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A novel link between the proteasome pathway and the signal transduction pathway of the bone morphogenetic proteins (BMPs).

Lin Y, Martin J, Gruendler C, Farley J, Meng X, Li BY, Lechleider R, Huff C, Kim RH, Grasser WA, Paralkar V, Wang T - BMC Cell Biol. (2002)

Bottom Line: Furthermore, BMPs trigger the translocation of Smad1, HsN3 and Az into the nucleus, where the novel CBP/p300 repressor protein SNIP1 is further recruited to Smad1/HsN3/Az complex and degraded in a Smad1-, Smad4- and Az-dependent fashion.The degradation of the CBP/p300 repressor SNIP1 is likely an essential step for Smad1-, Smad4-mediated transcriptional activation, since increased SNIP1 expression inhibits BMP-induced gene responses.Our studies thus add two additional important functional partners of Smad1 into the signaling web of BMPs and also suggest a novel mechanism for Smad1 and Smad4 to co-modulate transcription via regulating proteasomal degradation of CBP/p300 repressor SNIP1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virginia Mason Research Center, 1201 Ninth Ave, Seattle WA 98101, USA. yinlinn@yahoo.com

ABSTRACT

Background: The intracellular signaling events of the bone morphogenetic proteins (BMPs) involve the R-Smad family members Smad1, Smad5, Smad8 and the Co-Smad, Smad4. Smads are currently considered to be DNA-binding transcriptional modulators and shown to recruit the master transcriptional co-activator CBP/p300 for transcriptional activation. SNIP1 is a recently discovered novel repressor of CBP/p300. Currently, the detailed molecular mechanisms that allow R-Smads and Co-Smad to co-operatively modulate transcription events are not fully understood.

Results: Here we report a novel physical and functional link between Smad1 and the 26S proteasome that contributes to Smad1- and Smad4-mediated transcriptional regulation. Smad1 forms a complex with a proteasome beta subunit HsN3 and the ornithine decarboxylase antizyme (Az). The interaction is enhanced upon BMP type I receptor activation and occur prior to the incorporation of HsN3 into the mature 20S proteasome. Furthermore, BMPs trigger the translocation of Smad1, HsN3 and Az into the nucleus, where the novel CBP/p300 repressor protein SNIP1 is further recruited to Smad1/HsN3/Az complex and degraded in a Smad1-, Smad4- and Az-dependent fashion. The degradation of the CBP/p300 repressor SNIP1 is likely an essential step for Smad1-, Smad4-mediated transcriptional activation, since increased SNIP1 expression inhibits BMP-induced gene responses.

Conclusions: Our studies thus add two additional important functional partners of Smad1 into the signaling web of BMPs and also suggest a novel mechanism for Smad1 and Smad4 to co-modulate transcription via regulating proteasomal degradation of CBP/p300 repressor SNIP1.

