ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1

Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.     

Proteins induced by DNA-damaging agents in cultured Drosophila cells

In Drosophila cultured cells, the effects of several DNA-damaging agents on the expression of proteins were investigated. Poly(A+) RNA prepared from both untreated cells and cells treated with DNA-damaging agents was translated in vitro. The translation products were analyzed by two-dimensional electrophoresis. Methyl methanesulfonate, the most potent agent used, induced about 25 proteins, some new and some enhanced pre-existing proteins. Angelicin plus near UV irradiation, 4-nitroquinoline N-oxide and ethyl methanesulfonate were efficient inducers. Mitomycin C, UV irradiation and hydrogen peroxide were poor inducers, inducing only a few proteins at low levels. A tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, and a DNA gyrase inhibitor, nalidixic acid, also were used. In this system they were weak inducers of new proteins. Several of the new or enhanced proteins were common to several agents, but others were agent specific. The distribution of mutagen-induced proteins was compared with that of proteins induced in cells heated at 37 degrees C. Some of the proteins induced by DNA-damaging agents were found to overlap heat-shock proteins. These results suggest that there are sets of induced genes that are regulated differently.     

Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents

UV and ionizing radiation (IR) activate DNA damage checkpoints and induce Cdc25A degradation (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429; Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J., and Lukas J. (2001) Nature 410, 842-847). The degradation of Cdc25A is abrogated by caffeine, which implicates Chk1 as the potential mediator (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429). However, the involvement of Chk1 is far from clear, because caffeine is a rather nonspecific inhibitor of the ATR/Chk1 signaling pathway. Additionally, it is not known whether DNA-damaging drugs commonly used in chemotherapy, which may activate different signal transduction pathways than UV or IR, also confer Cdc25A degradation. Herein, we show that camptothecin and doxorubicin, two widely used topoisomerase inhibitors conferring S and G2 arrest, respectively, cause the degradation of Cdc25A. Using a small interfering RNA that enables the specific elimination of Chk1 expression, we show that the observed proteolysis of Cdc25A is mediated through Chk1. Moreover, Cdc25A overexpression abrogates the Chk1-mediated degradation and overcomes the doxorubicin-induced G2 arrest through dephosphorylation and activation of Cdc2/Cdk1 in a dose-dependent manner. These results suggest that: (a) Cdc25A is involved in the G2/M transition in addition to its commonly accepted effect on G1/S progression, and (b) Chk1 mediates both S and G2 checkpoint and is thus a more ubiquitous cell cycle checkpoint mediator than previously thought.     

Distinct signaling pathways regulate TLR2 co-stimulatory function in human T cells

Toll-like receptor 2 (TLR2) serves as a co-stimulatory receptor for human T cells by enhancing T cell receptor (TCR)-induced cytokine production and proliferation. However, it is unknown where signals from the TCR and TLR2 converge to enhance T cell activation. To address this gap, we examined changes in TCR-induced signaling following concurrent TLR2 activation in human T cells. Both proximal TCR-mediated signaling and early NFκB activation were not enhanced by TCR andTLR2 co-activation, potentially due to the association of TLR2 with TLR10. Instead, TLR2 co-induction did augment Akt and Erk1/Erk2 activation in human T cells. These findings demonstrate that TLR2 activates distinct signaling pathways in human T cells and suggest that alterations in expression of TLR2 co-receptors may contribute to aberrant T cell responses.     

p21CIP1 is dispensable for the G2 arrest caused by genistein in human melanoma cells

We have investigated the effect of genistein on cell cycle distribution of the human choroidal melanoma cell line OCM-1. We report that this isoflavonoid arrested cells in G2. This effect was correlated with the induction of the CDK inhibitor p21CIP1. However, while CDK1 activity was markedly reduced following genistein treatment, CDK2 activity was not affected. This was in agreement with the absence of G1 arrest that we observed but caused some doubt about the functionality of p21CIP1. Attempts to demonstrate mutation or post-translational modification of p21CIP1 from OCM-1 cells were unsuccessful. In fact, the level of p21CIP1 induced by genistein was shown to be insufficient to cause CDK2 inhibition. The role of p21CIP1 in the inhibition of CDK1 was questionable, as we demonstrated that genistein impaired Tyr15 dephosphorylation of CDK1 and because CDK1-cyclin B1 complexes from treated cells could be reactivated upon exposure to CDC25 phosphatase. Finally, we report that p21CIP1 was not absolutely required for the genistein-induced G2 arrest, as the isoflavone caused at least partial G2 arrest in p21-deficient Rat-1 fibroblasts as well as in p21-/- mouse embryo fibroblasts.     

