The isolation of Smad1 interacting proteins reveals a physical interaction between Smad1 and components in proteasome-mediated degradation pathways
We applied the yeast "Protein Trap" system to isolate Smad1 interactors[26, 27]. Full length Smad1 was fused with the DNA-binding domain of LexA to serve as bait. A human fetal brain cDNA library with cDNA inserts fused with the transcriptional activation domain B42 was used for the screen. From screening one million cDNA clones, 40 clones exhibited strong activation of both of the reporter genes (LacZ and Leu2). Thirteen different cDNA inserts were identified from these 40 clones. The ability of these thirteen candidate Smad1 interactors to bind Smad1, Smad2, Smad3 and Smad4 were directly tested in the yeast two-hybrid system (Fig. 1A). Among the thirteen isolated Smad1 interactors, only clones 13, 17, 19 and 21 bind Smad2, whereas only clones 1, 17 and 21 exhibit strong interaction with Smad3. Only one clone (clone 17) among the thirteen clones interacts with Smad4. Interestingly, this clone encodes a truncated Smad1 lacking its MH1 domain. Such an interaction is consistent with the known ability of Smad1 to bind Smad4 upon its activation in mammalian cells [9].
The identities of the thirteen interactors are summarized (Fig. 1B, top panel). We divided the interactors into three groups (Fig. 1B, top panel). The first group contains proteins that have a functional link to the degradation pathways of the 26S proteasome. They are: two ubiquitin precursors UBA80 (clone 1) and UBA52 (clone 21), the ornithine decarboxylase antizyme (Az) (clone 15) and the proteasome β subunit HsN3 (clone 18). The second group contains four known proteins: GST (clone 13), MBP-1 (c-Myc promoter Binding Protein, clone 14) [28], Smad1 (clone 17), TAG (Tumor Associated Gene, clone 31) [29] and PAG (Proliferation Associated Gene, clone 35) [30]. The third group contains four novel proteins: clone 19, 12, 27 and 28, which we named as SNIP1, SIP2, SIP3 and SIP4, respectively. The functional characterization of clone 19 (SNIP1) as a novel CBP/p300 interactor and repressor has been reported recently in the signaling pathways of TGF-β [25]. The functional characterization of clone 12 (SIP2) as a new member of the sorting nexin family member has also been reported [31]. Studies of the novel protein SIP3 will be reported separately (Paralkar et al, manuscript in preparation).
We were intrigued by the fact that Smad1 binds to Ub, Az and HsN3, all of which are involved in proteasome-mediated degradation pathways, as illustrated in Fig. 1B, bottom panel. The well-known ubiquitin-dependent pathway involves the covalent attachment of ubiquitin to substrate proteins to form polyubiquitin chain, which then targets the marked substrates to proteasome for degradation [17, 19, 20]. The ubiquitin-independent pathway is not well defined, but involves targeting proteins other than ubiquitin. The best-characterized example of ubiquitin-independent degradation is the degradation of ornithine decarboxylase (ODC), which is dependent upon ODC interaction with the targeting protein antizyme (Az) [18]. How ubiquitinated substrates or Az-bound ODC is recognized by the 26S proteasome is not well understood. HsN3 is a β subunit of the 20S core of the 26S proteasome and previously has also been implicated in targeting its interacting protein p105 subunit of NF-κB into the 26S proteasome [22–24, 32]. Thus, it is interesting that Smad1 can bind to two types of proteasome substrate-targeting proteins (ubiquitin and Az) as well as a proteasome component (HsN3) that has a possible substrate-targeting role. Studies were then carried out to further characterize Smad1 interaction with HsN3 and Az in mammalian cells and test a functional role of these interactions in the signaling pathways of BMPs as detailed below.
