Cancer-predisposing mutations within the RING domain of BRCA1: Loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity

BRCA1 is a breast and ovarian cancer-specific tumor suppressor that seems to be involved in transcription and DNA repair. Here we report that BRCA1 exhibits a bona fide ubiquitin (Ub) protein ligase (E3) activity, and that cancer-predisposing mutations within the BRCA1 RING domain abolish its Ub ligase activity. Furthermore, these mutants are unable to reverse γ-radiation hypersensitivity of BRCA1-null human breast cancer cells, HCC1937. Additionally, these mutations within the BRCA1 RING domain are not capable of restoring a G2 + M checkpoint in HCC1937 cells. These results establish a link between Ub protein ligase activity and γ-radiation protection function of BRCA1, and provide an explanation for why mutations within the BRCA1 RING domain predispose to cancer. Furthermore, we propose that the analysis of the Ub ligase activity of RING-domain mutations identified in patients may constitute an assay to predict predisposition to cancer.

Maintenance of caspase-3 proenzyme dormancy by an intrinsic “safety catch” regulatory tripeptide

Caspase-3 is synthesized as a dormant proenzyme and is maintained in an inactive conformation by an Asp-Asp-Asp “safety-catch” regulatory tripeptide contained within a flexible loop near the large-subunit/small-subunit junction. Removal of this “safety catch” results in substantially enhanced autocatalytic maturation as well as increased vulnerability to proteolytic activation by upstream proteases in the apoptotic pathway such as caspase-9 and granzyme B. The safety catch functions through multiple ionic interactions that are disrupted by acidification, which occurs in the cytosol of cells during the early stages of apoptosis. We propose that the caspase-3 safety catch is a key regulatory checkpoint in the apoptotic cascade that regulates terminal events in the caspase cascade by modulating the triggering of caspase-3 activation.

Tetraploid State Induces p53-dependent Arrest of Nontransformed Mammalian Cells in G1

A “spindle assembly” checkpoint has been described that arrests cells in G1 following inappropriate exit from mitosis in the presence of microtubule inhibitors. We have here addressed the question of whether the resulting tetraploid state itself, rather than failure of spindle function or induction of spindle damage, acts as a checkpoint to arrest cells in G1. Dihydrocytochalasin B induces cleavage failure in cells where spindle function and chromatid segregation are both normal. Notably, we show here that nontransformed REF-52 cells arrest indefinitely in tetraploid G1 following cleavage failure. The spindle assembly checkpoint and the tetraploidization checkpoint that we describe here are likely to be equivalent. Both involve arrest in G1 with inactive cdk2 kinase, hypophosphorylated retinoblastoma protein, and elevated levels of p21WAF1 and cyclin E. Furthermore, both require p53. We show that failure to arrest in G1 following tetraploidization rapidly results in aneuploidy. Similar tetraploid G1 arrest results have been obtained with mouse NIH3T3 and human IMR-90 cells. Thus, we propose that a general checkpoint control acts in G1 to recognize tetraploid cells and induce their arrest and thereby prevents the propagation of errors of late mitosis and the generation of aneuploidy. As such, the tetraploidy checkpoint may be a critical activity of p53 in its role of ensuring genomic integrity.

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Δ cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Δ cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.

Identification of a Saccharomyces cerevisiae Gene that Is Required for G1 Arrest in Response to the Lipid Oxidation Product Linoleic Acid Hydroperoxide*

Reactive oxygen species cause damage to all of the major cellular constituents, including peroxidation of lipids. Previous studies have revealed that oxidative stress, including exposure to oxidation products, affects the progression of cells through the cell division cycle. This study examined the effect of linoleic acid hydroperoxide, a lipid peroxidation product, on the yeast cell cycle. Treatment with this peroxide led to accumulation of unbudded cells in asynchronous populations, together with a budding and replication delay in synchronous ones. This observed modulation of G1 progression could be distinguished from the lethal effects of the treatment and may have been due to a checkpoint mechanism, analogous to that known to be involved in effecting cell cycle arrest in response to DNA damage. By examining several mutants sensitive to linoleic acid hydroperoxide, the YNL099c open reading frame was found to be required for the arrest. This gene (designated OCA1) encodes a putative protein tyrosine phosphatase of previously unknown function. Cells lacking OCA1 did not accumulate in G1 on treatment with linoleic acid hydroperoxide, nor did they show a budding, replication, or Start delay in synchronous cultures. Although not essential for adaptation or immediate cellular survival, OCA1 was required for growth in the presence of linoleic acid hydroperoxide, thus indicating that it may function in linking growth, stress responses, and the cell cycle. Identification of OCA1 establishes cell cycle arrest as an actively regulated response to oxidative stress and will enable further elucidation of oxidative stress-responsive signaling pathways in yeast.

