Death-associated protein kinase (DAPK) is a death domain-containing serine/threonine kinase, and participates in various apoptotic paradigms. Here, we identify the extracellular signal-regulated kinase (ERK) as a DAPKinteracting protein. DAPK interacts with ERK through a docking sequence within its death domain and is a substrate of ERK. Phosphorylation of DAPK at Ser 735 by ERK increases the catalytic activity of DAPK both in vitro and in vivo. Conversely, DAPK promotes the cytoplasmic retention of ERK, thereby inhibiting ERK signaling in the nucleus. This reciprocal regulation between DAPK and ERK constitutes a positive feedback loop that ultimately promotes the apoptotic activity of DAPK. In a physiological apoptosis system where ERK-DAPK interplay is reinforced, downregulation of either ERK or DAPK suppresses such apoptosis. These results indicate that bidirectional signalings between DAPK and ERK may contribute to the apoptosis-promoting function of the death domain of DAPK.

The synthesis of dTDP is unique because there is a requirement for thymidylate kinase (TMPK). All other dNDPs including dUDP are directly produced by ribonucleotide reductase (RNR). We report the binding of TMPK and RNR at sites of DNA damage. In tumor cells, when TMPK function is blocked, dUTP is incorporated during DNA double-strand break (DSB) repair. Disrupting RNR recruitment to damage sites or reducing the expression of the R2 subunit of RNR prevents the impairment of DNA repair by TMPK intervention, indicating that RNR contributes to dUTP incorporation during DSB repair. We identified a cell-permeable nontoxic inhibitor of TMPK that sensitizes tumor cells to doxorubicin in vitro and in vivo, suggesting its potential as a therapeutic option.

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G 2 phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G 1 /S progression, resulting in a smaller G 2 fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53 ؊/؊ cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G 2 arrest.The synthesis of dTTP is highly regulated and is important for DNA replication and repair in all living cells (1, 2). There are two pathways for dTTP synthesis in cells. In the de novo pathway, ribonucleotide reductase, which is composed of two pairs of R1 and R2 subunits, converts CDP and UDP to dCDP and dUDP, respectively. Both dCDP and dUDP can be metabolically converted to dUMP, and thymidylate synthase (TS) 3 catalyzes the reaction of dTMP formation from dUMP. In the salvage pathway, thymidine kinases (TKs), TK1 in cytosol and TK2 in mitochondria (3, 4), are responsible for dTMP production from thymidine (5). Phosphorylation of dTMP from either the salvage or de novo pathway to dTDP is catalyzed by thymidylate kinase, and nucleoside diphosphate kinase then converts dTDP to dTTP (6). Unlike TK2, the expression of TK1, TS, and thymidylate kinase is cell cycle-dependent (7-13).In response to DNA damage, cells trigger multifaceted responses such as cell cycle arrest, DNA repair, or apoptosis (14, 15). In Saccharomyces cerevisiae, it has been reported that DNA damage by ␥-irradiation, UV, or methyl methane sulfonate leads to increases in the levels of the four dNTPs through ribonucleotide reductase-mediated de novo synthesis, indicating a close relationship between the regulation of dNTP synthesis and DNA damage response (16,17). In mammalian cells, expression of p53-inducible R2 (p53R2), a homolog of the R2 subunit, is increased due to p53-dependent transcriptional activation upon DNA damage, suggesting a master role of p53 in integrating regulation of dNTP pools through the de novo pathway (18 -21). However, it is known that more than 50% human cancer cells harbor mutated or deleted p53, and these tumors are more resistant to chemotherapy due to the loss of p53-dependent apoptosis (22,23). This evoked the question as how p53-deficie...

Abbreviations used in this paper: FN, fi bronectin; MLC, myosin light chain; PTP, protein tyrosine phosphatase; RBD, rho binding domain; ROCK, rho kinase; SRE, serum-responsive element; SRF, serum response factor.The online version of this paper contains supplemental material.

Treatment of cells with phorbol ester, phorbol-12-myristate-13-acetate(PMA), triggers differentiation or apoptosis, depending on the cell type. In this study, we used an erythroblastic cell line, TF-1, to investigate the molecular mechanism that determines the cell fate in response to PMA exposure. Upon PMA treatment in the presence of serum or lysophosphatidic acid (LPA),TF-1 cells exhibited contraction followed by apoptosis. By contrast, under serum-free conditions, cells became adherent and survived after PMA treatment. Here, we show that the pathway of Rho kinase (ROCK)/myosin light chain (MLC)phosphorylation/myosin-mediated contraction was activated in PMA-induced apoptotic cells in serum-containing medium, but not in the adherent and survived cells. Pretreatment of cells with a specific ROCK inhibitor, Y27632,not only abrogated MLC phosphorylation and membrane contraction, but also prevented PMA-induced activation of caspase-3 and subsequent cell death,indicating that ROCK-dependent myosin-mediated contraction elicits an upstream signal required for caspase-3 activation in PMA-induced apoptosis. Interestingly, we further found that caspases-8 and -10 are the initiator caspases in PMA-induced apoptosis and a ROCK-dependent enhancement of specific complex formation between the Fas-associated death domain (FADD) and pro-caspase-10 in pro-apoptotic cells. In summary, these results revealed that, following PMA treatment, the upregulation of the RhoA/ROCK pathway contributes to a cellular context that switches-on myosin-mediated contraction, which provides a mechanism for triggering apoptotic induction mediated by caspase-8 and -10.