Smad1 interacts with prosequence-containing HsN3 but not processed HsN3 in the 20S proteasome and the activation of the BMP type I receptor enhances the interaction
We first focused upon the physical interaction between Smad1 and HsN3. As one of the seven β subunits of the 20S proteasome, the newly translated HsN3 has an N-terminal 44 amino-acid prosequence, which is processed upon the complete assembly of HsN3 into the mature 20S proteasome [22–24]. The assembly of the 20S proteasome involves multiple steps and the formation of assembly intermediates [33–36]. As illustrated in Fig. 2A, seven single α subunits first form the α ring, which serves as the template for seven β subunits to assemble the β ring. The half proteasomes containing one α and one β rings are further assembled into 20S proteasome, a process involving the proteolytic cleavage of the prosequences of five β subunits, including HsN3. The 20S proteasome then combines with two 19S regulatory complex to form the 26S proteasome, which is the degradation machinery responsible for both ubiquitin-dependent and ubiquitin-independent degradation [37]. The prosequences of the proteolytic β subunits are important to keep the proteolytic sites inaccessible to the substrates. In addition, the prosequences still bind to the β subunits after its cleavage and may serve important chaperon roles for the β subunits to be properly positioned into assembly complex. This was demonstrated previously by artificially removing the prosequence of one β subunit and showed that the assembly of this β subunit is completely blocked while separate expression of the prosequence was enough to restore the assembly[19]. The defective assembly of a β subunit due to the lack of prosequence is illustrated in Fig. 2B.
To follow up the observed interaction between Smad1 and HsN3 in mammalian cells, we first constructed three HsN3 expression constructs. The first construct contains a Flag-epitope placed at the C-terminus of HsN3 (N3-F), since the C-terminus of HsN3 is not buried inside of the mature proteasome, according to the crystal structure of HsN3 [38]. The second and the third constructs contain either the Flag or the T7 epitopes placed at the N-terminus of HsN3 (F-ΔN3, T7-ΔN3). Since the N-terminal prosequence of HsN3 is processed upon assembly into the 20S proteasome, we deleted the prosequence of HsN3 and replaced it with the Flag/T7-epitopes to assure that the epitopes will not be lost due to prosequence-processing. We first expressed these constructs into COS1 cells and examined their expression and assembly properties by immunoprecipitation of each expressed protein from metabolically labeled COS1 cells (Fig. 2C). Multiple endogenous proteins from COS1 cells were co-precipitated with Flag tagged wild type HsN3 (Fig. 2C, lane 2), while much few proteins were detected in the immunoprecipitates of the two deletion versions of HsN3 lacking the prosequence (Fig. 2C, lanes 3 & 4). The predominant band in the immunoprecipitation of wild type HsN3 is the prosequence-containing HsN3 (Pro-N3-F), as indicated by its molecular weight and the ability of anti-Flag antibody to detect it in a parallel Western blot. The other predominant band immediately below Pro-N3-F is the mature form of Pro-N3-F, as indicated by the ability of this band to be also detected by anti-Flag on the Western blot and by its co-migration with the deletion version of N3 lacking the prosequence (F-ΔN3 in lane 3). The rest of the proteins detected in the immunoprecipitates of wild type full length HsN3 (Fig. 2C, lane 2) are predominantly different subunits of the 20S proteasome and 19S regulators, as indicated by their molecular weights and their ability to be recognized by anti-20S and anti-19S antibodies (data not shown). The lack of most of these endogenous proteasome components in the immunoprecipitates of the two deletion version of HsN3 whose prosequences have been artificially removed via deletions indicates that the prosequence of HsN3 is also important for its successful assembly into the mature 20S proteasome. The slight different band patterns detected in the immunoprecipitates of F-ΔN3 and T7-ΔN3 suggests that different epitope tags may influence N3 interaction with endogenous proteasome components. Since mature β subunits are only found in the 20S proteasome [19], the detection of the mature form of the Flag-tagged HsN3 assured us that the exogenous Flag-tagged HsN3 proteins are normal in their ability to be incorporated into the 20S proteasome.