Signalling pathways evoked by alpha1-adrenoceptors in human melanoma cells

The biological effects of catecholamines in mammalian pigment cells are poorly understood, but in poikilothermic vertebrates they regulate the translocation of pigment granules. We have previously demonstrated in SK-Mel 23-human melanoma cells the presence of low affinity alpha(1)-adrenoceptors, which mediate a decrease in cell proliferation and increase in tyrosinase activity, with no change of tyrosinase expression. In this report, we investigated the signalling pathways involved in these responses. Calcium mobilization in response to phenylephrine (PHE), an alpha(1)-adrenergic agonist, was investigated by confocal microscopy, and no change of fluorescence during the treatment was observed, suggesting that calcium is not involved in the signalling pathway activated by alpha(1)-adrenoceptors in SK-Mel 23 cells. cAMP levels, determined by enzyme-immunoassay, were significantly increased by PHE (10(-5)-10(-4)M), that could be blocked by the alpha(1)-adrenergic antagonist benoxathian (10(-5)-10(-4)M). Several biological assays were then performed with PHE, for 72 h, in the absence or presence of various signalling pathway inhibitors, in an attempt to determine the intracellular messengers involved in the responses of proliferation and tyrosinase activity. Our results suggest the participation of p38 and ERKs in PHE-induced decrease of proliferation, and possibly also of cAMP and protein kinase A. Regarding PHE-induced increase of tyrosinase activity, it is suggested that the following signalling components are involved: cAMP/PKA, PKC, PI3K, p38 and ERKs.     

Sensitization of human cells by inhibitors of DNA synthesis following the action of DNA-damaging agents

Inhibitors of DNA synthesis 1-β-arabinofuranosylcytosine (Ac) and hydroxyurea (Hu) taken together drastically sensitized human cells to the killing effect of DNA-damaging agents. For UV-irradiation this sensitization depended on the cells′ ability for excision repair.

By using viscoelastometric methods of measurement of double-strand breaks (DSB) in the genome, it was established that the first DSB were generated after incubation of the damaged cells in the mixture of inhibitors at about the same dose when sensitization appeared.

A scheme is proposed to described molecular events associated with the phenomenon studied.     

Dichotomy of Ca2+ signals triggered by different phospholipid pathways in antigen stimulation of human mast cells

Mast cell activation triggers Ca(2+) signals and the release of enzyme-containing granules, events that play a major role in allergic/hypersensitivity reactions. However, the precise molecular mechanisms that regulate antigen-triggered degranulation and Ca(2+) fluxes in human mast cells are still poorly understood. Here we show, for the first time, that a receptor can trigger Ca(2+) via two separate molecular mechanisms. Using an antisense approach, we show that IgE-antigen stimulation of human bone marrow-derived mast cells triggers a sphingosine kinase (SPHK) 1-mediated fast and transient Ca(2+) release from intracellular stores. However, phospholipase C (PLC) gamma1 triggers a second (slower) wave of calcium release from intracellular stores, and it is this PLCgamma1-generated signal that is responsible for Ca(2+) entry. Surprisingly, FcepsilonRI (a high affinity receptor for IgE)-triggered mast cell degranulation depends on the first, sphingosine kinase-mediated Ca(2+) signal. These two pathways act independently because antisense knock down of either enzyme does not interfere with the activity of the other enzyme. Of interest, similar to PLCgamma1, SPHK1 translocates rapidly to the membrane after FcepsilonRI cross-linking. Here we also show that SPHK1 activity depends on phospholipase D1 and that FcepsilonRI-triggered mast cell degranulation depends primarily on the activation of both phospholipase D1 and SPHK1.     

Per3, a circadian gene, is required for Chk2 activation in human cells

PER3 is a member of the PERIOD genes, but does not play essential roles in the circadian clock. Depletion of Per3 by siRNA almost completely abolished activation of checkpoint kinase 2 (Chk2) after inducing DNA damage in human cells. In addition, Per3 physically interacted with ATM and Chk2. Per3 overexpression induced Chk2 activation in the absence of exogenous DNA damage, and this activation depended on ATM. Per3 overexpression also led to the inhibition of cell proliferation and apoptotic cell death. These combined results suggest that Per3 is a checkpoint protein that plays important roles in checkpoint activation, cell proliferation and apoptosis.

