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WD repeat protein WDR48 in complex with deubiquitinase USP12 suppresses Akt-dependent cell survival signaling by stabilizing PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1).

Gangula NR, Maddika S - J. Biol. Chem. (2013)

Bottom Line: The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability.Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis.Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression.

View Article: PubMed Central - PubMed

Affiliation: From the Laboratory of Cell Death and Cell Survival, Centre for DNA Fingerprinting and Diagnostics (CDFD), Nampally, Hyderabad 500001, India.

ABSTRACT
PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1) is a protein-serine/threonine phosphatase and a negative regulator of the PI3-kinase/Akt pathway. Although its function as a suppressor of tumor cell growth has been established, the mechanism of its regulation is not completely understood. In this study, by utilizing the tandem affinity purification approach we have identified WDR48 and USP12 as novel PHLPP1-associated proteins. The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability. Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis. Functionally, we show that WDR48 and USP12 suppress proliferation of tumor cells. Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression. Together, our results reveal WDR48 and USP12 as novel PHLPP1 regulators and potential suppressors of tumor cell survival.

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WDR48·USP12 complex regulates PHLPP1 protein stability by deubiquitination.A, HCT116 cells were transfected with either control shRNA or shRNA against WDR48. Cell lysates prepared after 6 h of MG132 (10 μm) treatment were subjected to immunoprecipitation (IP) with streptavidin beads (SBP), and the ubiquitinated PHLPP1 was detected with anti-ubiquitin (Ub) antibody. The protein expression and the efficiency of shRNA were confirmed by immunoblotting (WB) of cell extracts using antibodies as indicated. B, HCT116 cells were transfected with control shRNA or shRNA against USP12 shRNA. Ubiquitination of PHLPP1 was detected as described in A. C, Myc-tagged wild type USP12 or catalytically inactive C48S mutant of USP12 was expressed in HCT116 cells along with FLAG-PHLPP1. The levels of PHLPP1 ubiquitination were evaluated by immunoprecipitation of PHLPP1 using anti-FLAG antibody followed by immunoblotting with anti-ubiquitin antibody. The values presented below the ubiquitin blot were normalized ubiquitinated PHLPP1/input PHLPP1 derived from the quantification of the blots by using Image Lab software. D, Myc-tagged wild type WDR48 was expressed in HCT116 cells along with either full-length FLAG-PHLPP1 or PHLPP1 ΔWIR mutant (WDR48 interaction-deficient mutant). PHLPP1 ubiquitination was evaluated using anti-ubiquitin antibody after immunoprecipitation with FLAG antibody. The values presented below the ubiquitin blot were normalized ubiquitinated PHLPP1/input PHLPP1 derived from the quantification of the blots by using Image Lab software. E, HEK293T cells were transfected with plasmids encoding SFB-tagged WDR48 and USP12. Twenty four hours after transfection, cells were treated with cycloheximide (CHX; 50 μg/ml) and collected at the indicated times. The protein levels of PHLPP1 were determined by immunoblotting with PHLPP1 antibody. F, HCT116 cells were transfected with control and WDR48 shRNA. The levels of PHLPP1 were detected using specific antibody at the indicated times after cycloheximide treatment.
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Figure 3: WDR48·USP12 complex regulates PHLPP1 protein stability by deubiquitination.A, HCT116 cells were transfected with either control shRNA or shRNA against WDR48. Cell lysates prepared after 6 h of MG132 (10 μm) treatment were subjected to immunoprecipitation (IP) with streptavidin beads (SBP), and the ubiquitinated PHLPP1 was detected with anti-ubiquitin (Ub) antibody. The protein expression and the efficiency of shRNA were confirmed by immunoblotting (WB) of cell extracts using antibodies as indicated. B, HCT116 cells were transfected with control shRNA or shRNA against USP12 shRNA. Ubiquitination of PHLPP1 was detected as described in A. C, Myc-tagged wild type USP12 or catalytically inactive C48S mutant of USP12 was expressed in HCT116 cells along with FLAG-PHLPP1. The levels of PHLPP1 ubiquitination were evaluated by immunoprecipitation of PHLPP1 using anti-FLAG antibody followed by immunoblotting with anti-ubiquitin antibody. The values presented below the ubiquitin blot were normalized ubiquitinated PHLPP1/input PHLPP1 derived from the quantification of the blots by using Image Lab software. D, Myc-tagged wild type WDR48 was expressed in HCT116 cells along with either full-length FLAG-PHLPP1 or PHLPP1 ΔWIR mutant (WDR48 interaction-deficient mutant). PHLPP1 ubiquitination was evaluated using anti-ubiquitin antibody after immunoprecipitation with FLAG antibody. The values presented below the ubiquitin blot were normalized ubiquitinated PHLPP1/input PHLPP1 derived from the quantification of the blots by using Image Lab software. E, HEK293T cells were transfected with plasmids encoding SFB-tagged WDR48 and USP12. Twenty four hours after transfection, cells were treated with cycloheximide (CHX; 50 μg/ml) and collected at the indicated times. The protein levels of PHLPP1 were determined by immunoblotting with PHLPP1 antibody. F, HCT116 cells were transfected with control and WDR48 shRNA. The levels of PHLPP1 were detected using specific antibody at the indicated times after cycloheximide treatment.

