In vivo phosphorylation of T81 in response to stress and DNA damage. (a) NHF cells were subjected to the indicated treatments (heat shock [HS], 42°C for 1 h; UV-C (254 nm), 10 or 50 J/m² [UV]; H₂O₂ [HP], 100 μM; cis-platinum [CP], 0.6 μM; sorbitol [S], 0.6 M), and proteins were prepared 1 h later unless indicated otherwise. The quantities of p53 used for the Western blotting were prenormalized to assure equal loading of p53 and thus do not reflect changes in the amount of p53 seen after these treatments. Upper lane, analysis with antibodies to phospho-T81; lower lane, analysis with DO1 antibodies to p53. (b) JNK activity in the protein samples analyzed in panel a, upper lane (1-h time point), was monitored via the solid-phase kinase reaction using GST-Jun^1–87 as a substrate.

Activity of p53 mutated at T81. (a) p53 null cells were transfected with p21^WAF1/CIP1-Luc, a constitutively active form of MEKK1 or JNKK2 and wt or mutant forms of p53. (Inset) Expression levels of the p53 forms in extracts used for transcriptional analysis. Basal transcriptional activities of wt p53 were five times higher than those seen with either the T81A mutant or empty vector. The data shown reflect fold increases over basal activity of the construct. C, control; M, ΔMEKK1; J, JNKK^CAA. (b) Comparison of T81A transcriptional activities with those of different transcriptionally dead p53 mutants. Mouse 10.1 p53 null cells were transfected with p21^WAF1/CIP1-Luc and wt or mutant forms of p53. Cells were treated with UV (15 J/m²) 36 h later, and proteins were prepared after an additional 6 h. Analysis was carried out as indicated in panel a. Bottom, Western blots of proteins prepared from the same experiment using antibodies to p53 and p21^WAF1/CIP. (c) p53 vectors and GFP were transfected into 10.1 cells, and 24 h later cells were treated with Taxol or nocodazole (5 μg/ml). PI staining and flow cytometry analyses of GFP⁺ cells were performed 16 h after treatment as detailed in Materials and Methods. (d) H1299 cells were transfected with the constructs and 24 h later were treated with anisomycin (10 μg/ml) or UV-C irradiation (15 J/m²). Analysis of cell death was performed after an additional 36 h. The fraction of dead cells shown includes annexin-V- and PI-positive cells, thereby including apoptotic and necrotic death. (e) 3T3 mouse fibroblasts were transfected with P7 (corresponding to the JNK binding site on p53) or C7 (P7 sequence in scrambled order) cloned into pcDNA3_HA. UV irradiation was administered 24 h later. The degree of apoptosis was calculated 24 or 72 h after irradiation based on fluorescence-activated cell sorter analysis of PI-positive cells. The inset depicts immunocytochemistry analysis of P7 expression.

Cellular localization of wt and T81A p53 forms. p53 null 10.1 cells were transfected with wt p53, p53 plus JNKK^CAA, p53 plus JNKK^CAA plus MKP5, p53^T81A, p53^T81A plus JNKK^CAA, or p53^T81A plus JNKK^CAA plus MKP5. Cells were immunolabeled with a rabbit polyclonal antibody to p53 (FL-393) (Santa Cruz Biotechnology) (diluted 1:500) followed by detection with fluorescein-conjugated anti-rabbit immunoglobulin G (heavy plus light chains) (Vector Laboratories). Cells were illuminated with the 488-nm line of the argon laser of a Leica TCS-SP (UV) confocal laser scanning microscope and examined using a 100×, 1.4 -numerical-aperture objective lens. For wt p53 (top three panels), labeling is restricted primarily to the nucleus (excluding the nucleoli) and nuclear bodies. Little or no cytoplasmic labeling is seen. For p53^T81A (bottom three panels), labeling is divided between the nucleus and the cytoplasm. The lower left panel shows a pair of cells that are completing division. In the lower right panel, p53^T81A is divided evenly between the nucleus and the cytoplasm in cells that express the protein at higher levels; at lower expression levels, it is found primarily in the nucleus. In all cases, p53 labeling is restricted from the nucleolus. Bars, 10 nm