Structured summary

MINT-8052850: Chk2 (uniprotkb:O96017) physically interacts (MI:0915) with Per3 (uniprotkb:P56645) by anti bait coimmunoprecipitation (MI:0006)MINT-8052875: Per3 (uniprotkb:P56645) physically interacts (MI:0914) with Chk2 (uniprotkb:O96017) and ATM (uniprotkb:Q13315) by anti tag coimmunoprecipitation (MI:0007)     

Chk2 activation and phosphorylation-dependent oligomerization

The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. We show here that autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto- and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways.     

Sensitivity to DNA-damaging agents and mutation induction by UV light in UV-sensitive CHO cells

Three UV-sensitive (UVs) mutants isolated from a CHO cell line were analyzed for survival after exposure to H2O2, EMS, MMC, CCNU, X-rays and for mutation induction after UV-irradiation. The UVs mutants showed normal sensitivities to EMS and H2O2, whereas they were hypersensitive to the bifunctional alkylating agents MMC and CCNU and to hypoxic X-irradiation. Compared to parental cells, one of the UV-sensitive clones showed approximately 3- and 7-fold enhancement in the mutagenic response per unit UV dose for 6-thioguanine and ouabain resistance, respectively.     

Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein

In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane-cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.     

Repair of single-strand DNA interruptions by redundant pathways and its implication in cellular sensitivity to DNA-damaging agents

Single-strand DNA interruptions (SSIs) are produced during the process of base excision repair (BER). Through biochemical studies, two SSI repair subpathways have been identified: a pathway mediated by DNA polymerase β (Pol β) and DNA ligase III (Lig III), and a pathway mediated by DNA polymerase δ/ε (Pol δ/ε) and DNA ligase I (Lig I). In addition, the existence of another pathway, mediated by Pol β and DNA Lig I, has been suggested. Although each pathway may play a unique role in cellular DNA damage response, the functional implications of SSI repair by these three pathways are not clearly understood. To obtain a better understanding of the functional relevance of SSI repair by these pathways, we investigated the involvement of each pathway by monitoring the utilization of DNA ligases in cell-free extracts. Our results suggest that the majority of SSIs produced during the repair of alkylated DNA bases are repaired by the pathway mediated by Pol β and either Lig I or Lig III, although some SSIs are repaired by Pol δ/ε and Lig I. At a cellular level, we found that Lig III over-expression increased the resistance of cells to DNA-damaging agents, while Lig I over-expression had little effect. Thus, repair pathways mediated by Lig III may have a role in the regulation of cellular sensitivity to DNA-damaging agents.     

Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells.

Only a few of the genes involved in DNA repair in mammalian cells have been isolated, and induction of a DNA repair gene in response to DNA damage has not yet been established. DNA polymerase beta (beta-polymerase) appears to have a synthetic role in DNA repair after certain types of DNA damage. Here we show that the level of beta-polymerase mRNA is increased in CHO cells after treatment with several DNA-damaging agents.     

Death receptor-induced activation of the Chk2- and histone H2AX-associated DNA damage response pathways

TRAIL is an endogenous death receptor ligand also used therapeutically because of its selective proapoptotic activity in cancer cells. In the present study, we examined chromatin alterations induced by TRAIL and show that TRAIL induces a rapid activation of DNA damage response (DDR) pathways with histone H2AX, Chk2, ATM, and DNA-PK phosphorylations. Within 1 h of TRAIL exposure, immunofluorescence confocal microscopy revealed γ-H2AX peripheral nuclear staining (γ-H2AX ring) colocalizing with phosphorylated/activated Chk2, ATM, and DNA-PK inside heterochromatin regions. The marginal distribution of DDR proteins in early apoptotic cells is remarkably different from the focal staining seen after DNA damage. TRAIL-induced DDR was suppressed upon caspase inhibition or Bax inactivation, demonstrating that the DDR activated by TRAIL is downstream from the mitochondrial death pathway. H2AX phosphorylation was dependent on DNA-PK, while Chk2 phosphorylation was dependent on both ATM and DNA-PK. Downregulation of Chk2 decreased TRAIL-induced cell detachment; delayed the activation of caspases 2, 3, 8, and 9; and reduced TRAIL-induced cell killing. Together, our findings suggest that nuclear activation of Chk2 by TRAIL acts as a positive feedback loop involving the mitochondrion-dependent activation of caspases, independently of p53.     

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca²⁺ ([Ca²⁺]_i) by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca²⁺]_i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M₃) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M₁) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M₃, but not M₁, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms—pilocarpine acting via M₃ receptors and Src-dependent transactivation of EGF receptors, and carbachol via M₁/M₃ receptors and PKC—converging on the ERK pathway. muscarinic receptor; epidermal growth factor receptor; protein kinase C