Figure 1.
p19 phosphorylation is induced in response to DNA damage.
(A, B) WI-38 fibroblasts were labeled with [32P]-orthophosphate and treated with β-amyloid peptide (20 µM), cisplatin (10 µM) or UV light (4 mJ/cm2) for the indicated times. Equal amounts of whole cell extracts were subjected to immunoprecipitation with anti-p19 antibody and the immune complexes were analyzed by SDS-PAGE and autoradiography (upper panels; P-p19, phosphorylated p19) or immunoblotting (lower panels; p19). (C; Control, untreated cells).
Figure 2.
Sequential phosphorylation of p19 at S76 and T141 following DNA damage.
(A, B) Phosphorylation ability of p19 mutants. WI-38 fibroblasts were transfected with expression vectors encoding the V5 epitope tag in frame with wild type p19 (p19wt) or p19 mutants, in which the potential phosphorylation sites were replaced by alanine (p19S13A, p19S66A, p19S76A, p19T89A, p19T141A). Transfected cells were labeled with [32P]-orthophosphate, treated with UV light (4 mJ/cm2) and collected 3 hours after treatment. Extracts were subjected to immunoprecipitation with anti-V5 antibody and analyzed by autoradiography (upper panels, P-p19) or immunoblotting (lower panels, V5). Unstransfected cells were used as a control to monitor immunoprecipitation specificity. (C) In a similar experiment, in vivo phosphorylation of two p19 phosphomimetic mutants (p19S76E and p19S76E/T141E) was tested. (D, E) Structural changes promoted by the sequential phosphorylation were analyzed by molecular dynamics simulation. The images show the comparison between the structures of p19 (cyan) and p19 phosphorylated on S76 (p19p, red) (D), or between p19p and p19 phosphorylated on sites S76 and T141 (p19pp, yellow) (E). Graphs show CA-distance between p19 and p19p (D), or p19p and p19pp (E) average structures. Arrows indicate domains with predicted structural changes. (ANK 1–5, ankyrin domains 1 to 5).
Figure 3.
ATM/ATR signaling pathways are differentially involved in p19 phosphorylation.
(A) Inhibition of p19 phosphorylation by caffeine treatment. WI-38 fibroblasts were incubated with caffeine (5 mM) for 1 hour, then treated with cisplatin (10 µM) or β-amyloid peptide (20 µM) for the indicated times and endogenous p19 phosphorylation analyzed by autoradiography. (B) Evaluation of ATM/ATR involvement in p19 phosphorylation by wortmannin treatment. WI-38 fibroblasts were incubated with the indicated doses of wortmannin for 1 hour, followed by treatment with cisplatin (10 µM) or β-amyloid peptide (20 µM) for 2 hours. (C) Effect of Chk1 and Chk2 inhibitors on p19 phosphorylation. WI-38 fibroblasts were incubated with SB-218078 (SB, 15 nM) or dopamine ß-hidroxylase inhibitor (DBH, 3 µM), both Chk1 inhibitors, or with Chk2 Inhibitor Calbiochem (ICHK2, 20 nM) for 1 hour before treatment with UV light (4 mJ/cm2), cisplatin (10 µM) or β-amyloid peptide (20 µM). After 2 hours, cell extracts were analyzed as in A.
Figure 4.
CDK2 and PKA participate in p19 sequential phosphorylation.
(A) CDK and PKA involvement in endogenous p19 phosphorylation. WI-38 fibroblasts were incubated with roscovitine (RSC, 10 µM), or with H-89 (1 µM) for 1 hour before the damaging treatments (4 mJ/cm2 UV light, 10 µM cisplatin or 20 µM ß-amyloid peptide). p19 phosphorylation was analyzed by autoradiography. (B, C) Effect of CDK and PKA inhibition on the phosphorylation of T141 mutants. WI-38 cells were transfected with the indicated p19 constructs expression plasmids, incubated with roscovitine or H-89 for 1 hour and then treated with UV light (4 mJ/cm2) or β-amyloid peptide (20 µM) for 2 hours. p19wt or the mutants were immunoprecipitated with anti-V5 antibody and the immunocomplexes were analyzed by autorradiography and immunoblotting. (D) Measurement of CDK1 and CDK2 activities in the phosphorylation process of endogenous p19. WI-38 fibroblasts were incubated for 24 hours with specific CDK1 or CDK2 antisense oligonucleotides before treatment with UV radiation (4 mJ/cm2). After 2 hours, p19 was immunoprecipitated and phosphorylation observed by autoradiography as mentioned before (upper panel). Northern blot results show the efficiency of the antisense oligonucleotides (lower panel).
Figure 5.
CDK2 and PKA phosphorylates p19 in vitro.
