Phosphorylation at serine 331 is required for Aurora B activation.

Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora B(S331A), in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation.

Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2.

Using chemical genetics to reversibly inhibit Cdk1, we find that cells arrested in late G2 are unable to delay mitotic entry after irradiation. Late G2 cells detect DNA damage lesions and form gamma-H2AX foci but fail to activate Chk1. This reflects a lack of DNA double-strand break processing because late G2 cells fail to recruit RPA (replication protein A), ATR (ataxia telangiectasia and Rad3 related), Rad51, or CtIP (C-terminal interacting protein) to sites of radiation-induced damage, events essential for both checkpoint activation and initiation of DNA repair by homologous recombination. Remarkably, inhibition of Akt/PKB (protein kinase B) restores DNA damage processing and Chk1 activation after irradiation in late G2. These data demonstrate a previously unrecognized role for Akt in cell cycle regulation of DNA repair and checkpoint activation. Because Akt/PKB is frequently activated in many tumor types, these findings have important implications for the evolution and therapy of such cancers.

DNA mismatch repair and Chk1-dependent centrosome amplification in response to DNA alkylation damage.

Centrosome amplification is frequently observed in tumor cells exposed to genotoxic stress, however the underlying mechanisms and biological consequences are poorly understood. Here, we show that the anti-metabolite and alkylating agent 6-thioguanine (6-TG) induces centrosome amplification resulting in the formation of multi-polar spindles when damaged cells subsequently enter mitosis. These aberrant, multi-polar mitoses are frequently resolved by asymmetric cell divisions causing unequal segregation of genetic material and cell death in one or both daughter products. We show that this phenomenon is associated with transient cell cycle delay in S- and G(2)-phase and is dependent on DNA mismatch repair (DNA MMR) proficiency and Chk1 protein kinase activity. Although Chk1-deficient cells do not exhibit cell cycle delay, centrosome amplification, or multi-polar spindle formation, continued cell cycle progression in the presence of 6-TG eventually results in increased levels of mitotic catastrophe, most probably due to mitosis with incompletely replicated DNA. Taken together, these results reveal novel mechanisms of cell killing by 6-TG and underscore the importance of interactions between cell cycle checkpoints and DNA MMR in determining the fate of cells bearing DNA damage.

Chk1 is required for spindle checkpoint function.

The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Here, we show that Chk1, a component of the DNA damage and replication checkpoints, protects vertebrate cells against spontaneous chromosome missegregation and is required to sustain anaphase delay when spindle function is disrupted by taxol, but not when microtubules are completely depolymerized by nocodazole. Spindle checkpoint failure in Chk1-deficient cells correlates with decreased Aurora-B kinase activity and impaired phosphorylation and kinetochore localization of BubR1. Furthermore, Chk1 phosphorylates Aurora-B and enhances its catalytic activity in vitro. We propose that Chk1 augments spindle checkpoint signaling and is required for optimal regulation of Aurora-B and BubR1 when kinetochores produce a weakened signal. In addition, Chk1-deficient cells exhibit increased resistance to taxol. These results suggest a mechanism through which Chk1 could protect against tumorigenesis through its role in spindle checkpoint signaling.

c-Jun-deficient cells undergo premature senescence as a result of spontaneous DNA damage accumulation.

Mouse embryo fibroblasts deficient for the c-Jun proto-oncogene (c-Jun-/- MEF) undergo p53-dependent premature senescence in conventional culture. This phenotype becomes evident only after several cell divisions, suggesting that senescence may result from exposure to unknown environmental factors. Here, we show that c-Jun-/- MEF can proliferate successfully in low oxygen (3% O2), indicating that premature senescence under conventional culture conditions is a consequence of hyperoxic stress. c-Jun-/- MEF exhibit higher basal levels of DNA damage compared to normal fibroblasts in high but not low oxygen, implying that senescence results from chronic accumulation of spontaneous DNA damage. This accumulation may be attributable, at least in part, to inefficient repair, since DNA damage induced by gamma ionizing radiation and H2O2 persists for longer in c-Jun-/- MEF than in wild-type MEF. Unexpectedly, p53 expression, phosphorylation, and transcriptional activity are largely unaffected by oxygen exposure, indicating that the accumulation of spontaneous DNA damage does not result in chronic activation of p53 as judged by conventional criteria. Finally, we find that c-Jun associates with nuclear foci containing gammaH2AX and ATM following irradiation, suggesting a potential role for c-Jun in DNA repair processes per se.

