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

et al. (2012) CDK2 and PKA Mediated-Sequential Phosphorylation Is Critical for p19INK4d Function in the DNA Damage Response. PLoS ONE 7(4): e35638. doi:10.1371/journal.pone.0035638 CDK2 and PKA Mediated-Sequential Phosphorylation Is Critical for p19INK4d Function in the DNA Damage Response Mariela C. Marazita 0 M. Florencia Ogara 0 Silvina V. Sonzogni 0 Marcelo Mart 0 Nelson J. Dusetti 0 Omar P. Pignataro 0 Eduardo T. Ca nepa 0 Ferenc Gallyas, University of Pecs Medical School, Hungary 0 1 Laboratorio de Biolog a Molecular, Departamento de Qu mica Biolo gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires , Ciudad de Buenos Aires, Argentina, 2 INSERM, U624 Stress Cellulaire, Marseille , France , 3 Laboratorio de Endocrinolog a Molecular y Transduccio n de sen ales, Instituto de Biolog a y Medicina Experimental-CONICET , Ciudad de Buenos Aires , Argentina 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, b-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 32Porthophosphate. 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. - Funding: This work was supported by research grants from Consejo Nacional de Investigaciones Cientficas y Tecnicas (CONICET), Agencia Nacional de Promocio n Cientfica y Tecnol ogica (ANCYPT) and Universidad de Buenos Aires. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. DNA damage response (DDR) mechanisms are essential for maintaining genomic integrity and an accurate transmission of genetic information. DDR consists of an intricate signaling network in which complex DNA surveillance programs play a key role [13]. These control programs or checkpoints respond to a variety of lesions including stalled replication forks and DNA damage induced by both internal and external sources like reactive cellular metabolites, ionizing or UV radiation and chemotherapeutic agents [2,4,5]. After sensing the damage, the activation of the checkpoints modulate cell cycle arrest, DNA repair systems and cell death mechanisms to repair or to eliminate hazardous, genetically unstable cells [6,7]. Although DDR components have not yet been completely described the canonical checkpoint signaling is composed by two major transduction pathways initiated by the upstream PI3K-like kinases Ataxia-telangiectasia Mutated (ATM) and ATM and Rad3-related (ATR). ATM is predominantly activated by double strand break lesions (DSBs) while ATR responds fundamentally to single strand breaks or bulky lesions. ATM and ATR activate their downstream kinases Chk1 and Chk2 amplifying the initial signal and modulating the G1/S, intra-S and G2/M checkpoints [4,8]. While ATM and ATR were initially reported to activate Chk2 and Chk1 respectively, this concept was challenged by studies that show crosstalks between these kinases [9]. Chk1 activation by ATM was reported in cells exposed to ionizing radiation treatment [10,11] and ATM and ATR were required for Chk2 activation in response to replication stress [12]. Moreover, it was shown that both ATR and ATM were able to target the SQ-rich C terminus of Chk1 on serine 317 and 345 leading to its activation [10,1315]. Following Chk1 and Chk2 activation, these kinases phosphorylate a wide range of downstream effectors which prevent further progression through the cell cycle and initiate DNA repair mechanisms but also modulate the trigger of cell death pathways if the insult exceeds the repair capacity [2,16]. Among these effector proteins, Chk1 phosphorylates TLK12 and RAD51, while BRCA, PIK3, PML and E2F1 are Chk2 substrates. They also share target proteins like Mdm2, p53, cdc25A and cdc25C [5,1720] The cell cycle progression is driven by the activity of cyclindependent kinases (CDKs) and is negatively regulated by INK4 and Cip/Kip inhibitory proteins [2124]. INK4 family consists of four members, p16INK4a, p15INK4b, p18INK4c and p19INK4d which play a redundant role as CDK4/6 inhibitors. However, novel cell cycle independent functions were recently described for some of them [25]. Interestingly, p16INK4a and p19INK4d (p19) were linked to the cellular response to genotoxic agents [2628]. In particular, extensive data points out that p19 is a critical factor in the maintenance of genomic integrity and cell survival. It was reported that UV light, cisplatin and b-amyloid peptide promoted p19INK4d transcriptional induction and nuclear translocation [27]. Adding to this, p19 overexpression significantly enhanced DNA repair and diminished apoptosis in different cell lines. More important, physiological p19 levels are necessary for an appropriate response to the damage. In this way, p19 deficient cells display an impaired DNA repair activity and enhanced apoptosis [2729]. Consistent with these findings, other studies described enhanced sensitivity of cells to apoptosis and autophagic cell death in p19 null mice [30]. p19 expression status directly correlates with cell resistance and survival to DNA damage. Finally, p19 activity protects from UV-induced chromosomal aberrations and spontaneous chromosome abnormalities as well [27,29]. These facts uncovered a novel p19 function in regulating genomic stability and overall cell viability under conditions of genotoxic stress. Despite these findings, the regulation of p19 activity in the DDR remains unknown. We hypothesized that post-translational modifications on p19 could be taking part in regulating this specific function. Here, we report (...truncated)