CDK2 and PKA Mediated-Sequential Phosphorylation Is Critical for p19INK4d Function in the DNA Damage Response

DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with 32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage

Phosphorylation of serine 76 and threonine 141 is required for p19 function linked to the response to DNA damage

<p>(<b>A</b>) 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 <i>t</i>-test was used to compare UV-treated control sample (none) with UV-treated p19wt or p19 mutant samples. (*<i>p</i><0,005). Protein expression was analyzed by immunoblot. (<b>B</b>) Similarly as in (<b>A</b>) but overexpressing the phosphomimetic p19 mutants. (<b>C</b>) 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 <i>t</i>-test was used to compare, UV-treated control sample (none) with UV-treated p19wt or p19 mutant samples (^*<i>p</i><0.005). (<b>D</b>) Similarly as in (<b>C)</b> but overexpressing the phosphomimetic p19 mutants.</p

p19 phosphorylation is induced in response to DNA damage.

<p>(<b>A, B</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).</p

ATM/ATR signaling pathways are differentially involved in p19 phosphorylation.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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.</p

CDK2 and PKA phosphorylates p19 <i>in vitro.</i>

<p>(<b>A, B</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 <i>in vitro</i> 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). (<b>C</b>) CDK2 phosphorylates p19. <i>In vitro</i> kinase assays were performed using immunoprecipitated CDK2 and recombinant GST-p19. Histone H1 was used as a control for CDK2 activity. (<b>D</b>) PKA phosphorylates p19. <i>In vitro</i> 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 (<b>E</b>) Analysis of the interaction between PKA and p19 <i>in vivo</i>. 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.</p

CDK2 and PKA participate in p19 sequential phosphorylation.

<p>(<b>A</b>) 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>B, C</b>) 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. (<b>D</b>) 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).</p

DNA damage induced p19 nuclear translocation is dependent on S76 phosphorylation.

<p>(<b>A</b>) Distribution of phosphorylated p19 in the cytoplasmic and nuclear fractions after DNA damage. <i>In vivo</i> 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>B</b>) Subcellular distribution of the phosphorylation deficient mutant p19T141A. For <i>in vivo</i> 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 (<b>A</b>). p19wt or p19T141A subcellular distributions were also studied by immunoblot (<b>C</b>) Subcellular localization of endogenous deficiently phosphorylated-p19 after PKA inhibition. For <i>in vivo</i> phosphorylation assays, cells were processed as in (<b>A</b>) but, before UV irradiation, they were incubated with H-89 for 1 hour. Endogenous distribution of p19 was also studied by immunoblot. (<b>D</b>) 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.</p

Whole genome sequencing reveals a de novo SHANK3 mutation in familial autism spectrum disorder.

Clinical genomics promise to be especially suitable for the study of etiologically heterogeneous conditions such as Autism Spectrum Disorder (ASD). Here we present three siblings with ASD where we evaluated the usefulness of Whole Genome Sequencing (WGS) for the diagnostic approach to ASD.We identified a family segregating ASD in three siblings with an unidentified cause. We performed WGS in the three probands and used a state-of-the-art comprehensive bioinformatic analysis pipeline and prioritized the identified variants located in genes likely to be related to ASD. We validated the finding by Sanger sequencing in the probands and their parents.Three male siblings presented a syndrome characterized by severe intellectual disability, absence of language, autism spectrum symptoms and epilepsy with negative family history for mental retardation, language disorders, ASD or other psychiatric disorders. We found germline mosaicism for a heterozygous deletion of a cytosine in the exon 21 of the SHANK3 gene, resulting in a missense sequence of 5 codons followed by a premature stop codon (NM_033517:c.3259_3259delC, p.Ser1088Profs*6).We reported an infrequent form of familial ASD where WGS proved useful in the clinic. We identified a mutation in SHANK3 that underscores its relevance in Autism Spectrum Disorder

DNA binding and chromatin recruitment of the GR mutants.

<p>(A) GR loading at the MMTV promoter array (white arrow) in the 3617 cell line (single cell analysis). Scale bar, 4 µm. (B–E) ChIPs using a GFP antibody in the 3134 cell line previously transfected with pEGFP-GR mutants. qPCR data as ChIP pulldown/input normalized to vehicle-treated cells (<i>n</i> = 4) for the MMTV promoter array (B) or endogenous GR binding sites, either <i>de novo</i> (C), pre-programmed (D), or nGRE (E). Means ± SEM from four independent experiments are shown. Bars with different superscript letters are significantly different from each other (<i>p</i><0.05). If at least one superscript letter is shared between treatments, then no significant differences were found.</p

GR does not change its oligomerization state in the presence of NF-κB.

<p>3617 cells transiently expressing the indicated combination of mCherry, mCherryGRwt, GFP, and/or GFP-P65 were treated with 300 nM Cort and/or 10 ng/ml TNF-α (TNF). (A) Fold-increase of the nuclear brightness (ε) relative to the control (total <i>n</i> = 215) for the green or red channels (color coded). Parenthesis means “in the presence of.” Bars with different superscript letters are significantly different from each other (<i>p</i><0.05). (B) subcelluar distribution of eGFP of mCherry in one representative cell for each condition. Scale bar = 10 µm. (C–D) Cross correlation analysis of the fluorescence fluctuations. The cross correlation brightness (Bcc) plot for each pixel (blue dots) of a representative nucleus as defined in Digman and colleagues <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001813#pbio.1001813-Digman2" target="_blank">[35]</a> as well as a histogram of the Bcc are shown.</p