Microarray analysis identifies Autotaxin, a tumour cell motility and angiogenic factor with lysophospholipase D activity, as a specific target of cell transformation by v-Jun.

We have used chicken cDNA microarrays to investigate gene-expression changes induced during transformation of chick embryo fibroblasts (CEF) by the viral Jun oncoprotein encoded by ASV17. This analysis reveals that v-Jun induces increases and decreases of varying magnitude in the expression of genes involved in diverse cellular functions, most of which have not been detected in previous screens for putative v-Jun targets. In all, 27 individual genes were identified, whose expression is increased threefold or more in v-Jun-transformed cells, including genes involved in energy generation, protein synthesis, and gene transcription. Interestingly, this group includes the hypoxia-inducible factor-1 alpha (Hif-1alpha) transcription factor and the glycolytic enzyme enolase, suggesting that adaptation to hypoxia could play a role in tumorigenesis by v-Jun. We also identified 32 genes whose expression is decreased threefold or more, including chaperones, components of the cytoskeleton, and, unexpectedly, DNA replication factors. The gene whose expression is upregulated most dramatically (approximately 100-fold) encodes Autotaxin (ATX), a secreted tumor motility-promoting factor with lysophospholipase D activity. Strikingly, v-Jun-transformed CEF secrete catalytically active ATX and chemotactic activity, which can be detected in conditioned medium. ATX is not detectably expressed in normal CEF or CEF transformed by the v-Src or v-Myc oncoproteins, indicating that induction of this putative autocrine/paracrine factor is a specific consequence of cell transformation by v-Jun. ATX has been implicated in both angiogenesis and invasion, and could therefore play an important role in tumorigenesis by v-Jun in vivo.

Estrogen receptor activation function 2 (AF-2) is essential for hormone-dependent transactivation and cell transformation induced by a v-Jun DNA binding domain-estrogen receptor chimera.

A chimeric protein consisting of the estrogen receptor alpha ligand binding domain (ER-alpha LBD) fused to the DNA binding domain (DBD) of the v-Jun oncoprotein, deltavJ-hER, was previously shown to elicit estradiol-dependent transcriptional activation and cell transformation. Remarkably, in the unliganded state deltavJ-hER is not inert, but rather inhibits cell proliferation. To understand the molecular basis for these opposite effects on cell growth, we investigated the effect of estradiol on deltavJ-hER function. We find that deltavJ-hER is localised to the cell nucleus and capable of binding TPA-response element (TRE) DNA recognition sites in the presence and absence of estradiol, indicating that these properties are unlikely to be the targets of hormonal regulation. In contrast, a mutant derivative of deltavJ-hER in which amino acid substitutions selectively disrupt activation function 2 (AF-2) function is unable to elicit estradiol-dependent transcription or cell transformation, even though DNA binding is not impaired. Taken together, these observations establish that estrogen receptor AF-2 activity is essential for cell transformation by deltavJ-hER.

Cell transformation by v-Jun deactivates ERK MAP kinase signalling.

Previous studies have shown that v-Jun accelerates G1 progression and enables cells to sustain S phase entry in the absence of serum growth factors. Since growth factor-dependent ERK MAP kinase signalling plays an important role in regulating the G1/S transition, we investigated whether aberrant ERK regulation might contribute to cell cycle deregulation by v-Jun. Contrary to expectation, we find that cells transformed by v-Jun exhibit a profound reduction in the basal level of active, dual-phosphorylated ERK. In addition, ERK becomes refractory to stimulation by a subset of agonists including serum, LPA, and EGF, but remains partially responsive to the phorbol ester, TPA. Biochemical analysis indicates that these defects are attributable to a combination of inefficient signal propagation between Ras and Raf within the ERK pathway and increased tonic deactivation by MAP kinase phosphatases. Taken together, these results demonstrate that cell transformation by v-Jun induces alterations in cell physiology which antagonize ERK signalling at multiple levels. The potential significance of this phenotype for oncogenesis by v-Jun is discussed.