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The expression and assembly of HsN3 in transfected COS1 cells.A. A cartoon that illustrates the multiple steps known to involve in the assembly of the 26S proteasome. See text for details. B. A cartoon that illustrate the importance of β subunit prosequence in proteasome assembly. See text for details. C. The different assembly properties of wild type HsN3 and mutant HsN3 lacking its prosequence in transfected COS1 cells. COS1 was transfected with N3-F, a construct that contains the full-length wild type HsN3 and a Flag-epitope at the C-terminus (lane 2), or with F-ΔN3 (lane 3), a construct that lacks its prosequence at the N-terminus, instead contains an N-terminal Flag-epitope, or with T7-ΔN3, a construct that similarly lacks the prosequence but contains an N-terminal T7-epitope (lane 4). Cells were metabolically labeled with 35S-methionine for 4 hrs before cells were harvested and analyzed by immunoprecipitation, as indicated. The two major bands in lane 2 were detected by Western blot using anti-Flag and marked out as prosequence-containing HsN3 (Pro-N3-F) and mature HsN3 (mature N3-F). The endogenous proteasome components at the molecular weight range of 19S proteasome (marked by small dots) or 20S proteasome in lane 2 are marked with brackets. A distinct set of eight bands that commonly detected to be associated with F-ΔN3 or T7-ΔN3 is marked with short lines at the right side of the panel. The transfection efficiency was monitored by co-transfection with a GFP construct. Equal transfection efficiency (70–80%) among different samples in a single experiment was the pre-requisite for metabolic labeling and the subsequent immunoprecipitation assays. Equal total amount of proteins were used for immunoprecipitation.
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Figure 2: The expression and assembly of HsN3 in transfected COS1 cells.A. A cartoon that illustrates the multiple steps known to involve in the assembly of the 26S proteasome. See text for details. B. A cartoon that illustrate the importance of β subunit prosequence in proteasome assembly. See text for details. C. The different assembly properties of wild type HsN3 and mutant HsN3 lacking its prosequence in transfected COS1 cells. COS1 was transfected with N3-F, a construct that contains the full-length wild type HsN3 and a Flag-epitope at the C-terminus (lane 2), or with F-ΔN3 (lane 3), a construct that lacks its prosequence at the N-terminus, instead contains an N-terminal Flag-epitope, or with T7-ΔN3, a construct that similarly lacks the prosequence but contains an N-terminal T7-epitope (lane 4). Cells were metabolically labeled with 35S-methionine for 4 hrs before cells were harvested and analyzed by immunoprecipitation, as indicated. The two major bands in lane 2 were detected by Western blot using anti-Flag and marked out as prosequence-containing HsN3 (Pro-N3-F) and mature HsN3 (mature N3-F). The endogenous proteasome components at the molecular weight range of 19S proteasome (marked by small dots) or 20S proteasome in lane 2 are marked with brackets. A distinct set of eight bands that commonly detected to be associated with F-ΔN3 or T7-ΔN3 is marked with short lines at the right side of the panel. The transfection efficiency was monitored by co-transfection with a GFP construct. Equal transfection efficiency (70–80%) among different samples in a single experiment was the pre-requisite for metabolic labeling and the subsequent immunoprecipitation assays. Equal total amount of proteins were used for immunoprecipitation.

Mentions: 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.


A novel link between the proteasome pathway and the signal transduction pathway of the bone morphogenetic proteins (BMPs).

Lin Y, Martin J, Gruendler C, Farley J, Meng X, Li BY, Lechleider R, Huff C, Kim RH, Grasser WA, Paralkar V, Wang T - BMC Cell Biol. (2002)

The expression and assembly of HsN3 in transfected COS1 cells.A. A cartoon that illustrates the multiple steps known to involve in the assembly of the 26S proteasome. See text for details. B. A cartoon that illustrate the importance of β subunit prosequence in proteasome assembly. See text for details. C. The different assembly properties of wild type HsN3 and mutant HsN3 lacking its prosequence in transfected COS1 cells. COS1 was transfected with N3-F, a construct that contains the full-length wild type HsN3 and a Flag-epitope at the C-terminus (lane 2), or with F-ΔN3 (lane 3), a construct that lacks its prosequence at the N-terminus, instead contains an N-terminal Flag-epitope, or with T7-ΔN3, a construct that similarly lacks the prosequence but contains an N-terminal T7-epitope (lane 4). Cells were metabolically labeled with 35S-methionine for 4 hrs before cells were harvested and analyzed by immunoprecipitation, as indicated. The two major bands in lane 2 were detected by Western blot using anti-Flag and marked out as prosequence-containing HsN3 (Pro-N3-F) and mature HsN3 (mature N3-F). The endogenous proteasome components at the molecular weight range of 19S proteasome (marked by small dots) or 20S proteasome in lane 2 are marked with brackets. A distinct set of eight bands that commonly detected to be associated with F-ΔN3 or T7-ΔN3 is marked with short lines at the right side of the panel. The transfection efficiency was monitored by co-transfection with a GFP construct. Equal transfection efficiency (70–80%) among different samples in a single experiment was the pre-requisite for metabolic labeling and the subsequent immunoprecipitation assays. Equal total amount of proteins were used for immunoprecipitation.
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Related In: Results  -  Collection

