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E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif.

Ferreira RT, Menezes RA, Rodrigues-Pousada C - Biol Open (2015)

Bottom Line: Here, we show that Ufd2, an E4-Ubiquitin (Ub) ligase, is upregulated by arsenic compounds both at mRNA and protein levels.Thus, our data disclose a novel Ufd2 role beyond degradation.This finding is further supported by genetic analyses showing that proteins belonging to Ufd2 proteolytic pathways, namely Ubc4, Rad23 and Dsk2, mediate Yap8 degradation.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, EAN, Oeiras 2781-901, Portugal.

No MeSH data available.


Related in: MedlinePlus

Yap8 interacts with Ufd2 upon arsenic stress. (A) Yeast two-hybrid assays reveal Ufd2 as a Yap8-interaction partner. The Y187 strain was co-transformed with plasmids encoding GAL4DBDLam/GAL4ADT, GAL4DBDp53/GAL4ADT, GAL4DBD/GAL4ADUFD2, GAL4DBDYAP8/GAL4ADor GAL4DBDYAP8/GAL4ADUFD2 and β-galactosidase activity was measured in cells challenged or not with 2 mM As(V) (MU, Miller Units). Values represent the mean±standard deviation (s.d.) of three biological replicates and statistical differences denoted as *P<0.05. (B) Ufd2 co-immunoprecipitates together with Yap8. Y187 cells co-transformed with GAL4DBD/GAL4ADUFD2 (lanes 1 and 4), GAL4DBDYAP8/GAL4ADYAP8 (lanes 2 and 5) or GAL4DBDYAP8/GAL4ADUFD2 (lanes 3 and 6) were exposed to 2 mM As(V) for 60 min and Gal4DBDYap8, bearing a c-myc epitope, was immunoprecipitated with anti-c-myc antibody. Immunoblotting was performed using anti-HA, anti-c-myc and anti-Pgk1 antibodies. A representative experiment is shown. IP, immunoprecipitation; IB, immunoblotting.
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BIO010405F2: Yap8 interacts with Ufd2 upon arsenic stress. (A) Yeast two-hybrid assays reveal Ufd2 as a Yap8-interaction partner. The Y187 strain was co-transformed with plasmids encoding GAL4DBDLam/GAL4ADT, GAL4DBDp53/GAL4ADT, GAL4DBD/GAL4ADUFD2, GAL4DBDYAP8/GAL4ADor GAL4DBDYAP8/GAL4ADUFD2 and β-galactosidase activity was measured in cells challenged or not with 2 mM As(V) (MU, Miller Units). Values represent the mean±standard deviation (s.d.) of three biological replicates and statistical differences denoted as *P<0.05. (B) Ufd2 co-immunoprecipitates together with Yap8. Y187 cells co-transformed with GAL4DBD/GAL4ADUFD2 (lanes 1 and 4), GAL4DBDYAP8/GAL4ADYAP8 (lanes 2 and 5) or GAL4DBDYAP8/GAL4ADUFD2 (lanes 3 and 6) were exposed to 2 mM As(V) for 60 min and Gal4DBDYap8, bearing a c-myc epitope, was immunoprecipitated with anti-c-myc antibody. Immunoblotting was performed using anti-HA, anti-c-myc and anti-Pgk1 antibodies. A representative experiment is shown. IP, immunoprecipitation; IB, immunoblotting.

Mentions: To get further insights into the molecular basis of Yap8 stabilization that circumvents degradation upon arsenic stress, we have screened a yeast two-hybrid cDNA library fused to the Gal4 activation domain (Gal4AD), using Gal4 DNA binding domain-Yap8 (Gal4DBDYap8) fusion as a bait protein. Yap8 is strongly activated by arsenic, therefore, to increase the likelihood of identifying new Yap8-interaction partners, the cDNA library was generated from cells induced with a sub-lethal dose of pentavalent inorganic arsenic As(V). Performing the screening in the presence of 0.5 mM As(V) allowed us to identify new Yap8-interaction partners (data not shown), among them the ubiquitin fusion degradation enzyme Ufd2. In order to assess the specific interaction between Yap8 and Ufd2, cells co-expressing Gal4DBDYap8 and Gal4ADUfd2 along with the respective controls, were treated or not with 2 mM As(V) for 60 min, and interaction was followed through induction of lacZ reporter gene in quantitative β-galactosidase assays (Fig. 2A). A high β-galactosidase activity was detected in Gal4DBDYap8/Gal4ADUfd2-expressing cells only under conditions where Yap8 is activated, i.e. in the presence of arsenic. The β-galactosidase signal observed in control cells expressing Gal4DBDYap8/Gal4AD and challenged with arsenic is due to the Yap8 transactivation potential (Menezes et al., 2004). Notwithstanding, Gal4DBDYap8/Gal4ADUfd2 interaction has yielded β-galactosidase activity values significantly higher than those determined for Gal4DBDYap8/Gal4AD. As a positive control we have used cells expressing the well-known interacting proteins, p53 and SV40 T-antigen, fused to the Gal4DBD and Gal4AD, respectively (Gal4DBDp53/Gal4ADT) (Li and Fields, 1993). Similarly to what we observed for non-stressed cells co-expressing Gal4DBDYap8/Gal4ADUfd2, we did not detect β-galactosidase activity for Gal4DBD/Gal4ADUfd2 and Gal4DBDLamC/Gal4ADT control cells.Fig. 2.


