Limits...
Notch3 interactome analysis identified WWP2 as a negative regulator of Notch3 signaling in ovarian cancer.

Jung JG, Stoeck A, Guan B, Wu RC, Zhu H, Blackshaw S, Shih IeM, Wang TL - PLoS Genet. (2014)

Bottom Line: The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor.Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas.Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.

Show MeSH

Related in: MedlinePlus

WWP2 ubiquitinates Notch3 receptor and regulates its activity.(A) Schematic representation of N3-TM, N3-NEXT, and N3-ICD constructs. Signal peptide sequences were inserted into N3-TM and N3-NEXT constructs. To facilitate detection, all Notch3 variant constructs contained a V5 epitope tag at the C-terminus. (B) 293T cells were transfected with HA-tagged ubiquitin together WWP2 and different Notch3 variant constructs: N3-TM, N3-NEXT, or N3-ICD. To determine the expression of different constructs, Western blot analysis was performed with anti-V5, anti-FLAG, or anti-HA, antibody using input lysates. Equal loading was determined with an anti-GAPDH antibody. (C) To detect ubiquitinated Notch3, immunoprecipitation (IP) was performed on the cell lysates using anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. To detect the interaction between Notch3 variant and WWP2, IP was performed by using anti-Flag agarose, and western blot was performed using anti-V5 antibody. Left panel: short exposure; right panel: longer exposure. (D) Schematic representation of wild type WWP2, a catalytically inactive C838A mutant of WWP2 (WWP2-CA), and a deletion construct of WWP2 lacking the HECT ubiquitination domain and three WW domains (WWP2ΔHECT). All of the WWP2 expression constructs contained a flag epitope tag at the C-terminal end. (E) 293 cells were transfected with N3-NEXT, ubiquitin-HA, and pLPC control plasmid, WWP2, WWP2-CA, or WWP2ΔHECT. To determine expression of the different constructs, western blot analysis of lysates was performed with anti-FLAG or anti-V5 antibody (input lysate). (F) To detect ubiquitinated N3-NEXT, immunoprecipitation (IP) was performed on the cell lysates using the anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. Reciprocal co-IP was performed by using anti-HA agarose beads for pull-down and using the anti-V5 antibody for western blot.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214668&req=5

pgen-1004751-g003: WWP2 ubiquitinates Notch3 receptor and regulates its activity.(A) Schematic representation of N3-TM, N3-NEXT, and N3-ICD constructs. Signal peptide sequences were inserted into N3-TM and N3-NEXT constructs. To facilitate detection, all Notch3 variant constructs contained a V5 epitope tag at the C-terminus. (B) 293T cells were transfected with HA-tagged ubiquitin together WWP2 and different Notch3 variant constructs: N3-TM, N3-NEXT, or N3-ICD. To determine the expression of different constructs, Western blot analysis was performed with anti-V5, anti-FLAG, or anti-HA, antibody using input lysates. Equal loading was determined with an anti-GAPDH antibody. (C) To detect ubiquitinated Notch3, immunoprecipitation (IP) was performed on the cell lysates using anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. To detect the interaction between Notch3 variant and WWP2, IP was performed by using anti-Flag agarose, and western blot was performed using anti-V5 antibody. Left panel: short exposure; right panel: longer exposure. (D) Schematic representation of wild type WWP2, a catalytically inactive C838A mutant of WWP2 (WWP2-CA), and a deletion construct of WWP2 lacking the HECT ubiquitination domain and three WW domains (WWP2ΔHECT). All of the WWP2 expression constructs contained a flag epitope tag at the C-terminal end. (E) 293 cells were transfected with N3-NEXT, ubiquitin-HA, and pLPC control plasmid, WWP2, WWP2-CA, or WWP2ΔHECT. To determine expression of the different constructs, western blot analysis of lysates was performed with anti-FLAG or anti-V5 antibody (input lysate). (F) To detect ubiquitinated N3-NEXT, immunoprecipitation (IP) was performed on the cell lysates using the anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. Reciprocal co-IP was performed by using anti-HA agarose beads for pull-down and using the anti-V5 antibody for western blot.