We then tested the ability of HsN3 to bind Smad1 in COS1 cells by transient transfection followed by immunoprecipitation assays. To monitor the co-precipitation of Smad1 with HsN3-containing proteasome complexes, immunoprecipitation were carried out using total cellular proteins from metabolically labeled COS1 cells. Upon the immunoprecipitation of Smad1, only the full-length HsN3 protein, but not the processed mature form of HsN3, was detected to co-precipitate with Smad1 (Fig. 3A, right panel, lane 4). Although the overexpressed HsN3 is associated with many other endogenous proteasome components from the COS cells (Fig. 3A, left panel, lane 4), these endogenous proteins also were not detected in the immunoprecipitates of Smad1 (Fig. 3A, right panel, lane 4). Thus, Smad1 only binds the prosequence-containing HsN3 (pro-N3-F), which, as illustrated in Fig. 2A, only exists transiently before the formation of 20S proteasome. Our domain mapping studies in yeast two-hybrid system have previously suggested that the MH2 domain of Smad1 is necessary for its interaction with HsN3 [39]. Thus we also tested the interaction between HsN3 and Smad1 MH2 domain. Pro-N3-F was also detected in the immunoprecipitates of Smad1MH2 (Fig. 3A, right panel, lane 3), suggesting that the MH2 domain is sufficient to bind to the pre-assembled HsN3.
To test whether the observed interaction between Smad1 and Pro-N3-F, the pre-assembled form of HsN3, is regulated by BMP type I receptor activation, we co-expressed Smad1 and HsN3 with a mutant type I receptor, ALK3Q233D. ALK3Q233D is constitutively active in inducing downstream signaling events in the absence of BMPs or the type II receptor [40]. The co-expression of ALK3Q233D with Smad1 and HsN3 significantly increased the amount of the pre-assembled HsN3 that was co-precipitated with Smad1 (Fig. 3B, right panel, compare lanes 2 & 3). Smad1 was also detected in the immunoprecipitates of HsN3 (Fig. 3B, lanes 2 & 3). Since equal transfection efficiency was monitored and equal total proteins were used for the immunoprecipitation, the increased signals of pro-N3-F and its associated proteins in Fig. 3B, lane 3, left panel, may reflect an increased stability of HsN3 due to complex formation with Smad1, upon BMP receptor activation.
The association between Smad1 and HsN3 was further confirmed by immunoprecipitation followed by Western blot analyses. As shown in Fig. 4A and Fig. 4B, Pro-N3-F was specifically detected in the immunoprecipitates of Smad1 but not those of Smad2. When the activated BMP type I receptor was co-expressed with Smad1 and HsN3, a reduction of Smad1 level was detected and the reduction is blocked by the addition of the proteasome inhibitor lactacystin, indicating the proteasomal degradation of Smad1 induced by BMP type I receptor activation. This phenomenon was further investigated and confirmed in a separate study [39]. The interaction between Pro-N3-F and Smad1 was also stabilized in the presence of proteasome inhibitor lactacystin (Fig. 4B, lanes 4). Western blot using anti-26S proteasome was also carried out to show that only Pro-N3-F, not mature N3, nor other proteasome components, was co-precipitated with Smad1 (data not shown).
