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The Role of the Ubiquitin Ligase E6-AP in Human Papillomavirus E6-mediated Degradation of PDZ Domain-containing Proteins

Kuballa, Petric, Matentzoglu, Konstantin, Scheffner, Martin·2007·Journal of Biological Chemistry
DOI 10.1074/jbc.m605117200
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FIGURE 1 E6-AP facilitates E6-mediated ubiquitination of Dlg in vitro . A , schematic representation of the E6 proteins used and their ability to bind and degrade p53 and Dlg, respectively. 18C11, chimeric protein consisting of the 10 C-terminal amino acid residues of HPV11 E6 fused to amino acid residues 1-141 of HV18 E6 (consists of 158 residues); 11C18, chimeric protein consisting of the 17 C-terminal amino acid residues of HPV18 E6 fused to amino acid residues 1-140 of HV11 E6 (consists of 150 residues). + , binds to p53 and Dlg, respectively; -, does not bind to p53 and Dlg, respectively. B , bacterially expressed GST fusion proteins of the E6 proteins indicated were incubated with in vitro translated radiolabeled Dlg under standardubiquitinationconditionsfor2hinthepresenceorabsenceofbaculovirus-expressed E6-AP and ubiquitin ( ubi ), respectively, as indicated. The reaction products were analyzed by SDS-PAGE followed by fluorography. Running positions of the nonmodified form and the ubiquitinated forms of Dlg are indicated by an arrowhead and an asterisk , respectively.
FIGURE 1 E6-AP facilitates E6-mediated ubiquitination of Dlg in vitro . A , schematic representation of the E6 proteins used and their ability to bind and degrade p53 and Dlg, respectively. 18C11, chimeric protein consisting of the 10 C-terminal amino acid residues of HPV11 E6 fused to amino acid residues 1-141 of HV18 E6 (consists of 158 residues); 11C18, chimeric protein consisting of the 17 C-terminal amino acid residues of HPV18 E6 fused to amino acid residues 1-140 of HV11 E6 (consists of 150 residues). + , binds to p53 and Dlg, respectively; -, does not bind to p53 and Dlg, respectively. B , bacterially expressed GST fusion proteins of the E6 proteins indicated were incubated with in vitro translated radiolabeled Dlg under standardubiquitinationconditionsfor2hinthepresenceorabsenceofbaculovirus-expressed E6-AP and ubiquitin ( ubi ), respectively, as indicated. The reaction products were analyzed by SDS-PAGE followed by fluorography. Running positions of the nonmodified form and the ubiquitinated forms of Dlg are indicated by an arrowhead and an asterisk , respectively.CC-BY · From the PDF
FIGURE 2 E6-AP is required for E6-induced degradation of Dlg in MEFs derived from E6-AP null mice. A-C , mouse embryo fibroblasts derived from E6-AP null mice (24) were cotransfected with expression constructs for p53 and HA-tagged Dlg in the absence and presence of expression constructs encoding various E6 proteins and different forms of E6-AP isoform 1 (28) as indicated. Protein extracts were prepared 24 h after transfection, and levels of p53, Dlg, and E6-AP were determined by Western blot analysis as indicated. Notethat the anti-p53 antibody used (DO1) recognizes ectopically expressed human p53 but not endogenous murine p53. wt , wild-type E6-AP isoform 1; C820A , catalytically inactive E6-AP with a substitution of the catalytic site cysteine residue at position 820 (numbering is according to E6-AP isoform 1) by alanine; 𝚫 E6bs , E6-AP isoform 1 with a deletion of the E6 binding region (amino acid residues 378-395). *, denotes a cross-reactive band serving as loading control.
