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Quantitative proteomics identifies the Myb-binding protein p160 as a novel target of the von Hippel-Lindau tumor suppressor.

Lai Y, Qiao M, Song M, Weintraub ST, Shiio Y - PLoS ONE (2011)

Bottom Line: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates.The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.

View Article: PubMed Central - PubMed

Affiliation: Greehey Children's Cancer Research Institute, San Antonio, Texas, United States of America.

ABSTRACT

Background: The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex, which is best understood as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the α subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. However, several lines of evidence suggest that there are unidentified substrates or targets for VHL that play important roles in tumor suppression.

Methodology/principal findings: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.

Conclusions/significance: A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates. The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.

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Related in: MedlinePlus

Proteomic analysis of p160-interacting proteins.(A) Outline of the IP-ICAT approach. To identify p160-interacting proteins, FLAG-p160 is immunoprecipitated from transfected 293T cells, which results in isolation of FLAG-p160, its associated proteins “X” and “Y,” and non-specifically contaminating protein “E.” As a control, lysate of 293T cells transfected with empty FLAG vector is also immunoprecipitated, which isolates non-specifically contaminating protein “E.” The relative abundance of the protein components in the two immunoprecipitates can be determined by the ICAT quantitative proteomics approach. The specific components of the FLAG-p160 complex display enrichment in the FLAG-p160 IP sample whereas non-specific contaminant does not. In this way, specific components of the protein complex can be distinguished from non-specific contaminant. (B) Comparison of the conventional SDS-PAGE-based approach and IP-ICAT approach for the analysis of protein complexes. In the conventional approach, immunopurified protein complex is fractionated by SDS-PAGE and the protein bands specific to test IP sample (absent in control IP sample) are excised and analyzed for peptide sequence determination. The protein ‘Y’ is specific to test IP sample, but can be missed by this approach due to a co-migrating background band. In the IP-ICAT approach, the components of the test IP and control IP samples are compared without using a gel. (C) Partial list of proteins identified in the p160 complex. Components of the VHL E3 ligase complex as well as several nucleolar proteins were identified as p160-interacting proteins. VHL displayed obvious enrichment in the FLAG-p160 IP sample, but was difficult to quantify, which is indicated by “>”.
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pone-0016975-g003: Proteomic analysis of p160-interacting proteins.(A) Outline of the IP-ICAT approach. To identify p160-interacting proteins, FLAG-p160 is immunoprecipitated from transfected 293T cells, which results in isolation of FLAG-p160, its associated proteins “X” and “Y,” and non-specifically contaminating protein “E.” As a control, lysate of 293T cells transfected with empty FLAG vector is also immunoprecipitated, which isolates non-specifically contaminating protein “E.” The relative abundance of the protein components in the two immunoprecipitates can be determined by the ICAT quantitative proteomics approach. The specific components of the FLAG-p160 complex display enrichment in the FLAG-p160 IP sample whereas non-specific contaminant does not. In this way, specific components of the protein complex can be distinguished from non-specific contaminant. (B) Comparison of the conventional SDS-PAGE-based approach and IP-ICAT approach for the analysis of protein complexes. In the conventional approach, immunopurified protein complex is fractionated by SDS-PAGE and the protein bands specific to test IP sample (absent in control IP sample) are excised and analyzed for peptide sequence determination. The protein ‘Y’ is specific to test IP sample, but can be missed by this approach due to a co-migrating background band. In the IP-ICAT approach, the components of the test IP and control IP samples are compared without using a gel. (C) Partial list of proteins identified in the p160 complex. Components of the VHL E3 ligase complex as well as several nucleolar proteins were identified as p160-interacting proteins. VHL displayed obvious enrichment in the FLAG-p160 IP sample, but was difficult to quantify, which is indicated by “>”.

Mentions: The proteomic analysis of the p160-containing protein complex also confirmed the physical interaction of p160 and the VHL ubiquitin ligase complex (Figure 3): FLAG-p160 was transfected into 293T cells and the protein complex containing FLAG-p160 was purified by anti-FLAG immunoprecipitation under non-denaturing conditions. As a control, we used 293T cells transfected with FLAG empty vector. The protein components in the two immunoprecipitation samples (FLAG-p160 and FLAG-vector) were compared by the ICAT approach and the specific components of the FLAG-p160 complex were identified by their increased abundance in the FLAG-p160 immunoprecipitate compared with FLAG-vector immunoprecipitate (Figure 3A). This analysis identified the co-immunoprecipitation of FLAG-p160 with the components of the VHL ubiquitin ligase complex (VHL, elongin B, and elongin C) as well as a number of nucleolar proteins (Figure 3C, for a complete list of proteins displaying more than 2-fold enrichment in FLAG-p160 immunoprecipitate, see Table S3). Collectively, these results suggest that VHL induces proteasome- and iron-dependent degradation of p160 through direct physical interaction.