Smad1 can interact with prosequence-deleted HsN3 in HsN3 assembly intermediates
The lack of mature HsN3 and other proteasome components in the immunoprecipitates of Smad1 could be mediated by two different mechanisms. First, the prosequence of HsN3, which is processed from the mature HsN3, is necessary for Smad1 to bind to HsN3. Second, the final maturation of the 20S proteasome resulted from the assembly of two half proteasomes into the four stacked ring structure could trap the already bound Smad1 inside the proteasome for rapid degradation or lead to the dissociation of Smad1 from mature 20S proteasome due to the inaccessibility of Smad1 binding site on the incorporated HsN3. We first tested the role of the prosequence of HsN3 in binding to Smad1. As shown in Fig. 4C, artificial removal of the prosequence of HsN3 (ΔN3) did not abolish Smad1 binding to HsN3. In fact, the interaction between ΔN3 and Smad1 was even stronger when directly compared with the interaction between wild type HsN3 and Smad1 (data not shown). Thus, HsN3 does not require the prosequence to bind Smad1. Since we have shown that artificial removal of the prosequence of HsN3 leads to a blockage of the normal assembly of HsN3 into the mature proteasome (Fig. 2C), we suspect that the detected strong signals of ΔN3 from Smad1 immunoprecipitates reflect the accumulated HsN3 within the defective assembly intermediates, as illustrated in Fig. 2B. To test this possibility, we directly compared the ability of Smad1 MH2 domain to bind wild type HsN3 and prosequence-less mutant HsN3 in metabolically labeled COS 1 cells (Fig. 4D &4E). When COS1 cells were expressing wild type HsN3, only the Pro-N3-F was detected in the immunoprecipitates of Smad1MH2 (Fig. 4D, lane 4). When COS1 cells were expressing the mutant HsN3 lacking the prosequence, the mutant N3 (ΔN3) and ΔN3 associated endogenous proteins were detected in the immunoprecipitates of Smad1MH2 (Fig. 4E, lane 4). In addition, four distinct small molecular weight proteins with unknown identity were also recruited into the ΔN3/MH2-containing complex (Fig. 4E, lane 2 and lane 4). These four bands were specifically detected only when Smad1MH2 and ΔN3 were co-expressed. When Smad1MH2 was replaced with full length Smad1, the ΔN3-containing complex was also detected in the immunoprecipitates of Smad1 (Fig. 4F). Thus, a blockage of HsN3 assembly resulted from deleting the prosequence of HsN3 allows the detection of a stable complex between Smad1 and HsN3 assembly intermediates. These data suggest that Smad1 interacts with HsN3 transiently when HsN3 is either in single Pro-HsN3 form, or in Pro-HsN3 assembly intermediates. The lack of mature HsN3 in Smad1 immunoprecipitates does not reflect the inability of Smad1 to bind prosequence-less HsN3, but is likely due to either the trapping/degradation of Smad1 inside of the proteasome, or the rapid dissociation of Smad1 from HsN3. The degradation of Smad1 by proteasome is reported in a separate study [39]. The dissociation of Smad1 from the mature 20S proteasome could be due to the competition of Smad1 binding by a 19S regulator protein or simply due to the inaccessibility of the binding site on HsN3 when two half proteasomes assembly into the mature proteasome.
Two separate domains on Az mediate interaction with Smad1 and HsN3 prior to HsN3 incorporation into the 20S proteasome
We next examined the interaction between Az and Smad1 in COS1 cells. In the absence of the co-expressed HsN3, Smad1 was co-precipitated with Az (Fig. 5A, top panel, lane 1). The interaction was diminished when cells were co-transfected with the activated BMP type I receptor ALK3Q233D (Fig. 5A, top panel, lane 3). However, the addition of proteasome inhibitor LLnL stabilizes the interaction (Fig. 5A, top panel, lane 2). Thus, Smad1 interacts with Az in mammalian cells and the interaction is also enhanced upon BMP type I receptor activation. In the yeast two-hybrid tests, we also observed the ability of Az to bind HsN3 [39]. Domain mapping analyses of Az reveals two separate domains on Az that are involved in binding to Smad1 and HsN3 (Fig. 5B). In mammalian cells, co-expression of Az with Smad1 and HsN3 leads to the detection of both Smad1 and Pro-N3 in the immunoprecipitates of Az (Fig. 5C, top panel, lane 6). There appears to be a stoichiomatric relationship between these three proteins, since the relative levels of these three proteins influence the interaction properties (Fig. 5C). These data suggest that Smad1, Az and HsN3 might form a ternary complex. Since we again only detected the pro-sequence form of HsN3 in Az precipitates, such a ternary complex is likely formed along the assembly pathways of HsN3.