Mentions: Notch3 receptor is activated through two juxta-membrane protease cleavages: upon ligand stimulation, the alteration in configuration of membrane tethered Notch3 fragment (N3-TM) triggers the first cleavage by α-secretase of the ADMA family to generate N3-NEXT, which contains N3-ICD plus the transmembrane domain (Fig. 3A). Membrane-tethered N3-NEXT is then cleaved by γ-secretase within the transmembrane region to release the soluble intracellular fragment, N3-ICD. N3-ICD quickly translocates into the nucleus and regulates transcription of target genes through its interaction with co-factors including RBPJ. Although our protein microarray screen which used recombinant protein in a cell-free system suggests that WWP2 directly interacts with the final secretase cleavage product of Notch3, N3-ICD, at the cellular level, WWP2 is likely to interact with and ubiquitinate intermediate Notch3 fragments including N3-TM and N3-NEXT. To assess this possibility, we generated N3-TM-V5 and N3-NEXT-V5 expressing constructs and inserted a signal peptide sequence to the N-terminus of these constructs to ensure correct protein topology of these Notch3 fragments (Fig. 3A). In vivo ubiquitination status of these fragments was examined by performing co-transfection with flag-tagged WWP2 along with HA-tagged ubiquitin plasmids in 293T cells. The expression of each construct was confirmed by western blot, and the results demonstrated that similar amounts of Notch3 fragments were present in each experimental group (Fig. 3B). WWP2 and ubiquitin were also expressed at comparable levels (Fig. 3B). The level of ubiquitination of Notch3 fragments was measured by reciprocal immunoprecipitation with HA and V5 antibodies. The results demonstrated that all tested Notch3 fragments were ubiquitinated in cells co-transfected with WWP2; however, the ubiquitination level of N3-NEXT was most apparent (Top, Fig. 3C and Fig. S1A). There was a prominent band representing mono-ubiquitinated Notch3 protein and a weak high molecular weight smear corresponding to poly-ubiquitinated products. To test the possibility that the differential ubiquitination levels among the Notch3 fragments was due to their ability to encounter and interact with WWP2 in vivo, we performed co-immunoprecipitation experiments in 293T cells following co-transfection of the V5-tagged Notch3 fragment and flag-tagged WWP2. The results demonstrated that although protein expression levels were comparable among the three Notch3 fragments, N3-NEXT protein was more abundantly bound to WWP2 than N3-TM and N3-ICD, reflected by an intense protein pull-down band (Bottom, Fig. 3C). Reciprocal co-immunoprecipitation assays also confirmed the above finding (Fig. S1B).


Notch3 interactome analysis identified WWP2 as a negative regulator of Notch3 signaling in ovarian cancer.

Jung JG, Stoeck A, Guan B, Wu RC, Zhu H, Blackshaw S, Shih IeM, Wang TL - PLoS Genet. (2014)

WWP2 ubiquitinates Notch3 receptor and regulates its activity.(A) Schematic representation of N3-TM, N3-NEXT, and N3-ICD constructs. Signal peptide sequences were inserted into N3-TM and N3-NEXT constructs. To facilitate detection, all Notch3 variant constructs contained a V5 epitope tag at the C-terminus. (B) 293T cells were transfected with HA-tagged ubiquitin together WWP2 and different Notch3 variant constructs: N3-TM, N3-NEXT, or N3-ICD. To determine the expression of different constructs, Western blot analysis was performed with anti-V5, anti-FLAG, or anti-HA, antibody using input lysates. Equal loading was determined with an anti-GAPDH antibody. (C) To detect ubiquitinated Notch3, immunoprecipitation (IP) was performed on the cell lysates using anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. To detect the interaction between Notch3 variant and WWP2, IP was performed by using anti-Flag agarose, and western blot was performed using anti-V5 antibody. Left panel: short exposure; right panel: longer exposure. (D) Schematic representation of wild type WWP2, a catalytically inactive C838A mutant of WWP2 (WWP2-CA), and a deletion construct of WWP2 lacking the HECT ubiquitination domain and three WW domains (WWP2ΔHECT). All of the WWP2 expression constructs contained a flag epitope tag at the C-terminal end. (E) 293 cells were transfected with N3-NEXT, ubiquitin-HA, and pLPC control plasmid, WWP2, WWP2-CA, or WWP2ΔHECT. To determine expression of the different constructs, western blot analysis of lysates was performed with anti-FLAG or anti-V5 antibody (input lysate). (F) To detect ubiquitinated N3-NEXT, immunoprecipitation (IP) was performed on the cell lysates using the anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. Reciprocal co-IP was performed by using anti-HA agarose beads for pull-down and using the anti-V5 antibody for western blot.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214668&req=5

