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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 isolation of Smad1 interacting proteins suggests a functional link between Smad1 and the 26S proteasome-mediated protein degradation.A. Yeast two-hybrid test of the interaction specificity between thirteen isolated Smad1 interactors and Smad1, Smad2, Smad3 and Smad4. The "Protein Trap" system was used for the test [27]. Yeast EGY48 (leu2,his3,trp1,ura3) was first transformed with LexA fusion constructs of Smads in pEG202 vector and then transformed again with each of the thirteen different cDNA clones in pJG4-5 vector. The transformants were streaked onto selective plates of either galactose/raffinose (top) or glucose (bottom) lacking uracil, histidine and tryptophan (U-H-W-) but containing X-Gal. The expression of the cDNA encoded fusion proteins is under the control of the GAL1 promoter. The light blue detected on the glucose plates reflects a basal level of transcriptional activation by the LexA-Smad fusion proteins. B. The thirteen Smad1 interactors were grouped into three groups based upon their known functions (top panel). The four clones that have functions along the proteasome-mediated degradation pathways are marked by different colors in the top panel and matches with the colored symbol in the bottom panel, which illustrates the proteasomal targeting pathways. See text for details.
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Figure 1: The isolation of Smad1 interacting proteins suggests a functional link between Smad1 and the 26S proteasome-mediated protein degradation.A. Yeast two-hybrid test of the interaction specificity between thirteen isolated Smad1 interactors and Smad1, Smad2, Smad3 and Smad4. The "Protein Trap" system was used for the test [27]. Yeast EGY48 (leu2,his3,trp1,ura3) was first transformed with LexA fusion constructs of Smads in pEG202 vector and then transformed again with each of the thirteen different cDNA clones in pJG4-5 vector. The transformants were streaked onto selective plates of either galactose/raffinose (top) or glucose (bottom) lacking uracil, histidine and tryptophan (U-H-W-) but containing X-Gal. The expression of the cDNA encoded fusion proteins is under the control of the GAL1 promoter. The light blue detected on the glucose plates reflects a basal level of transcriptional activation by the LexA-Smad fusion proteins. B. The thirteen Smad1 interactors were grouped into three groups based upon their known functions (top panel). The four clones that have functions along the proteasome-mediated degradation pathways are marked by different colors in the top panel and matches with the colored symbol in the bottom panel, which illustrates the proteasomal targeting pathways. See text for details.

Mentions: 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].


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 isolation of Smad1 interacting proteins suggests a functional link between Smad1 and the 26S proteasome-mediated protein degradation.A. Yeast two-hybrid test of the interaction specificity between thirteen isolated Smad1 interactors and Smad1, Smad2, Smad3 and Smad4. The "Protein Trap" system was used for the test [27]. Yeast EGY48 (leu2,his3,trp1,ura3) was first transformed with LexA fusion constructs of Smads in pEG202 vector and then transformed again with each of the thirteen different cDNA clones in pJG4-5 vector. The transformants were streaked onto selective plates of either galactose/raffinose (top) or glucose (bottom) lacking uracil, histidine and tryptophan (U-H-W-) but containing X-Gal. The expression of the cDNA encoded fusion proteins is under the control of the GAL1 promoter. The light blue detected on the glucose plates reflects a basal level of transcriptional activation by the LexA-Smad fusion proteins. B. The thirteen Smad1 interactors were grouped into three groups based upon their known functions (top panel). The four clones that have functions along the proteasome-mediated degradation pathways are marked by different colors in the top panel and matches with the colored symbol in the bottom panel, which illustrates the proteasomal targeting pathways. See text for details.
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Related In: Results  -  Collection

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Figure 1: The isolation of Smad1 interacting proteins suggests a functional link between Smad1 and the 26S proteasome-mediated protein degradation.A. Yeast two-hybrid test of the interaction specificity between thirteen isolated Smad1 interactors and Smad1, Smad2, Smad3 and Smad4. The "Protein Trap" system was used for the test [27]. Yeast EGY48 (leu2,his3,trp1,ura3) was first transformed with LexA fusion constructs of Smads in pEG202 vector and then transformed again with each of the thirteen different cDNA clones in pJG4-5 vector. The transformants were streaked onto selective plates of either galactose/raffinose (top) or glucose (bottom) lacking uracil, histidine and tryptophan (U-H-W-) but containing X-Gal. The expression of the cDNA encoded fusion proteins is under the control of the GAL1 promoter. The light blue detected on the glucose plates reflects a basal level of transcriptional activation by the LexA-Smad fusion proteins. B. The thirteen Smad1 interactors were grouped into three groups based upon their known functions (top panel). The four clones that have functions along the proteasome-mediated degradation pathways are marked by different colors in the top panel and matches with the colored symbol in the bottom panel, which illustrates the proteasomal targeting pathways. See text for details.
Mentions: 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].

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
Related in: MedlinePlus