Mentions: Cells were transfected with various combinations of plasmids as indicated in Figs. 3 and 6, and 24 h after transfection cells were treated with MG132 (10 μm) for 6 h. The whole cell extracts prepared by NETN lysis were subjected to immunoprecipitation of the substrate protein. The analysis of ubiquitination was performed by immunoblotting with ubiquitin antibody.


WD repeat protein WDR48 in complex with deubiquitinase USP12 suppresses Akt-dependent cell survival signaling by stabilizing PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1).

Gangula NR, Maddika S - J. Biol. Chem. (2013)

WDR48·USP12 complex regulates PHLPP1 protein stability by deubiquitination.A, HCT116 cells were transfected with either control shRNA or shRNA against WDR48. Cell lysates prepared after 6 h of MG132 (10 μm) treatment were subjected to immunoprecipitation (IP) with streptavidin beads (SBP), and the ubiquitinated PHLPP1 was detected with anti-ubiquitin (Ub) antibody. The protein expression and the efficiency of shRNA were confirmed by immunoblotting (WB) of cell extracts using antibodies as indicated. B, HCT116 cells were transfected with control shRNA or shRNA against USP12 shRNA. Ubiquitination of PHLPP1 was detected as described in A. C, Myc-tagged wild type USP12 or catalytically inactive C48S mutant of USP12 was expressed in HCT116 cells along with FLAG-PHLPP1. The levels of PHLPP1 ubiquitination were evaluated by immunoprecipitation of PHLPP1 using anti-FLAG antibody followed by immunoblotting with anti-ubiquitin antibody. The values presented below the ubiquitin blot were normalized ubiquitinated PHLPP1/input PHLPP1 derived from the quantification of the blots by using Image Lab software. D, Myc-tagged wild type WDR48 was expressed in HCT116 cells along with either full-length FLAG-PHLPP1 or PHLPP1 ΔWIR mutant (WDR48 interaction-deficient mutant). PHLPP1 ubiquitination was evaluated using anti-ubiquitin antibody after immunoprecipitation with FLAG antibody. The values presented below the ubiquitin blot were normalized ubiquitinated PHLPP1/input PHLPP1 derived from the quantification of the blots by using Image Lab software. E, HEK293T cells were transfected with plasmids encoding SFB-tagged WDR48 and USP12. Twenty four hours after transfection, cells were treated with cycloheximide (CHX; 50 μg/ml) and collected at the indicated times. The protein levels of PHLPP1 were determined by immunoblotting with PHLPP1 antibody. F, HCT116 cells were transfected with control and WDR48 shRNA. The levels of PHLPP1 were detected using specific antibody at the indicated times after cycloheximide treatment.
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Figure 3: WDR48·USP12 complex regulates PHLPP1 protein stability by deubiquitination.A, HCT116 cells were transfected with either control shRNA or shRNA against WDR48. Cell lysates prepared after 6 h of MG132 (10 μm) treatment were subjected to immunoprecipitation (IP) with streptavidin beads (SBP), and the ubiquitinated PHLPP1 was detected with anti-ubiquitin (Ub) antibody. The protein expression and the efficiency of shRNA were confirmed by immunoblotting (WB) of cell extracts using antibodies as indicated. B, HCT116 cells were transfected with control shRNA or shRNA against USP12 