(A, B) S76 and T141 as suitable sites for CDK2 and PKA action. Two synthetic peptides containing the sequence in which S76 (p-S76) or T141 (p-T141) are positioned, were used to performed in vitro kinase assays. p-S76 or p-T141 peptides were incubated with CDK2 (immunoprecipitated from HEK-293 cells) or the catalytic subunit of PKA (cPKA, purified from bovine heart), respectively. A histone H1 peptide (p-H1) or kemptide (Kemp) were used as specific subtrates for CDK2 and PKA, respectively, as a control of enzymatic activity. Kinase activity specificity was tested by substituting one substrate to the other. Measurements were done in triplicates and bars show the mean ± s.e.m. (n = 3). (C) CDK2 phosphorylates p19. In vitro kinase assays were performed using immunoprecipitated CDK2 and recombinant GST-p19. Histone H1 was used as a control for CDK2 activity. (D) PKA phosphorylates p19. In vitro kinase assays were performed using cPKA and recombinant GST-p19 as substrate, with or without H-89 inhibitor. CREB protein was used as a control for cPKA activity (E) Analysis of the interaction between PKA and p19 in vivo. Co-immunoprecipitation assays were performed transfecting p19-V5 (p19wt) in WI-38 cells. Cells were irradiated with UV light. At the indicated times following irradiation treatment cells were collected and the extracts immunoprecipitated with anti-V5 antibody (IP:V5). The immune complexes were analyzed by immunoblot with anti-cPKA and anti-V5 antibodies. Expression of p19-V5 and cPKA was analyzed in the inputs by immunoblot.
Figure 6.
DNA damage induced p19 nuclear translocation is dependent on S76 phosphorylation.
(A) Distribution of phosphorylated p19 in the cytoplasmic and nuclear fractions after DNA damage. In vivo phosphorylation assays were performed in WI-38 fibroblasts. Cells were treated with UV (4 mJ/cm2), collected at the indicated times, and the extracts subjected to a subcellular fractionation protocol. Either the cytoplasmic (C) or nuclear fractions (N) were immunoprecipitated with anti-p19 antibody, and the immunocomplexes analyzed by SDS-PAGE and autoradiography (upper panel). (B) Subcellular distribution of the phosphorylation deficient mutant p19T141A. For in vivo phosphorylation assays, WI-38 cells were transfected with p19wt or p19T141A, treated with UV radiation and collected at the indicated times. After subcellular fractionation, extracts were immunoprecipitated with an anti-V5 antibody and analyzed as in (A). p19wt or p19T141A subcellular distributions were also studied by immunoblot (C) Subcellular localization of endogenous deficiently phosphorylated-p19 after PKA inhibition. For in vivo phosphorylation assays, cells were processed as in (A) but, before UV irradiation, they were incubated with H-89 for 1 hour. Endogenous distribution of p19 was also studied by immunoblot. (D) Subcellular localization of p19S76A mutant following DNA damage. WI-38 cells were transfected with p19S76A and treated with UV radiation. At the indicated times, extracts were prepared by subcellular fractionation and analized by immunoblot with anti V5-antibody.
Figure 7.
Phosphorylation of serine 76 and threonine 141 is required for p19 function linked to the response to DNA damage
(A) DNA repair ability of cells overexpressing p19wt or p19 phosphorylation deficient mutants. WI-38 fibroblasts were transfected with p19wt or the indicated p19 mutants. Cells were maintained in an arginine-free medium containing 1% fetal bovine serum during 48 h, damage with 4 mJ/cm2 UV and incubated with [3H]-thymidine. Following 10 h, cell lysates were tested for Unscheduled DNA Synthesis assay (UDS). Bars represent the mean ± s.e.m of three independent experiments performed in triplicate. Student's t-test was used to compare UV-treated control sample (none) with UV-treated p19wt or p19 mutant samples. (*p<0,005). Protein expression was analyzed by immunoblot. (B) Similarly as in (A) but overexpressing the phosphomimetic p19 mutants. (C) UV-dependent apoptotic response of cells overexpressing p19wt or phosphorylation deficient mutants of p19. WI-38 fibroblasts were transfected with p19wt or the indicated p19 mutants. Twelve hours following UV irradiation, cell lysates were tested for caspase-3 activity. Results are expressed as percentage of caspase-3 activity with respect to basal activity of cell lysates nontransfected and without UV-treatment, which was set to 100. Bars represent the mean ± s.e.m. of three independent experiments performed in triplicate. Student t-test was used to compare, UV-treated control sample (none) with UV-treated p19wt or p19 mutant samples (*p<0.005). (D) Similarly as in (C) but overexpressing the phosphomimetic p19 mutants.