Rad52 forms DNA repair and recombination centers during S phase

Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by γ-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.

Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.

G1 arrest and down-regulation of cyclin E/cyclin-dependent kinase 2 by the protein kinase inhibitor staurosporine are dependent on the retinoblastoma protein in the bladder carcinoma cell line 5637.

The protein kinase inhibitor staurosporine has been shown to induce G1 phase arrest in normal cells but not in most transformed cells. Staurosporine did not induce G1 phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB-). However, when infected with a pRB-expressing retrovirus [Goodrich, D. W., Chen, Y., Scully, P. & Lee, W.-H. (1992) Cancer Res. 52, 1968-1973], these cells, now pRB+, were arrested by staurosporine in G1 phase. This arrest was accompanied by the accumulation of hypophosphorylated pRB. In both the pRB+ and pRB- cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment. In contrast, cyclin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB+ cells. Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E. The CDK inhibitor proteins p21(Waf1/Cip1) and p27 (Kip1) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p2l from staurosporine-treated pRB+ cells revealed a 2.5- to 3-fold higher ratio of p2l bound to CDK2 compared with staurosporine-treated pRB- cells. In pRB+ cells, p2l was preferentially associated with Thrl6O phosphorylated active CDK2. In pRB- cells, however, p2l was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant. This is the first evidence suggesting that G1 arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB- dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p2l and p27.

Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA.

Kinetochores are DNA-protein structures that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Because of their simplicity, the 125-bp centromeres of Saccharomyces cerevisiae are particularly amenable to molecular analysis. Budding yeast centromeres contain three sequence elements of which centromere DNA sequence element III (CDEIII) appears to be particularly important. cis-acting mutations in CDEIII and trans-acting mutations in genes encoding subunits of the CDEIII-binding complex (CBF3) prevent correct chromosome transmission. Using temperature-sensitive mutations in CBF3 subunits, we show a strong correlation between DNA-binding activity measured in vitro and kinetochore activity in vivo. We extend previous findings by Goh and Kilmartin [Goh, P.-Y. & Kilmartin, J.V. (1993) J. Cell Biol. 121, 503-512] to argue that DNA-bound CBF3 may be involved in the operation of a mitotic checkpoint but that functional CBF3 is not required for the assembly of a bipolar spindle.

Platelet-derived growth factor induces apoptosis in growth-arrested murine fibroblasts.

The platelet-derived growth factor (PDGF) is a potent mitogen for murine fibroblasts. PDGF-stimulated cells express a set of immediate-early-response genes but require additional (progression) factors in serum to progress through the cell cycle. Serum-deprived cells are reversibly arrested in G0 phase and fail to fully traverse the G1 phase of the cell cycle when stimulated by PDGF alone. We now report that serum-deprived normal rat kidney fibroblast (NRK) cells stimulated by either PDGF AA or PDGF BB homodimers undergo apoptotic cell death. Furthermore, we show that epidermal growth factor also induces apoptotic cell death in serum-deprived NRK cells, epidermal growth factor enhances the rate of apoptosis in PDGF-treated cells, and a progression factor (insulin) but not endogenously expressed Bc1-2 fully protects NRK cells from PDGF-stimulated apoptosis. The results indicate that PDGF induces apoptosis in growth-arrested NRK cells and that the inability of NRK cells to transit the G1/S checkpoint is the critical determinant in establishing the genetic program(s) to direct the PDGF signal to apoptosis. The results suggest that polypeptide growth factors in vivo may signal cell fate positively or negatively in settings that limit the potential of cells to completely transit the cell cycle.

Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis.

