Genotoxic stress exerts biological activity by activating downstream effectors, including the p53 tumor suppressor. p53 regulates cell-cycle checkpoint and induction of apoptosis in response to DNA damage; however, molecular mechanisms responsible for committing to these distinct functions remain to be elucidated. Recent studies demonstrated that phosphorylation of p53 at Ser46 is associated with induction of p53AIP1 expression, resulting in commitment to apoptotic cell death. In this regard, the role for Ser46 kinases in p53-dependent apoptosis has been established; however, the kinases responsible for Ser46 phosphorylation have yet to be identified. Here, we demonstrate that the dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) directly phosphorylates p53 at Ser46. Upon exposure to genotoxic stress, DYRK2 translocates into the nucleus for Ser46 phosphorylation. Consistent with these results, DYRK2 induces p53AIP1 expression and apoptosis in a Ser46 phosphorylation-dependent manner. These findings indicate that DYRK2 regulates p53 to induce apoptosis in response to DNA damage.

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The tumor suppressor p53 is a transcription factor that regulates cell cycle, DNA repair, senescence, and apoptosis in response to DNA damage. Phosphorylation of p53 at Ser-46 is indispensable for the commitment to apoptotic cell death. A previous study has shown that upon exposure to genotoxic stress, DYRK2 translocates into the nucleus and phosphorylates p53 at Ser-46, thereby inducing apoptosis. However, less is known about mechanisms responsible for intracellular control of DYRK2. Here we show the functional nuclear localization signal at N-terminal domain of DYRK2. Under normal conditions, nuclear and not cytoplasmic DYRK2 is ubiquitinated by MDM2, resulting in its constitutive degradation. In the presence of proteasome inhibitors, we detected a stable complex of DYRK2 with MDM2 at the nucleus. Upon exposure to genotoxic stress, ATM phosphorylates DYRK2 at Thr-33 and Ser-369, which enables DYRK2 to escape from degradation by dissociation from MDM2 and to induce the kinase activity toward p53 at Ser-46 in the nucleus. These findings indicate that ATM controls stability and pro-apoptotic function of DYRK2 in response to DNA damage.Dual-specificity tyrosine-regulated kinases (DYRKs) 3 are a novel subfamily of protein kinases that catalyze their autophosphorylation on tyrosine residues and the phosphorylation of serine/threonine residues on exogenous substrates (1-3). DYRK2 shares a conserved kinase domain and adjacent N-terminal DH box but does not contain a C-terminal PEST (the proline-, glutamic acid-, serine-and threonine-rich) domain. DYRK2 is presumed to be involved in regulating key developmental and cellular processes such as neurogenesis, cell proliferation, cytokinesis, and cellular differentiation. Recent findings have shown that DYRK1A and DYRK2 phosphorylate NFATc, which regulates calcium signaling, to lead NFATc inactivation by its cytoplasmic sequestration (4, 5).Upon exposure to genotoxic stress, p53 is stabilized and activated by phosphorylation at Ser-15 and Ser-20 to regulate a cell cycle checkpoint and DNA repair. In case of the lesion for irreparable DNA damage, p53 induces apoptotic cell death by a mechanism in which an additional phosphorylation increases the binding affinity of p53 to promoters of pro-apoptotic genes, such as p53AIP1. In this context previous studies have established the mechanism in which p53 transactivates p53AIP1 by its additional phosphorylation at Ser-46; thereby, this phosphorylation is essential for p53-dependent apoptosis (6, 7). We recently demonstrated that DYRK2 is a novel Ser-46 kinase (8 -10). Ataxia telangiectasia mutated (ATM) was involved in DYRK2 activation and Ser-46 phosphorylation. Furthermore, DYRK2 accumulated in the nucleus after DNA damage. Significantly, DYRK2 phosphorylation of Ser-46 was associated with the induction of apoptosis. These findings provide a novel signaling mechanism in which phosphorylation of p53 at Ser-46 by DYRK2 regulates apoptotic cell death in response to DNA damage. Although certain insights are, thus, available regardin...

