated RanBP3-mediated Smad2 export in a dose-dependent manner (). Consistently, NLS-R enhanced the TGF-β-induced SBE-luc response in HaCaT cells (). Collectively, these data suggest that through its direct interaction with Smad2/3, RanBP3 mediates the nuclear export of Smad2/3 and hence function as a negative modulator in TGF-β signaling.
RanBP3 controls Smad2/3 export and TGF-β signaling via its contact with Smad2/3