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First published online 18 March 2008
doi: 10.1242/jcs.021105

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A20 is a negative regulator of BCL10- and CARMA3-mediated activation of NF-B

¹ Dip. Scienze Biologiche ed Ambientali, Università degli Studi del Sannio di Benevento, Via Port'Arsa 11, 82100 Benevento, Italy
² Centro di Endocrinologia ed Oncologia Sperimentale, Napoli, Italy
³ Dip. di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli `Federico II', Via S. Pansini 5, 80131, Napoli, Italy
⁴ BioGeM Consortium, Via Camporeale, 83031 Ariano Irpino, Italy

View larger version (14K): [in this window] [in a new window]   Fig. 1. Selective inhibition of BCL10- and CARMA3-induced NF-B activation by A20. (A,B) HEK293 cells were transiently cotransfected with an expression vector encoding the indicated polypeptides, together with NF-B–luciferase and β-galactosidase reporter vectors. The total amount of transfected plasmid DNA was maintained constant by adding empty vector. 16 hours after transfection, cell lysates were prepared and luciferase activity was measured. The data shown represent the relative luciferase activity normalized against β-galactosidase activity and are representative of six independent experiments performed in triplicate. Bottom panels: a fraction of the transfection reaction mixture was separated by SDS-PAGE, blotted onto nitrocellulose membrane and analyzed by immunoblotting to monitor protein expression.

View larger version (27K): [in this window] [in a new window]   Fig. 2. The deubiquitylation activity of A20 inhibits interaction of BCL10-IKK/NEMO in mammalian cells. (A) Lysates from HEK293 cells transfected with the indicated expression vectors were immunoprecipitated with mAb against IKK/NEMO. Immunocomplexes were separated by SDS-PAGE, transferred onto membranes and subsequently probed with antisera specific for BCL10 and IKK/NEMO. A fraction of the transfection reaction mixture was separated by SDS-PAGE, blotted onto nitrocellulose membranes and analyzed by immunoblotting to visualize protein expression. The results shown are representative of five separate experiments. (B,C) HEK293 cells were transiently cotransfected with an expression vector encoding the indicated polypeptides, together with NF-B–luciferase and β-galactosidase reporter vectors. The total amount of transfected plasmid DNA was maintained constant by adding empty vector. 16 hours after transfection, cell lysates were prepared and luciferase activity was measured. The data shown represent the relative luciferase activity normalized against β-galactosidase activity and are representative of six independent experiments performed in triplicate. Right panels: a fraction of the transfection reaction mixture was separated by SDS-PAGE, blotted onto nitrocellulose membranes and analyzed by immunoblotting to monitor protein expression. (D,E) Lysates from HEK293 cells transfected with the indicated expression vectors were immunoprecipitated with mAb against IKK/NEMO. Immunocomplexes were separated by SDS-PAGE, transferred onto membranes and subsequently probed with antibodies against ubiquitin and IKK/NEMO. The results shown are representative of four separate experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 3. A20 inhibits PKC-mediated NF-B activation. (A) HEK293 cells were transfected with empty vector or with the indicated expression vectors, together with an NF-B–luciferase reporter plasmid. 24 hours later, cells were left untreated or treated with PMA (40 ng/ml) plus ionomycin (1 µM) for 5 hours and luciferase activity was measured. The data shown are representative of six independent experiments. (B) HEK293 cells were transfected with empty vector or with the indicated expression plasmids. 24 hours later, cells were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for 40 minutes. Lysates were then prepared and immunoprecipitated (IP) with mAb against IKK/NEMO. Immunocomplexes were separated by SDS-PAGE, transferred onto membranes and subsequently probed with antibodies (WB) against ubiquitin and IKK/NEMO. (C) HEK293 cells, transfected with an empty expression vector or vector encoding A20wt or A20 mut, were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for the indicated periods of time. Cell lysates were then prepared and analyzed for degradation of the inhibitory subunit I-B by immunoblotting (WP).