Upon exposure to genotoxic stress, the c-Abl tyrosine kinase is released from cytoplasmic 14-3-3 proteins and then is targeted to the nucleus. Phosphorylation of Thr735 in c-Abl is critical for binding to 14-3-3; however, kinases responsible for this phosphorylation are unknown. Here, we identify CLK1, CLK4, MST1, MST2 and TTK (also known as Mps1) as novel Thr735 kinases in vitro by expression cloning strategy using phosphospecific antibody. We also demonstrate that ectopic expression of these kinases is capable for phosphorylation of Thr735 in cells. Importantly, upon exposure to oxidative stress, phosphorylation of Thr735 is transiently upregulated, and the status of this phosphorylation remains unchanged in cells silenced for CLK1, CLK4, MST1 or MST2. By contrast, knockdown of TTK attenuates phosphorylation of Thr735, suggesting that TTK is a physiological kinase that phosphorylates Thr735. In concert with these results, we show that, in cells silenced for TTK, c-Abl is accumulated in the nucleus even in unstressed condition and no further targeting into the nucleus occurs after oxidative stress. Moreover, nuclear entrapment of c-Abl by knocking down TTK enhances oxidative stress-induced apoptosis. These findings provide evidence that TTK phosphorylates c-Abl at Thr735 and that this phosphorylation is of importance to the cytoplasmic sequestration of c-Abl.

Upon DNA damage, tumor suppressor p53 determines cell fate by repairing DNA lesions to survive or by inducing apoptosis to eliminate damaged cells. The decision is based on its posttranslational modifications. Especially, p53 phosphorylation at Ser46 exerts apoptotic cell death. However, little is known about the precise mechanism of p53 phosphorylation on the induction of apoptosis. Here, we show that amphiregulin (AREG) is identified for a direct target of Ser46 phosphorylation via the comprehensive expression analyses. Ser46-phosphorylated p53 selectively binds to the promoter region of AREG gene, indicating that the p53 modification changes target genes by altering its binding affinity to the promoter. Although AREG belongs to a family of the epidermal growth factor, it also emerges in the nucleus under DNA damage. To clarify nuclear function of AREG, we analyze AREG-binding proteins by mass spectrometry. AREG interacts with DEAD-box RNA helicase p68 (DDX5). Intriguingly, AREG regulates precursor microRNA processing (i.e., miR-15a) with DDX5 to reduce the expression of antiapoptotic protein Bcl-2. These findings collectively support a mechanism in which the induction of AREG by Ser46-phosphorylated p53 is required for the microRNA biogenesis in the apoptotic response to DNA damage. microarray | Drosha | miRNA processing

Evasion from apoptotic cell death is a characteristic of cancer; genes that modulate this process may be optimal for therapeutic attack. Identifying key regulators of apoptosis is thus a central goal in cancer therapy. Here, we describe a loss-of-function screen that uses RNA interference libraries to identify genes required for induction of apoptosis. We used a short-hairpin RNA expressing vector with high gene-expression silencing activity that contained fetal brain cDNAs. Survived cells from genotoxic stress were isolated to determine knock-down of molecules that are crucial for induction of apoptosis. We identified TBP-associated factor 1 (TAF1), a gene previously implicated as an essential component of transcription machinery. Depletion of TAF1 was associated with substantial attenuation of apoptosis induced by oxidative as well as genotoxic stress. Microarray analysis further demonstrated that a number of genes were transcriptionally declined in cells silenced for TAF1. Surprisingly, knocking down TAF1 exhibited a marked decrease in p27Kip1 expression, allowing cells resistant from oxidative stress-induced apoptosis. These results suggest that TAF1 regulates apoptosis by controlling p27Kip1 expression. Our system provides a novel approach to identifying candidate genes that modulate apoptosis.

The tumor suppressor gene p53 regulates apoptosis in response to DNA damage. Promoter selectivity of p53 depends on mainly its phosphorylation. Particularly, the phosphorylation at serine-46 of p53 is indispensable in promoting pro-apoptotic genes that are, however, poorly determined. In the current study, we identified palmdelphin as a pro-apoptotic gene induced by p53 in a phosphorylated serine-46-specific manner. Upregulation of palmdelphin was observed in wild-type p53-transfected cells, but not in serine-46-mutated cells. Expression of palmdelphin was induced by p53 in response to DNA damage. In turn, palmdelphin induced apoptosis. Intriguingly, downregulation of palmdelphin resulted in necroptosis-like cell death via ATP depletion. Upon DNA damage, palmdelphin dominantly accumulated in the nucleus to induce apoptosis. These findings define palmdelphin as a target of serine-46-phosphorylated p53 that controls cell death in response to DNA damage.