E4-Ubiquitin ligase Ufd2 stabilizes Yap8 and modulates arsenic stress responses independent of the U-box motif.

Ferreira RT, Menezes RA, Rodrigues-Pousada C - Biol Open (2015)

Yap8 interacts with Ufd2 upon arsenic stress. (A) Yeast two-hybrid assays reveal Ufd2 as a Yap8-interaction partner. The Y187 strain was co-transformed with plasmids encoding GAL4DBDLam/GAL4ADT, GAL4DBDp53/GAL4ADT, GAL4DBD/GAL4ADUFD2, GAL4DBDYAP8/GAL4ADor GAL4DBDYAP8/GAL4ADUFD2 and β-galactosidase activity was measured in cells challenged or not with 2 mM As(V) (MU, Miller Units). Values represent the mean±standard deviation (s.d.) of three biological replicates and statistical differences denoted as *P<0.05. (B) Ufd2 co-immunoprecipitates together with Yap8. Y187 cells co-transformed with GAL4DBD/GAL4ADUFD2 (lanes 1 and 4), GAL4DBDYAP8/GAL4ADYAP8 (lanes 2 and 5) or GAL4DBDYAP8/GAL4ADUFD2 (lanes 3 and 6) were exposed to 2 mM As(V) for 60 min and Gal4DBDYap8, bearing a c-myc epitope, was immunoprecipitated with anti-c-myc antibody. Immunoblotting was performed using anti-HA, anti-c-myc and anti-Pgk1 antibodies. A representative experiment is shown. IP, immunoprecipitation; IB, immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4582114&req=5

BIO010405F2: Yap8 interacts with Ufd2 upon arsenic stress. (A) Yeast two-hybrid assays reveal Ufd2 as a Yap8-interaction partner. The Y187 strain was co-transformed with plasmids encoding GAL4DBDLam/GAL4ADT, GAL4DBDp53/GAL4ADT, GAL4DBD/GAL4ADUFD2, GAL4DBDYAP8/GAL4ADor GAL4DBDYAP8/GAL4ADUFD2 and β-galactosidase activity was measured in cells challenged or not with 2 mM As(V) (MU, Miller Units). Values represent the mean±standard deviation (s.d.) of three biological replicates and statistical differences denoted as *P<0.05. (B) Ufd2 co-immunoprecipitates together with Yap8. Y187 cells co-transformed with GAL4DBD/GAL4ADUFD2 (lanes 1 and 4), GAL4DBDYAP8/GAL4ADYAP8 (lanes 2 and 5) or GAL4DBDYAP8/GAL4ADUFD2 (lanes 3 and 6) were exposed to 2 mM As(V) for 60 min and Gal4DBDYap8, bearing a c-myc epitope, was immunoprecipitated with anti-c-myc antibody. Immunoblotting was performed using anti-HA, anti-c-myc and anti-Pgk1 antibodies. A representative experiment is shown. IP, immunoprecipitation; IB, immunoblotting.
Mentions: To get further insights into the molecular basis of Yap8 stabilization that circumvents degradation upon arsenic stress, we have screened a yeast two-hybrid cDNA library fused to the Gal4 activation domain (Gal4AD), using Gal4 DNA binding domain-Yap8 (Gal4DBDYap8) fusion as a bait protein. Yap8 is strongly activated by arsenic, therefore, to increase the likelihood of identifying new Yap8-interaction partners, the cDNA library was generated from cells induced with a sub-lethal dose of pentavalent inorganic arsenic As(V). Performing the screening in the presence of 0.5 mM As(V) allowed us to identify new Yap8-interaction partners (data not shown), among them the ubiquitin fusion degradation enzyme Ufd2. In order to assess the specific interaction between Yap8 and Ufd2, cells co-expressing Gal4DBDYap8 and Gal4ADUfd2 along with the respective controls, were treated or not with 2 mM As(V) for 60 min, and interaction was followed through induction of lacZ reporter gene in quantitative β-galactosidase assays (Fig. 2A). A high β-galactosidase activity was detected in Gal4DBDYap8/Gal4ADUfd2-expressing cells only under conditions where Yap8 is activated, i.e. in the presence of arsenic. The β-galactosidase signal observed in control cells expressing Gal4DBDYap8/Gal4AD and challenged with arsenic is due to the Yap8 transactivation potential (Menezes et al., 2004). Notwithstanding, Gal4DBDYap8/Gal4ADUfd2 interaction has yielded β-galactosidase activity values significantly higher than those determined for Gal4DBDYap8/Gal4AD. As a positive control we have used cells expressing the well-known interacting proteins, p53 and SV40 T-antigen, fused to the Gal4DBD and Gal4AD, respectively (Gal4DBDp53/Gal4ADT) (Li and Fields, 1993). Similarly to what we observed for non-stressed cells co-expressing Gal4DBDYap8/Gal4ADUfd2, we did not detect β-galactosidase activity for Gal4DBD/Gal4ADUfd2 and Gal4DBDLamC/Gal4ADT control cells.Fig. 2.

Bottom Line: Here, we show that Ufd2, an E4-Ubiquitin (Ub) ligase, is upregulated by arsenic compounds both at mRNA and protein levels.Thus, our data disclose a novel Ufd2 role beyond degradation.This finding is further supported by genetic analyses showing that proteins belonging to Ufd2 proteolytic pathways, namely Ubc4, Rad23 and Dsk2, mediate Yap8 degradation.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, EAN, Oeiras 2781-901, Portugal.

No MeSH data available.


Related in: MedlinePlus