View larger version (26K): [in this window] [in a new window]   Fig. 4. A20 inhibits formation of molecular complexes following activation of PKC. HEK293 cells were transfected with an empty expression plasmid or plasmid encoding A20. 24 hours later, cells were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for 40 minutes. Cell extracts were immunoprecipitated with the indicated immunoprecipitating antibody (IP) and assayed for coprecipitating proteins by immunoblot experiments probed with the indicated antibody (WB).

View larger version (19K): [in this window] [in a new window]   Fig. 5. A20 inhibits LPA-mediated NF-B activation. (A) MEFs were infected in 100 mm Petri dishes with an empty retroviral construct or construct expressing hemagglutinin (HA)-flagged forms of A20wt and A20mut and subsequently transfected with a luciferase reporter plasmid for NF-B. Infected cells were left untreated or stimulated with LPA (20 µM) for 8 hours and luciferase activity was measured. The data shown represent the relative luciferase activity and are representative of four independent experiments performed in triplicate. (B) MEFs were infected as in panel A and left untreated or treated with LPA for 60 minutes. Lysates were then prepared and immunoprecipitated (IP) with antibody against IKK/NEMO. Immunocomplexes were separated by SDS-PAGE, transferred onto membranes and subsequently probed with antibodies (WB) against BCL10 and IKK/NEMO. A20 expression was monitored by immunoblotting with antibody against HA.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Negative regulatory activity of A20 on the NF-B activation mediated by PKC. (A) HEK293 and Jurkat cells were left untreated or treated with PMA (40 ng/ml) plus ionomycin (1 µM) or TNF (5 ng/ml) for 6 hours. Lysates (80 µg) extracted from treated and untreated cells were separated by SDS-PAGE, blotted onto nitrocellulose membranes and analyzed by immunoblotting (WB) to monitor A20 protein expression. (B) HEK293 cells were transfected with an empty plasmid or plasmid encoding a shRNA against human A20. 24 hours later, the expression level of A20 was monitored by western blot experiments in cells left untreated or stimulated with PMA (40 ng/ml) plus ionomycin (1 µM) for 6 hours. (C,D) HEK293 cells were transiently cotransfected with the indicated plasmid DNA, together with NF-B–luciferase and β-galactosidase reporter vectors. 16 hours after transfection, cell lysates were prepared and luciferase activity was measured. As a control, a plasmid expressing a shRNA validated to target expression of eGFP was used (shCTR). The data shown represent the relative luciferase activity normalized against β-galactosidase activity and are representative of five independent experiments performed in triplicate. (E) HEK293 cells were transiently cotransfected with the indicated plasmid DNA, together with NF-B–luciferase and β-galactosidase reporter vectors. 24 hours later, cells were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for 5 hours and luciferase activity was measured. The data shown represent the relative luciferase activity normalized against β-galactosidase activity and are representative of five independent experiments performed in triplicate.

View larger version (25K): [in this window] [in a new window]   Fig. 7. A20 inhibits CARMA1 and BCL10 function. (A) Jurkat cells were transfected with an empty vector or vector encoding A20wt and A20mut, together with an NF-B–luciferase reporter plasmid. 24 hours later, cells were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for 5 hours and luciferase activity was measured. The data shown are representative of five separate experiments. The expression level of cotransfected A20wt and A20mut, relative to one experiment, was verified by western blot and is shown in the bottom panel. (B) Jurkat cells were transfected as in panel A and then stimulated for 10 hours with antibodies against CD3 and CD28 (1 µg/ml of anti-CD3 and 2 µg/ml of anti-CD28), after which the luciferase activity was determined. The data shown are representative of four independent experiments. (C) Jurkat cells were transfected with empty vector or with an expression plasmid encoding A20. 24 hours later, cells were treated with PMA (40 ng/ml) plus ionomycin (1 µM) for the indicated periods of time. Lysates were then prepared and immunoprecipitated (IP) with mAb against IKK/NEMO. Immunocomplexes were separated by SDS-PAGE, transferred onto membranes and subsequently probed with antibodies (WB) against ubiquitin and IKK/NEMO.

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