DNA-damaging agents induce accumulation of the tumor suppressor and G1 checkpoint protein p53, leading cells to either growth arrest in G1 or apoptosis (programmed cell death). The p53-dependent G1 arrest involves induction of p21 (also called WAF1/CIP1/SDI1), which prevents cyclin kinase-mediated phosphorylation of retinoblastoma protein (RB). Recent studies suggest a p53-independent G1 checkpoint as well; however, little is known about its molecular mechanisms. We report that induction of a protein-serine/threonine phosphatase activity by DNA damage signals is at least one of the mechanisms responsible for p53-independent, RB-mediated G1 arrest and consequent apoptosis. When two p53-null human leukemic cell lines (HL-60 and U-937) were treated with a variety of anticancer agents, RB became hypophosphorylated, accompanied with G1 arrest. This was followed immediately (in less than 30 min) by apoptosis, as determined by the accumulation of pre-G1 apoptotic cells and the internucleosomal fragmentation of DNA. Addition of calyculin A or okadaic acid (specific serine/threonine phosphatase inhibitors) or zinc chloride (apoptosis inhibitor) prevented the G1 arrest- and apoptosis-specific RB dephosphorylation. The levels of cyclin E- and cyclin A-associated kinase activities remained high during RB dephosphorylation, supporting the involvement of a chemotherapy-induced serine/threonine phosphatase(s) rather than p21. Furthermore, the induced phosphatase activity coimmunoprecipitated with the hyperphosphorylated RB and was active in a cell-free system that reproduced the growth arrest- and apoptosis-specific RB dephosphorylation, which was inhibitable by calyculin A but not zinc. We propose that the RB phosphatase(s) might be one of the p53-independent G1 checkpoint regulators.

p53 deficiency does not affect the accumulation of point mutations in a transgene target.

DNA repair is required by organisms to prevent the accumulation of mutations and to maintain the integrity of genetic information. Mammalian cells that have been treated with agents that damage DNA have an increase in p53 levels, a p53-dependent arrest at G1 in the cell cycle, and a p53-dependent apoptotic response. It has been hypothesized that this block in cell cycle progression is necessary to allow time for DNA repair or to direct the damaged cell to an apoptotic pathway. This hypothesis predicts that p53-deficient cells would have an abnormal apoptotic response and exhibit a "mutator" phenotype. Using a sensitive assay for the accumulation of point mutations, small deletions, and insertions, we have directly tested whether p53-deficient cells exhibit an increased frequency of mutation before and after exposure to DNA-damaging agents. We report that wild-type and p53-deficient fibroblasts, thymocytes, and tumor tissue have indistinguishable rates of point mutation accumulation in a transgenic lacI target gene. These results suggest that the role of p53 in G1 checkpoint control and tumor suppression does not affect the accumulation of point mutations.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

Stage-Specific Plasticity in Ovary Size Is Regulated by Insulin/Insulin-Like Growth Factor and Ecdysone Signaling in Drosophila

Animals from flies to humans adjust their development in response to environmental conditions through a series of developmental checkpoints, which alter the sensitivity of organs to environmental perturbation. Despite their importance, we know little about the molecular mechanisms through which this change in sensitivity occurs. Here we identify two phases of sensitivity to larval nutrition that contribute to plasticity in ovariole number, an important determinant of fecundity, in Drosophila melanogaster. These two phases of sensitivity are separated by the developmental checkpoint called "critical weight"; poor nutrition has greater effects on ovariole number in larvae before critical weight than after. We find that this switch in sensitivity results from distinct developmental processes. In precritical weight larvae, poor nutrition delays the onset of terminal filament cell differentiation, the starting point for ovariole development, and strongly suppresses the rate of terminal filament addition and the rate of increase in ovary volume. Conversely, in postcritical weight larvae, poor nutrition affects only the rate of increase in ovary volume. Our results further indicate that two hormonal pathways, the insulin/insulin-like growth factor and the ecdysone-signaling pathways, modulate the timing and rates of all three developmental processes. The change in sensitivity in the ovary results from changes in the relative contribution of each pathway to the rates of terminal filament addition and increase in ovary volume before and after critical weight. Our work deepens our understanding of how hormones act to modify the sensitivity of organs to environmental conditions, thereby affecting their plasticity.

Structural basis and prediction of substrate specificity in protein serine/threonine kinases

The large number of protein kinases makes it impractical to determine their specificities and substrates experimentally. Using the available crystal structures, molecular modeling, and sequence analyses of kinases and substrates, we developed a set of rules governing the binding of a heptapeptide substrate motif (surrounding the phosphorylation site) to the kinase and implemented these rules in a web-interfaced program for automated prediction of optimal substrate peptides, taking only the amino acid sequence of a protein kinase as input. We show the utility of the method by analyzing yeast cell cycle control and DNA damage checkpoint pathways. Our method is the only available predictive method generally applicable for identifying possible substrate proteins for protein serine/threonine kinases and helps in silico construction of signaling pathways. The accuracy of prediction is comparable to the accuracy of data from systematic large-scale experimental approaches.