pgen-1004751-g003: WWP2 ubiquitinates Notch3 receptor and regulates its activity.(A) Schematic representation of N3-TM, N3-NEXT, and N3-ICD constructs. Signal peptide sequences were inserted into N3-TM and N3-NEXT constructs. To facilitate detection, all Notch3 variant constructs contained a V5 epitope tag at the C-terminus. (B) 293T cells were transfected with HA-tagged ubiquitin together WWP2 and different Notch3 variant constructs: N3-TM, N3-NEXT, or N3-ICD. To determine the expression of different constructs, Western blot analysis was performed with anti-V5, anti-FLAG, or anti-HA, antibody using input lysates. Equal loading was determined with an anti-GAPDH antibody. (C) To detect ubiquitinated Notch3, immunoprecipitation (IP) was performed on the cell lysates using anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. To detect the interaction between Notch3 variant and WWP2, IP was performed by using anti-Flag agarose, and western blot was performed using anti-V5 antibody. Left panel: short exposure; right panel: longer exposure. (D) Schematic representation of wild type WWP2, a catalytically inactive C838A mutant of WWP2 (WWP2-CA), and a deletion construct of WWP2 lacking the HECT ubiquitination domain and three WW domains (WWP2ΔHECT). All of the WWP2 expression constructs contained a flag epitope tag at the C-terminal end. (E) 293 cells were transfected with N3-NEXT, ubiquitin-HA, and pLPC control plasmid, WWP2, WWP2-CA, or WWP2ΔHECT. To determine expression of the different constructs, western blot analysis of lysates was performed with anti-FLAG or anti-V5 antibody (input lysate). (F) To detect ubiquitinated N3-NEXT, immunoprecipitation (IP) was performed on the cell lysates using the anti-V5 beads, and western blot (WB) was performed with an anti-HA antibody. Reciprocal co-IP was performed by using anti-HA agarose beads for pull-down and using the anti-V5 antibody for western blot.
Mentions: Notch3 receptor is activated through two juxta-membrane protease cleavages: upon ligand stimulation, the alteration in configuration of membrane tethered Notch3 fragment (N3-TM) triggers the first cleavage by α-secretase of the ADMA family to generate N3-NEXT, which contains N3-ICD plus the transmembrane domain (Fig. 3A). Membrane-tethered N3-NEXT is then cleaved by γ-secretase within the transmembrane region to release the soluble intracellular fragment, N3-ICD. N3-ICD quickly translocates into the nucleus and regulates transcription of target genes through its interaction with co-factors including RBPJ. Although our protein microarray screen which used recombinant protein in a cell-free system suggests that WWP2 directly interacts with the final secretase cleavage product of Notch3, N3-ICD, at the cellular level, WWP2 is likely to interact with and ubiquitinate intermediate Notch3 fragments including N3-TM and N3-NEXT. To assess this possibility, we generated N3-TM-V5 and N3-NEXT-V5 expressing constructs and inserted a signal peptide sequence to the N-terminus of these constructs to ensure correct protein topology of these Notch3 fragments (Fig. 3A). In vivo ubiquitination status of these fragments was examined by performing co-transfection with flag-tagged WWP2 along with HA-tagged ubiquitin plasmids in 293T cells. The expression of each construct was confirmed by western blot, and the results demonstrated that similar amounts of Notch3 fragments were present in each experimental group (Fig. 3B). WWP2 and ubiquitin were also expressed at comparable levels (Fig. 3B). The level of ubiquitination of Notch3 fragments was measured by reciprocal immunoprecipitation with HA and V5 antibodies. The results demonstrated that all tested Notch3 fragments were ubiquitinated in cells co-transfected with WWP2; however, the ubiquitination level of N3-NEXT was most apparent (Top, Fig. 3C and Fig. S1A). There was a prominent band representing mono-ubiquitinated Notch3 protein and a weak high molecular weight smear corresponding to poly-ubiquitinated products. To test the possibility that the differential ubiquitination levels among the Notch3 fragments was due to their ability to encounter and interact with WWP2 in vivo, we performed co-immunoprecipitation experiments in 293T cells following co-transfection of the V5-tagged Notch3 fragment and flag-tagged WWP2. The results demonstrated that although protein expression levels were comparable among the three Notch3 fragments, N3-NEXT protein was more abundantly bound to WWP2 than N3-TM and N3-ICD, reflected by an intense protein pull-down band (Bottom, Fig. 3C). Reciprocal co-immunoprecipitation assays also confirmed the above finding (Fig. S1B).

Bottom Line: The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor.Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas.Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pathology and Gynecology/Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.

Show MeSH
Related in: MedlinePlus