The Activation of the BMP type I receptor induces Smad1-dependent nuclear translocation of both HsN3 and Az
Smad1 is translocated from the cytoplasm to the nucleus upon its activation by the BMP type I receptors [40]. The physical interaction between Smad1, Az and HsN3 suggests that BMP signaling might regulate the intracellular localization of Az and HsN3. We tested the localization of endogenous HsN3 in COS1 cell line, which was transiently transfected with Smad1, ALK3 and the BMP type II receptor. Cells were either not exposed to BMP2 (Fig. 6A), or treated with BMP2 for 30 mins (Fig. 6B) or 60 mins (Fig. 6C). Cells were stained with a monoclonal anti-HsN3 antibody and FITC-conjugated anti-mouse secondary (Fig. 6A,6B,6C, panels 1) and simultaneously with a polyclonal anti-Smad1 antibody and rhodamine-conjugated anti-rabbit secondary antibody (Fig. 6A,6B,6C, panels 2). The co-localization of Smad1 and HsN3 was detected by overlaying the signals in panels 1 and panels 2 and shown in panels 3. Smad1 and HsN3 were detected in both the cytoplasm and the nucleus in cells that were not exposed to BMP2 (Fig. 6A, panels 1–3). In cells treated with BMP2 for 30 mins, most HsN3 and Smad1 were concentrated in the nucleus (Fig. 6B, panels 1 & 2) and co-localization of Smad1 and HsN3 was detected in the nucleus (Fig. 6B, panel 3). In cells treated with BMP2 for 60 mins, all signals of Smad1 and HsN3 were detected to be co-localized in the nucleus (Fig. 6C, panels 1–3).
We further tested the role of Smad1 in the nuclear translocation of HsN3. HsN3 was detected in speckled pattern throughout the cytoplasm and the nucleus when it was expressed alone in COS1 cells (Fig. 6D, panel 1). Similar localization pattern was detected when HsN3 was co-expressed with Smad1 (Fig. 6D, panel 2) or with ALK3Q233D (Fig. 6D, panel 3). However, when HsN3 was co-expressed with both Smad1 and ALK3Q233D, the majority of HsN3 signals were detected in the nucleus (Fig. 6D, panel 4). Thus, the nuclear translocation of HsN3 is dependent upon both the receptor activation and the presence of Smad1. Similarly, the localization of Az is also dependent upon the co-expression of Smad1 and the activated BMP type I receptor (Fig. 6E, panels 1–4). When Smad1 was replaced by Smad2, Az was found in both the cytoplasm and the nucleus upon the co-expression of ALK3Q233D (Fig. 6E, panel 4), although cytoplasmic signals of Az were slightly decreased. This could be due to the presence of endogenous Smad1 in COS1 cells.
Thus, the localization of both Az and HsN3 is regulated by the BMP type I receptor in a Smad1-dependent manner. This data confirms the physical interaction between Smad1, HsN3 and Az detected by immunoprecipitation and further suggests that the cytoplasmic Smad1 forms a complex with Az and HsN3 and brings these two proteins into the nucleus upon the activation of BMP type I receptor.
The novel CBP/p300 repressor SNIP1 is recruited to Az upon the activation of BMP type I receptor
The observed physical interaction between Smad1, HsN3 and Az and the regulation of the complex formation by BMPs suggests a functional role of Az and HsN3 in the signaling function of Smad1. In a separate study, we have shown that Az and HsN3 play targeting roles in the proteasomal degradation of Smad1 upon the activation of BMP type I receptors [39]. The observed proteasomal degradation of Smad1 upon the activation of BMP signaling could simply serve to irreversibly terminate Smad1-mediated signaling pathways of BMPs. However, we observed that Az and HsN3 also interact with several isolated Smad1 interactors. Furthermore, the nuclear co-translocation of Az and HsN3 with Smad1 also suggests potential proteasomal targeting role of Az and HsN3 in the nucleus. Thus, we tested the hypothesis that Az and HsN3 also target Smad1 interacting proteins in the nucleus.
