Viral oncoproteins discriminate between p53 and the p53 homolog p73 - PubMed (original) (raw)

Viral oncoproteins discriminate between p53 and the p53 homolog p73

M C Marin et al. Mol Cell Biol. 1998 Nov.

Abstract

p73 is a recently identified member of the p53 family. Previously it was shown that p73 can, when overproduced in p53-defective tumor cells, activate p53-responsive promoters and induce apoptosis. In this report we describe the generation of anti-p73 monoclonal antibodies and confirm that two previously described p73 isoforms are produced in mammalian cells. Furthermore, we show that these two isoforms can bind to canonical p53 DNA-binding sites in electrophoretic mobility shift assays. Despite the high degree of similarity between p53 and p73, we found that adenovirus E1B 55K, simian virus 40 T, and human papillomavirus E6 do not physically interact with p73. The observation that viral oncoproteins discriminate between p53 and p73 suggests that the functions of these two proteins may differ under physiological conditions. Furthermore, they suggest that inactivation of p73 may not be required for transformation.

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Figures

FIG. 1

FIG. 1

GST-p73 fusion proteins used to generate monoclonal antibodies. Shown schematically are p53, p73α, and p73β. Mice were immunized with GST fused to either the C terminus of p73α (ER mice) or the C terminus of p73β (GC mice). The p73β cDNA lacks exon 13, resulting in a frameshift at residue 495. p73β therefore contains five residues (black box) not present in p73α.

FIG. 2

FIG. 2

Characterization of anti-p73 antibodies. (A) p73α and p73β 35S-labelled in vitro translation products were immunoprecipitated with either control (anti-T; PAb419) or anti-p73 (ER13, ER15, and GC15) antibodies, as indicated, under native (lanes 3 to 6 and 11 to 14) or denaturing (lanes 7 to 10 and 15 to 18) conditions. Two microliters of the indicated in vitro translation product was loaded directly in lanes 1 and 2. (B) p73α and p73β were cotranslated in vitro in the presence of [35S]methionine and immunoprecipitated with either control (anti-T; PAb419) or anti-p73 (ER13, ER15, and GC15) antibodies, as indicated, under native (lanes 2 to 5) or denaturing (lanes 6 to 10) conditions. Two microliters of the indicated in vitro translation product was loaded directly in lanes 1, 11, and 12. (C) p51A, p53, and p73β 35S-labelled in vitro translation products were immunoprecipitated with either control (anti-T; PAb419) or anti-p73 (ER13, ER15, and GC15) antibodies, as indicated. Two microliters of the indicated in vitro translation product was loaded directly in lanes 1 to 3. For panels A to C, proteins were resolved by SDS-polyacrylamide gel electrophoresis and detected by fluorography. (D) GST-p53 (lanes 1 to 5), GST-p73β (aa 380 to 499) (lanes 6 to 10), and GST-p73α (aa 380 to 637) (lanes 11 to 15) were produced in bacteria, purified by using glutathione-Sepharose, resolved by SDS-polyacrylamide gel electrophoresis in preparative wells, and transferred to a membrane. The membrane was cut into strips and immunoblotted with either control (anti-T; PAb419) or anti-p73 (ER13, ER15, and GC15) antibodies, as indicated. Bound antibody was detected colorimetrically with an alkaline phosphatase-conjugated antimouse antibody.

FIG. 3

FIG. 3

Identification of p73α and p73β in cell extracts. (A) COS cells were lysed in the presence of 0.5% Nonidet P-40 (EBC buffer) (lanes 1 to 3), in radioimmunoprecipitation assay buffer (lanes 4 to 6), or in the presence of 1% Triton (lanes 7 to 9) and immunoprecipitated (IP) with a control antibody (anti-T [PAb419]), anti-p73 (ER13), or anti-p73 (GC15) as indicated. Antibodies were cross-linked to protein A-Sepharose prior to immunoprecipitation. (B) COS cells (lanes 1 to 4) and SK-N-AS cells (lanes 5 to 8) were lysed in EBC buffer and immunoprecipitated with a control antibody (anti-T [PAb419]), anti-p73 (ER13), anti-p73 (ER15), or anti-p73 (GC15) as indicated. For both panels, Bound proteins were resolved by electrophoresis in an SDS–8% polyacrylamide gel and immunoblotted with anti-p73 (ER15) by using a horseradish peroxidase-conjugated antimouse antibody and enhanced chemiluminescence.