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Figure 2: The expression and assembly of HsN3 in transfected COS1 cells.A. A cartoon that illustrates the multiple steps known to involve in the assembly of the 26S proteasome. See text for details. B. A cartoon that illustrate the importance of β subunit prosequence in proteasome assembly. See text for details. C. The different assembly properties of wild type HsN3 and mutant HsN3 lacking its prosequence in transfected COS1 cells. COS1 was transfected with N3-F, a construct that contains the full-length wild type HsN3 and a Flag-epitope at the C-terminus (lane 2), or with F-ΔN3 (lane 3), a construct that lacks its prosequence at the N-terminus, instead contains an N-terminal Flag-epitope, or with T7-ΔN3, a construct that similarly lacks the prosequence but contains an N-terminal T7-epitope (lane 4). Cells were metabolically labeled with 35S-methionine for 4 hrs before cells were harvested and analyzed by immunoprecipitation, as indicated. The two major bands in lane 2 were detected by Western blot using anti-Flag and marked out as prosequence-containing HsN3 (Pro-N3-F) and mature HsN3 (mature N3-F). The endogenous proteasome components at the molecular weight range of 19S proteasome (marked by small dots) or 20S proteasome in lane 2 are marked with brackets. A distinct set of eight bands that commonly detected to be associated with F-ΔN3 or T7-ΔN3 is marked with short lines at the right side of the panel. The transfection efficiency was monitored by co-transfection with a GFP construct. Equal transfection efficiency (70–80%) among different samples in a single experiment was the pre-requisite for metabolic labeling and the subsequent immunoprecipitation assays. Equal total amount of proteins were used for immunoprecipitation.
Mentions: 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.

Bottom Line: Furthermore, BMPs trigger the translocation of Smad1, HsN3 and Az into the nucleus, where the novel CBP/p300 repressor protein SNIP1 is further recruited to Smad1/HsN3/Az complex and degraded in a Smad1-, Smad4- and Az-dependent fashion.The degradation of the CBP/p300 repressor SNIP1 is likely an essential step for Smad1-, Smad4-mediated transcriptional activation, since increased SNIP1 expression inhibits BMP-induced gene responses.Our studies thus add two additional important functional partners of Smad1 into the signaling web of BMPs and also suggest a novel mechanism for Smad1 and Smad4 to co-modulate transcription via regulating proteasomal degradation of CBP/p300 repressor SNIP1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Virginia Mason Research Center, 1201 Ninth Ave, Seattle WA 98101, USA. yinlinn@yahoo.com

ABSTRACT

Background: The intracellular signaling events of the bone morphogenetic proteins (BMPs) involve the R-Smad family members Smad1, Smad5, Smad8 and the Co-Smad, Smad4. Smads are currently considered to be DNA-binding transcriptional modulators and shown to recruit the master transcriptional co-activator CBP/p300 for transcriptional activation. SNIP1 is a recently discovered novel repressor of CBP/p300. Currently, the detailed molecular mechanisms that allow R-Smads and Co-Smad to co-operatively modulate transcription events are not fully understood.

Results: Here we report a novel physical and functional link between Smad1 and the 26S proteasome that contributes to Smad1- and Smad4-mediated transcriptional regulation. Smad1 forms a complex with a proteasome beta subunit HsN3 and the ornithine decarboxylase antizyme (Az). The interaction is enhanced upon BMP type I receptor activation and occur prior to the incorporation of HsN3 into the mature 20S proteasome. Furthermore, BMPs trigger the translocation of Smad1, HsN3 and Az into the nucleus, where the novel CBP/p300 repressor protein SNIP1 is further recruited to Smad1/HsN3/Az complex and degraded in a Smad1-, Smad4- and Az-dependent fashion. The degradation of the CBP/p300 repressor SNIP1 is likely an essential step for Smad1-, Smad4-mediated transcriptional activation, since increased SNIP1 expression inhibits BMP-induced gene responses.

Conclusions: Our studies thus add two additional important functional partners of Smad1 into the signaling web of BMPs and also suggest a novel mechanism for Smad1 and Smad4 to co-modulate transcription via regulating proteasomal degradation of CBP/p300 repressor SNIP1.

Show MeSH