Among the isolated Smad1 interactors shown in Fig. 1, clone 19 encodes a novel nuclear protein named as Smad1 Nuclear Interacting Protein-1 (SNIP-1). Our recent studies suggested that SNIP1 is a novel CBP/p300 repressor [25]. SNIP1, when expressed in COS1 cells, is entirely localized to distinct areas within the nucleus (Fig. 7A). Interestingly, SNIP1 binds to Az and HsN3 in the yeast two-hybrid system, in addition to its ability to bind Smad1 (Fig. 7B). SNIP1 and ODC exhibited similar affinity to Az in the yeast two-hybrid test (Fig. 7B). Domain mapping studies showed that the linker region of Smad1 was sufficient to bind SNIP1 (Fig. 7C). To confirm the interaction between SNIP1 and Az or HsN3 seen in the yeast two-hybrid system, we transfected COS1 cells with each pair of proteins and tested the interactions by immunoprecipitation assays. Upon the co-expression with SNIP1, the pre-assembled form of HsN3 but not Az was co-precipitated with SNIP1 (Fig. 7D, lanes 1 & 2). Since SNIP1 is a nuclear protein, the interaction between SNIP1 and Az may require the nuclear translocation of Az. Based upon the observation that the nuclear translocation of Az is dependent upon Smad1 and BMP type I receptor activation (Fig. 6E), we therefore tested Az interaction with SNIP1 in the presence of the co-expression of Smad1, Smad4 and ALK3Q233D. In the absence of ALK3Q233D, no F-Az was detected in the precipitates of SNIP1 (Fig. 7E, top panel, lane 2). However, the co-expression of ALK3Q233D allowed the detection of F-Az in SNIP1 immunoprecipitates (Fig. 7E, top panel, lane 3). A weak Smad1 signal but no Smad4 signal was detected in SNIP1 immunoprecipitates.
Thus, in the mammalian overexpression system, the pre-assembled HsN3 exhibits constitutive interaction with SNIP1, whereas Az is only recruited to SNIP1 upon receptor activation. The lack of strong signals of Smad1 or Smad4 in the immunoprecipitates of SNIP1 in the presence of BMP type I receptor activation may be due to the transient nature of the complex, which, as shown below, is targeted to proteasome for degradation.
BMP type I receptor activation induces SNIP1 degradation that is regulated by Smad1, Smad4 and Az
To follow the observed reduction of SNIP1 level upon the expression of high level of ALK3Q233D, as mentioned above, we co-expressed SNIP1 together with Smad1, Smad4, Az and high dose (6 μg) of either the wild type ALK3 or the constitutive active ALK3Q233D. The protein levels of each protein were analyzed by Western blot (Fig. 8A). A dramatic decrease of SNIP1 level was detected in ALK3Q233D-transfected cells (Fig. 8A, panel 1, lanes 1 & 2). The receptor activation-induced decrease of SNIP1 is partially sensitive to the proteasome-specific inhibitor lactacystin, suggesting the involvement of proteasomal degradation (Fig. 8A, panel 1, lane 3). The incomplete rescue of SNIP1 level by lactacystin suggests the involvement of additional mechanisms other than 26S proteasome in SNIP1 degradation. To systematically demonstrate the role of Smad1, Smad4 and Az in the observed decrease of SNIP1 protein level, we sequentially left out one of these three proteins in the above assay. The ALK3Q233D-induced degradation of SNIP1 was significantly blocked in the absence of Az (Fig. 8A, panel 1, lanes 4 & 5), Smad4 (lanes 6 & 7), or Smad1 (lanes 8 & 9). Thus, the ALK3Q233D-induced degradation of SNIP1 is regulated by the protein levels of Smad1, Smad4, Az and is only induced by high dose of the activated BMP type I receptor. Interestingly, by simply replacing Smad1 with the Smad1 mutant Smad1G419S [40], which is defective in binding to BMP type I receptor for receptor-mediated phosphorylation and the subsequent nuclear translocation, SNIP1 degradation was also inhibited (Fig. 8A, panel 1, lanes 10 & 11), suggesting that the degradation of SNIP1 is dependent upon the type I receptor-mediated phosphorylation of Smad1. The levels of SNIP1 in Fig. 8A top panel are quantified and plotted as Integrated Optical Density (IOD) of SNIP1 (Fig. 8A, right panel).