JNK phosphorylates p53 on T81. (a) Wild-type or mutant (T81A) forms of human p53 were in vitro translated, purified, and subjected to phosphorylation using JNK immunopurified from UV-treated 293T cells. Middle, immunoblot analysis of material subjected to autoradiography using DO1 antibodies. The doublet represents the nonphosphorylated (lower band) and phosphorylated (upper band) forms of p53. The control reaction using c-Jun as a substrate (immunokinase [IK] reaction of GST-Jun) and the level of HA-JNK2 that was immunoprecipitated (IP-HA-JNK2) are shown (bottom). (b) Proteins prepared from NHF that were exposed to sham stress (control) or heat shock (HS; 42°C for 1 h) were subjected to immunoblot analysis using the antibodies to phosphorylated T81 in the presence of excess phospho or nonphospho peptides as indicated (top) or to Western blotting using DO1 antibodies (bottom). (c) T81 phosphorylation on wt or T81A forms of p53 was monitored after cotransfection of the respective construct (1 μg) with the indicated upstream kinases (3 μg) into H1299 cells. Analysis of T81 phosphorylation was carried out using the antibodies to phospho-T81. Bottom, p53 levels detected with DO1 antibodies. (d) T81 phosphorylation of p53 impaired in JNK association. Either wt rat p53 or p53 with deletions (ΔP7) or mutations (at aa 101, 105, and 108) within the region, which encompasses the JNK binding site (1, 18) was cotransfected with JNK2^CAA into H1299 cells. Proteins were prepared and subjected to Western blot analysis using phospho-T81 antibodies (top) or DO1 antibodies (bottom).

Stability of p53 mutated at T81. Pulse-chase analysis of p53 half-life was performed as indicated in Materials and Methods using 10.1 p53 null cells that had been transfected with either wt or T81A forms of p53. At the indicated time points, p53 was immunoprecipitated with DO1 antibodies followed by autoradiography. The half-lives of p53 under nonstressed conditions and in response to UV irradiation or following JNK activation (forced expression of JNKK2^CAA) are shown. Bottom, corresponding densitometry analysis, which was carried out in three independent experiments.

JNK phosphatase attenuates T81 phosphorylation and p53 activities. (a) p53 null cells were transfected with p53, a constitutively active form of JNKK, and different concentrations of MKP5. Cells were exposed to 45 J of UV-C/m², as indicated. Top, specific phosphorylation pattern of T81. Phosphorylation on serine 15 after UV treatment and the expression of p53 are shown. Controls revealing the expression of exogenously transfected MKP5-phosphorylated and nonphosphorylated forms of JNK1 and -2 are also shown. (b) p53 null cells were transfected with wt or T81A mutant forms of p53 together with JNKK^CAA and MKP5 as indicated. Analysis for expression of p53 and p53 phosphorylation on T81 and a control analysis for p21^WAF1/CIP1 expression and JNK phosphorylation (and nonphosphorylation) levels were carried out 36 h after transfection. (c) p53 null cells were transfected with either p21-luc or Bax-luc constructs, together with the indicated vectors and β-Gal. For transfection efficiency lysates were normalized with respect to β-Gal activity and subjected to a luciferase assay. The diagram reflects changes in luciferase activity in six independent transfections. (d) 10.1 cells were transfected with the indicated constructs together with GFP, and 24 h after transfection cells were treated with 45 J of UV-C PI staining and flow cytometry analysis of GFP⁺ cells were performed 16 h after UV treatment. The diagram shows changes in apoptotic cell death after six independent transfections.

JNK antisense oligonucleotides attenuate T81 phosphorylation in vivo. MCF7 cells were transfected with either specific antisense oligonucleotides directed against JNK1 and JNK2 or nonspecific antisense oligonucleotides (250 nM each). Forty-eight hours after transfection, cells were either sham treated or UV treated and harvested 2 h after treatment. Top, specific T81 phosphorylation; middle, expression level of p53 in response to indicated treatments; bottom, expression of JNK1 and -2 in the presence or absence of transfected oligonucleotides. Control lane, no treatment; mock lipofection, cells maintained in concentrations of Lipofectin equal to those for the oligonucleotide-transfected samples; JNK1AS and JNK2AS, JNK antisense oligonucleotides that were transfected; JNKScr, control “scrambled” oligonucleotides.