FIGURE 2 E6-AP is required for E6-induced degradation of Dlg in MEFs derived from E6-AP null mice. A-C , mouse embryo fibroblasts derived from E6-AP null mice (24) were cotransfected with expression constructs for p53 and HA-tagged Dlg in the absence and presence of expression constructs encoding various E6 proteins and different forms of E6-AP isoform 1 (28) as indicated. Protein extracts were prepared 24 h after transfection, and levels of p53, Dlg, and E6-AP were determined by Western blot analysis as indicated. Notethat the anti-p53 antibody used (DO1) recognizes ectopically expressed human p53 but not endogenous murine p53. wt , wild-type E6-AP isoform 1; C820A , catalytically inactive E6-AP with a substitution of the catalytic site cysteine residue at position 820 (numbering is according to E6-AP isoform 1) by alanine; 𝚫 E6bs , E6-AP isoform 1 with a deletion of the E6 binding region (amino acid residues 378-395). *, denotes a cross-reactive band serving as loading control.CC-BY · From the PDF
Image 3
CC-BY · From the PDF
FIGURE 4 Reconstitution of E6-induced degradation of Dlg and MAGI-1 in E6-AP null H1299 cells. A , the E6-AP null H1299 clone K3 was cotransfected with expression constructs for p53 and HA-tagged Dlg in the absence and presence of expression constructs encoding HPV18 E6 and 11C18, respectively, and different forms of E6-AP isoform 1 (28) as indicated. Protein extracts were prepared 24 h after transfection, and levels of p53, Dlg, and E6-AP were determined by Western blot analysis as indicated. B , K3 cells were cotransfectedwithexpressionconstructsforFLAG-taggedMAGI-1andGFPin the absence and presence of expression constructs encoding HPV18 E6 and 11C18, respectively, and different forms of E6-AP isoform 1 (28) as indicated. Protein extracts were prepared 24 h after transfection, and levels of MAGI-1, GFP, and E6-AP were determined by Western blot analysis as indicated. wt , wild-type E6-AP isoform 1; C820A , catalytically inactive E6-AP with a substitution of the catalytic site cysteine residue at position 820 (numbering is according to E6-AP isoform 1) by alanine; 𝚫 E6bs , E6-AP isoform 1 with a deletion of the E6 binding region (amino acid residues 378-395).
FIGURE 4 Reconstitution of E6-induced degradation of Dlg and MAGI-1 in E6-AP null H1299 cells. A , the E6-AP null H1299 clone K3 was cotransfected with expression constructs for p53 and HA-tagged Dlg in the absence and presence of expression constructs encoding HPV18 E6 and 11C18, respectively, and different forms of E6-AP isoform 1 (28) as indicated. Protein extracts were prepared 24 h after transfection, and levels of p53, Dlg, and E6-AP were determined by Western blot analysis as indicated. B , K3 cells were cotransfectedwithexpressionconstructsforFLAG-taggedMAGI-1andGFPin the absence and presence of expression constructs encoding HPV18 E6 and 11C18, respectively, and different forms of E6-AP isoform 1 (28) as indicated. Protein extracts were prepared 24 h after transfection, and levels of MAGI-1, GFP, and E6-AP were determined by Western blot analysis as indicated. wt , wild-type E6-AP isoform 1; C820A , catalytically inactive E6-AP with a substitution of the catalytic site cysteine residue at position 820 (numbering is according to E6-AP isoform 1) by alanine; 𝚫 E6bs , E6-AP isoform 1 with a deletion of the E6 binding region (amino acid residues 378-395).CC-BY · From the PDF
FIGURE 5 Down-regulation of E6-AP expression results in accumulation of Dlg in p53 null HeLa cells. The previously described p53 null HeLa clones HA1andHC2(9)weretransfected with chemically synthesized siRNAs specifically targeting HPV18 E6 ( E6 ) or E6-AP ( E6-AP ). As control, HA1 and HC2 cells were transfected with siRNA directed against Renilla luciferase ( con ). Protein extracts were prepared 72 h after transfection, and levels of Dlg, E6-AP, and tubulin ( tub ; loading control) were determined by Western blot analysis as indicated.