Quantitative proteomics identifies the Myb-binding protein p160 as a novel target of the von Hippel-Lindau tumor suppressor.

Lai Y, Qiao M, Song M, Weintraub ST, Shiio Y - PLoS ONE (2011)

Proteomic analysis of p160-interacting proteins.(A) Outline of the IP-ICAT approach. To identify p160-interacting proteins, FLAG-p160 is immunoprecipitated from transfected 293T cells, which results in isolation of FLAG-p160, its associated proteins “X” and “Y,” and non-specifically contaminating protein “E.” As a control, lysate of 293T cells transfected with empty FLAG vector is also immunoprecipitated, which isolates non-specifically contaminating protein “E.” The relative abundance of the protein components in the two immunoprecipitates can be determined by the ICAT quantitative proteomics approach. The specific components of the FLAG-p160 complex display enrichment in the FLAG-p160 IP sample whereas non-specific contaminant does not. In this way, specific components of the protein complex can be distinguished from non-specific contaminant. (B) Comparison of the conventional SDS-PAGE-based approach and IP-ICAT approach for the analysis of protein complexes. In the conventional approach, immunopurified protein complex is fractionated by SDS-PAGE and the protein bands specific to test IP sample (absent in control IP sample) are excised and analyzed for peptide sequence determination. The protein ‘Y’ is specific to test IP sample, but can be missed by this approach due to a co-migrating background band. In the IP-ICAT approach, the components of the test IP and control IP samples are compared without using a gel. (C) Partial list of proteins identified in the p160 complex. Components of the VHL E3 ligase complex as well as several nucleolar proteins were identified as p160-interacting proteins. VHL displayed obvious enrichment in the FLAG-p160 IP sample, but was difficult to quantify, which is indicated by “>”.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046137&req=5

pone-0016975-g003: Proteomic analysis of p160-interacting proteins.(A) Outline of the IP-ICAT approach. To identify p160-interacting proteins, FLAG-p160 is immunoprecipitated from transfected 293T cells, which results in isolation of FLAG-p160, its associated proteins “X” and “Y,” and non-specifically contaminating protein “E.” As a control, lysate of 293T cells transfected with empty FLAG vector is also immunoprecipitated, which isolates non-specifically contaminating protein “E.” The relative abundance of the protein components in the two immunoprecipitates can be determined by the ICAT quantitative proteomics approach. The specific components of the FLAG-p160 complex display enrichment in the FLAG-p160 IP sample whereas non-specific contaminant does not. In this way, specific components of the protein complex can be distinguished from non-specific contaminant. (B) Comparison of the conventional SDS-PAGE-based approach and IP-ICAT approach for the analysis of protein complexes. In the conventional approach, immunopurified protein complex is fractionated by SDS-PAGE and the protein bands specific to test IP sample (absent in control IP sample) are excised and analyzed for peptide sequence determination. The protein ‘Y’ is specific to test IP sample, but can be missed by this approach due to a co-migrating background band. In the IP-ICAT approach, the components of the test IP and control IP samples are compared without using a gel. (C) Partial list of proteins identified in the p160 complex. Components of the VHL E3 ligase complex as well as several nucleolar proteins were identified as p160-interacting proteins. VHL displayed obvious enrichment in the FLAG-p160 IP sample, but was difficult to quantify, which is indicated by “>”.
Mentions: The proteomic analysis of the p160-containing protein complex also confirmed the physical interaction of p160 and the VHL ubiquitin ligase complex (Figure 3): FLAG-p160 was transfected into 293T cells and the protein complex containing FLAG-p160 was purified by anti-FLAG immunoprecipitation under non-denaturing conditions. As a control, we used 293T cells transfected with FLAG empty vector. The protein components in the two immunoprecipitation samples (FLAG-p160 and FLAG-vector) were compared by the ICAT approach and the specific components of the FLAG-p160 complex were identified by their increased abundance in the FLAG-p160 immunoprecipitate compared with FLAG-vector immunoprecipitate (Figure 3A). This analysis identified the co-immunoprecipitation of FLAG-p160 with the components of the VHL ubiquitin ligase complex (VHL, elongin B, and elongin C) as well as a number of nucleolar proteins (Figure 3C, for a complete list of proteins displaying more than 2-fold enrichment in FLAG-p160 immunoprecipitate, see Table S3). Collectively, these results suggest that VHL induces proteasome- and iron-dependent degradation of p160 through direct physical interaction.

Bottom Line: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates.The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.

View Article: PubMed Central - PubMed

Affiliation: Greehey Children's Cancer Research Institute, San Antonio, Texas, United States of America.

ABSTRACT

Background: The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex, which is best understood as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the α subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. However, several lines of evidence suggest that there are unidentified substrates or targets for VHL that play important roles in tumor suppression.

Methodology/principal findings: Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.

Conclusions/significance: A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates. The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.

Show MeSH
Related in: MedlinePlus