We noted that the protein levels of Smad1, Smad4 and Az were co-regulated with that of SNIP1. For example, the protein levels of Smad1, Smad4 and Az were all sensitive to lactacystin (Fig. 8A, all panels, compare lane 2 and lane 3). Blocking SNIP1 degradation was accompanied by increased protein levels of Smad1, Smad4 and Az in cells expressing the activated ALK3 (Fig. 8A, lanes 5, 7, 9, 11). The lower levels of Smad1, Smad4, Az and SNIP1 in lane 1 compared with those in lane 10 suggest that the leaky signaling by the overexpressed ALK3, which we have demonstrated recently [39], caused a constitutive reduction of Smad4, Az and SNIP1 level that is dependent upon the activation of Smad1. These data thus suggest that SNIP1 is targeted to proteasome for degradation together with Smad1, Az and Smad4.
The ability of Smad1 in regulating SNIP1 degradation was further demonstrated by increased expression of Smad1 in COS1 cells that were transfected with a constant amount of Smad4, Az, ALK3Q233D and SNIP1 (Fig. 8B). A dose-dependent effect of Smad1 on receptor-induced degradation of SNIP1 was observed (Fig. 8B, compare lanes 1 & 2 with lanes 5 & 6).
To confirm SNIP1 degradation in non-overexpression systems, we monitored the steady state levels of endogenous SNIP1 in the human keratinocyte HaCaT and mouse myoblast L6. Cells were treated with BMP7 for different periods of time. Western blot analyses using an anti-SNIP1 antibody detected two forms of SNIP1 with slightly different mobility, which are named as SNIP1.A and SNIP1.B (Fig. 8C, top panel). The IOD of each form of SNIP1 as well as the total IOD of both forms are plotted (Fig. 8C, right panel). In HaCaT cells, the protein levels of both forms of SNIP1 were significantly decreased after 3 hrs of BMP7 treatment and were almost diminished by 22 hrs. Interestingly, a different response was observed in L6 cells. The levels of SNIP1 were somewhat increased after 3 hrs and peaked at 3.5 hrs, but then started to decline at 7 hrs and were diminished at 22 hrs.
SNIP1 is a nuclear repressor of the BMP-induced transcription responses
To determine the functional significance of the observed SNIP1 degradation induced by BMPs, we tested the role of SNIP1 in Smad1-regulated gene responses of BMPs. We first tested the effect of SNIP1 on the BMP-induced activation of the Tlx2 promoter, which involves Smad1 [41]. Full length SNIP1 exhibits dose-dependent inhibition of the BMP-induced gene activation, as monitored by the luciferase activities (Fig. 9A). Thus, SNIP1 is an inhibitor of BMP-induced gene response. Since we have previously shown that SNIP1 binds and inhibits CBP/p300 via its N-terminal domain [25], the deletion mutant of SNIP1 lacking the CBP/p300 binding site, SNIP1 (142–369), was tested in the same assay. SNIP1 (142–369) exhibits little inhibitory activity towards the gene response (Fig. 9A, see ΔN-SNIP1). This data further suggests that the inhibitory activity of SNIP1 is mediated by its interaction with CBP/p300, similar to what has been reported in the TGF-β pathways [25]. We next tested the inhibitory activity of SNIP1 on the transcriptional activity of Smad1, using the GAL4-Smad1 and GAL4-Luc reporter system, as described previously [42, 43]. SNIP1 inhibited BMP-induced activation of GAL4-Luc in p19 cells transfected with GAL4-Smad1 (Fig. 9B). The proteasome specific inhibitor lactacystin also inhibited the BMP-induced GAL4-Smad1 transcriptional activity (Fig. 9B), consistent with a role of proteasomal degradation in Smad1-mediated transcription. The effect of lactacystin is specific, since the control reporter gene β-Gal was not inhibited by lactacystin (data not shown). In a separate study, we also demonstrated that proteasomal degradation also plays an essential role in BMP induced dendritic formation of rat sympathetic neurons [44].