FIGURE 5 Down-regulation of E6-AP expression results in accumulation of Dlg in p53 null HeLa cells. The previously described p53 null HeLa clones HA1andHC2(9)weretransfected with chemically synthesized siRNAs specifically targeting HPV18 E6 ( E6 ) or E6-AP ( E6-AP ). As control, HA1 and HC2 cells were transfected with siRNA directed against Renilla luciferase ( con ). Protein extracts were prepared 72 h after transfection, and levels of Dlg, E6-AP, and tubulin ( tub ; loading control) were determined by Western blot analysis as indicated.CC-BY · From the PDF
FIGURE6 Functional interaction of low risk HPV E6 proteins with E6-AP. HEK293cells were transfected with expression constructs encoding various forms of HA-tagged E6 and FLAG-tagged E6-AP, respectively, as indicated. Protein extracts were prepared 24 h after transfection. 5% of the respective extracts were directly used for determination of the expression levels of the various forms of E6 and E6-AP, respectively ( Input ). The remaining extracts were used for immunoprecipitation of E6 with an anti-HA antibody. A , 10% of the precipitates were analyzed by Western blot analysis to determine the levels of the respective immunoprecipitated E6 and of potentially coprecipitating E6-AP. B , the remaining precipitates were used for in vitro ubiquitination assays using in vitro translated radiolabeled p53 or Dlg as substrate. The running position of the ubiquitinated form of p53 and Dlg, respectively, is indicated by an asterisk . 11 , HPV11E6; 18 , HPV18 E6; 11C18 , chimeric protein consisting of the 17 C-terminal amino acid residues of HPV18 E6 fused to amino acid residues 1-140 of HPV11 E6; 5C18 , chimeric protein consisting of the 17 C-terminal amino acid residues of HPV18 E6 fused to amino acid residues 1-150 of HPV5 E6; wt , wild-type E6-AP isoform 1; C820A , catalytically inactive E6-AP with a substitution of the catalytic site cysteine residue at position 820 (numbering is according to E6-AP isoform 1) by alanine; 𝚫 E6bs , E6-AP isoform 1 with a deletion of the E6 binding region (amino acid residues 378-395).
FIGURE6 Functional interaction of low risk HPV E6 proteins with E6-AP. HEK293cells were transfected with expression constructs encoding various forms of HA-tagged E6 and FLAG-tagged E6-AP, respectively, as indicated. Protein extracts were prepared 24 h after transfection. 5% of the respective extracts were directly used for determination of the expression levels of the various forms of E6 and E6-AP, respectively ( Input ). The remaining extracts were used for immunoprecipitation of E6 with an anti-HA antibody. A , 10% of the precipitates were analyzed by Western blot analysis to determine the levels of the respective immunoprecipitated E6 and of potentially coprecipitating E6-AP. B , the remaining precipitates were used for in vitro ubiquitination assays using in vitro translated radiolabeled p53 or Dlg as substrate. The running position of the ubiquitinated form of p53 and Dlg, respectively, is indicated by an asterisk . 11 , HPV11E6; 18 , HPV18 E6; 11C18 , chimeric protein consisting of the 17 C-terminal amino acid residues of HPV18 E6 fused to amino acid residues 1-140 of HPV11 E6; 5C18 , chimeric protein consisting of the 17 C-terminal amino acid residues of HPV18 E6 fused to amino acid residues 1-150 of HPV5 E6; wt , wild-type E6-AP isoform 1; C820A , catalytically inactive E6-AP with a substitution of the catalytic site cysteine residue at position 820 (numbering is according to E6-AP isoform 1) by alanine; 𝚫 E6bs , E6-AP isoform 1 with a deletion of the E6 binding region (amino acid residues 378-395).CC-BY · From the PDF

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Metadata

Authors
Kuballa, P., Matentzoglu, K., & Scheffner, M.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2007-01
Volume
282
Issue
1
Pages
65-71
ISSN
0021-9258
DOI
10.1074/jbc.m605117200
Cited by
55
References
1
Language
en
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