FIG. 4

FIG. 4

p73α and p73β protein levels in neuroblastoma cell lines. Cell extracts prepared from the indicated cell lines were resolved by electrophoresis in an SDS–8% polyacrylamide gel and immunoblotted with an anti-p73 antibody (ER15). Each lane contained ∼300 μg of total cell extract. Bound antibody was detected by using a horseradish peroxidase-conjugated antimouse antibody and enhanced chemiluminescence.

FIG. 5

FIG. 5

p73 binds to canonical p53 DNA-binding sites. A 32P-radiolabelled p53 DNA-binding site was incubated with p53 in vitro translation product (A, lanes 4 to 9), p73α in vitro translation product (A, lanes 10 to 15, and B, lanes 1 to 5), p73β in vitro translation product (B, lanes 6 to 10), or unprogrammed reticulocyte lysate (A, lanes 1 to 3) and subjected to electrophoretic mobility shift analysis. Binding reactions were carried out in the presence of a vast molar excess of unlabelled specific or nonspecific competitor DNA where indicated. Anti-p53, anti-p73, or a control antibody (anti-T) was added prior to the electrophoretic mobility shift assay as indicated. The asterisk indicates a nonspecific complex.

FIG. 6

FIG. 6

Differential binding of p53 and p73 to E1B 55K. (A) 293 human embryonic kidney cells were lysed and immunoprecipitated (IP) with the indicated antibodies. Specifically bound proteins were resolved by electrophoresis in an SDS–10% polyacrylamide gel and detected by immunoblotting with anti-p73 antibody (left panel) or anti-p53 antibody (right panel). Bound antibodies were detected by using a horseradish peroxidase-conjugated antimouse antibody and enhanced chemiluminescence. The asterisk indicates faster-migrating species, previously noted by others, that interact with anti-p53 antibodies. (B) 293 human embryonic kidney cells were transfected with 1 or 10 μg, as indicated by the triangles, of expression plasmids encoding HA-tagged p53 (lanes 2, 7, and 8) or HA-tagged p73α (lanes 4, 5, 9, and 10) or with 10 μg of the backbone expression plasmid (lanes 1 and 6). Cell extracts were prepared and immunoprecipitated with anti-HA (lanes 1 to 5) or anti-E1B 55K antibodies (lanes 6 to 10). Specifically bound proteins were resolved by electrophoresis in an SDS–10% polyacrylamide gel and detected by immunoblotting with anti-HA antibody (12CA5) by using a horseradish peroxidase-conjugated antimouse antibody and enhanced chemiluminescence. The asterisks indicate nonspecific bands.

FIG. 7

FIG. 7

E6 targets p53, but not p73, for degradation in vitro. (A) p53, p73α, and p73β 35S-labelled in vitro translation products were incubated with immobilized GST-E6 (lanes 4 to 12) or GST (lanes 13 to 15) as indicated. Each binding reaction mixture contained 40 μl (lanes 4, 7, 10, and 13 to 15), 20 μl (lanes 5, 8, and 11), or 10 μl (lanes 6, 9, and 12) of in vitro translation product. Specifically bound proteins were resolved by SDS-polyacrylamide gel electrophoresis and detected by fluorography. Five microliters of the indicated in vitro translation product was loaded directly in lanes 1 to 3. (B) p53, p73α, and p73β 35S-labelled in vitro translation products were incubated at 30°C with a lysate prepared from bacteria producing GST-E6 (right panel) or GST-E4 (left panel). The proteins were then resolved by electrophoresis in an SDS–10% polyacrylamide gel and detected by fluorography.

FIG. 8

FIG. 8

Differential binding of p53 and p73 to SV40 T. (A) COS cells, which stably produce SV40 T, were transfected with expression plasmids encoding the indicated HA-tagged proteins or with the backbone expression plasmid (Mock). Cell extracts were prepared and immunoprecipitated (IP) with anti-HA (lanes 1 to 5) or anti-T (lanes 6 to 10) antibodies. Specifically bound proteins were resolved by electrophoresis in an SDS–10% polyacrylamide gel and detected by immunoblotting with anti-HA antibody (12CA5) by using a horseradish peroxidase-conjugated antimouse antibody and enhanced chemiluminescence. (B) SAOS2 cells were transfected a CAT reporter plasmid containing a minimal promoter consisting of two p53-binding sites upstream of a TATA box together with expression plasmids for wild-type p53 or p73α. In each case, the amount of CAT activity, in the absence of T antigen, was set to 100%. Where indicated, the cells were also transfected with a plasmid encoding wild-type (wt) or mutant (mut) T antigen. Error bars represent one standard deviation.

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