shRNA. Ubiquitination of PHLPP1 was detected as described in A. C, Myc-tagged wild type USP12 or catalytically inactive C48S mutant of USP12 was expressed in HCT116 cells along with FLAG-PHLPP1. The levels of PHLPP1 ubiquitination were evaluated by immunoprecipitation of PHLPP1 using anti-FLAG antibody followed by immunoblotting with anti-ubiquitin antibody. The values presented below the ubiquitin blot were normalized ubiquitinated PHLPP1/input PHLPP1 derived from the quantification of the blots by using Image Lab software. D, Myc-tagged wild type WDR48 was expressed in HCT116 cells along with either full-length FLAG-PHLPP1 or PHLPP1 ΔWIR mutant (WDR48 interaction-deficient mutant). PHLPP1 ubiquitination was evaluated using anti-ubiquitin antibody after immunoprecipitation with FLAG antibody. The values presented below the ubiquitin blot were normalized ubiquitinated PHLPP1/input PHLPP1 derived from the quantification of the blots by using Image Lab software. E, HEK293T cells were transfected with plasmids encoding SFB-tagged WDR48 and USP12. Twenty four hours after transfection, cells were treated with cycloheximide (CHX; 50 μg/ml) and collected at the indicated times. The protein levels of PHLPP1 were determined by immunoblotting with PHLPP1 antibody. F, HCT116 cells were transfected with control and WDR48 shRNA. The levels of PHLPP1 were detected using specific antibody at the indicated times after cycloheximide treatment.
Mentions: Cells were transfected with various combinations of plasmids as indicated in Figs. 3 and 6, and 24 h after transfection cells were treated with MG132 (10 μm) for 6 h. The whole cell extracts prepared by NETN lysis were subjected to immunoprecipitation of the substrate protein. The analysis of ubiquitination was performed by immunoblotting with ubiquitin antibody.

Bottom Line: The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability.Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis.Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression.

View Article: PubMed Central - PubMed

Affiliation: From the Laboratory of Cell Death and Cell Survival, Centre for DNA Fingerprinting and Diagnostics (CDFD), Nampally, Hyderabad 500001, India.

ABSTRACT
PHLPP1 (PH domain leucine-rich repeat protein phosphatase 1) is a protein-serine/threonine phosphatase and a negative regulator of the PI3-kinase/Akt pathway. Although its function as a suppressor of tumor cell growth has been established, the mechanism of its regulation is not completely understood. In this study, by utilizing the tandem affinity purification approach we have identified WDR48 and USP12 as novel PHLPP1-associated proteins. The WDR48·USP12 complex deubiquitinates PHLPP1 and thereby enhances its protein stability. Similar to PHLPP1 function, WDR48 and USP12 negatively regulate Akt activation and thus promote cellular apoptosis. Functionally, we show that WDR48 and USP12 suppress proliferation of tumor cells. Importantly, we found a WDR48 somatic mutation (L580F) that is defective in stabilizing PHLPP1 in colorectal cancers, supporting a WDR48 role in tumor suppression. Together, our results reveal WDR48 and USP12 as novel PHLPP1 regulators and potential suppressors of tumor cell survival.

Show MeSH
Related in: MedlinePlus