Clare H. McGowan

Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15.

In fission yeast, the M‐phase inducing kinase, a complex of p34cdc2 and cyclin B, is maintained in an inhibited state during interphase due to the phosphorylation of Cdc2 at Tyr15. This phosphorylation is believed to be carried out primarily by the Wee1 kinase. In human cells the negative regulation of p34cdc2/cyclin B is more complex, in that Cdc2 is phosphorylated at two inhibitory sites, Thr14 and Tyr15. The identities of the kinases that phosphorylate these sites are unknown. Since fission yeast Wee1 kinase behaves as a dual‐specificity kinase in vitro, a popular hypothesis is that a human Wee1 homolog might phosphorylate p34cdc2 at both sites. We report here that a human gene, identified as a possible Wee1 homologue, blocks cell division when overexpressed in HeLa cells. This demonstrates functional conservation of the Wee1 mitotic inhibitor. Contrary to the dual‐specificity kinase hypothesis, purified human Wee1 phosphorylates p34cdc2 exclusively on Tyr15 in vitro; no Thr14 phosphorylation was detected. Human and fission yeast Wee1 also specifically phosphorylate synthetic peptides at sites equivalent to Tyr15. Mutation of a critical lysine codon (Lys114) believed to be essential for kinase activity abolished both the in vivo mitotic inhibitor function and in vitro kinase activities of human Wee1. These results conclusively prove that Wee1 kinases inhibit mitosis by directly phosphorylating p34cdc2 on Tyr15, and strongly indicate that human cells have independent kinase pathways directing the two inhibitor phosphorylations of p34cdc2.

A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase

BACKGROUND In human cells, the mitosis-inducing kinase Cdc2 is inhibited by phosphorylation on Thr14 and Tyr15. Disruption of these phosphorylation sites abrogates checkpoint-mediated regulation of Cdc2 and renders cells highly sensitive to agents that damage DNA. Phosphorylation of these sites is controlled by the opposing activities of the Wee1/Myt1 kinases and the Cdc25 phosphatase. The regulation of these enzymes is therefore likely to be crucial for the operation of the G2-M DNA-damage checkpoint. RESULTS Here, we show that the activity of Cdc25 decreased following exposure to ionizing radiation. The irradiation-induced decrease in Cdc25 activity was suppressed by wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinases, and was dependent on the function of the gene that is mutated in ataxia telangiectasia. We also identified two human kinases that phosphorylate and inactivate Cdc25 in vitro. One is the previously characterized Chk1 kinase. The second is novel and is homologous to the Cds1/Rad53 family of checkpoint kinases in yeast. Human Cds1 was found to be activated in response to DNA damage. CONCLUSIONS These results suggest that, in human cells, the DNA-damage checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1.

Caffeine inhibits the checkpoint kinase ATM

The basis of many anti-cancer therapies is the use of genotoxic agents that damage DNA and thus kill dividing cells. Agents that cause cells to override the DNA-damage checkpoint are predicted to sensitize cells to killing by genotoxic agents. They have therefore been sought as adjuncts in radiation therapy and chemotherapy. One such compound, caffeine, uncouples cell-cycle progression from the replication and repair of DNA [1] [2]. Caffeine therefore servers as a model compound in establishing the principle that agents that override DNA-damage checkpoints can be used to sensitize cells to the killing effects of genotoxic drugs [3]. But despite more than 20 years of use, the molecular mechanisms by which caffeine affects the cell cycle and checkpoint responses have not been identified. We investigated the effects of caffeine on the G2/M DNA-damage checkpoint in human cells. We report that the radiation-induced activation of the kinase Cds1 [4] (also known as Chk2 [5]) is inhibited by caffeine in vivo and that ATM kinase activity is directly inhibited by caffeine in vitro. Inhibition of ATM provides a molecular explanation of the attenuation of DNA-damage checkpoint responses and for the increased radiosensitivity of caffeine-treated cells [6] [7] [8].

Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1.

In response to DNA damage, eukaryotic cells use a system of checkpoint controls to delay cell-cycle progression. Checkpoint delays provide time for repair of damaged DNA before its replication in S phase and before segregation of chromatids in M phase. The Cds1 (Chk2) tumour-suppressor protein has been implicated in certain checkpoint responses in mammalian cells. It directly phosphorylates and inactivates the mitosis-inducing phosphatase Cdc25 in vitro and is required to maintain the G2 arrest that is observed in response to γ-irradiation. Cds1 also directly phosphorylates p53 in vitro at a site that is implicated in its stabilization, and is required for stabilization of p53 and induction of p53-dependent transcripts in vivo upon γ-ionizing radiation. Thus, Cds1 functions in both the G1 and G2 checkpoint responses. Like Cds1, the checkpoint protein kinase ATM (ataxia-telangiectasia-mutated) is required for correct operation of both the G1 and G2 damage checkpoints. ATM is necessary for phosphorylation and activation of Cds1 in vivo and can phosphorylate Cds1 in vitro, although evidence that the sites that are phosphorylated by ATM are required for activation is lacking. Here we show that threonine 68 of Cds1 is the preferred site of phosphorylation by ATM in vitro, and is the principal irradiation-induced site of phosphorylation in vivo. The importance of this phosphorylation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G1 arrest in response to ionising radiation.

SUMO-targeted ubiquitin ligases in genome stability

We identify the SUMO‐Targeted Ubiquitin Ligase (STUbL) family of proteins and propose that STUbLs selectively ubiquitinate sumoylated proteins and proteins that contain SUMO‐like domains (SLDs). STUbL recruitment to sumoylated/SLD proteins is mediated by tandem SUMO interaction motifs (SIMs) within the STUbLs N‐terminus. STUbL‐mediated ubiquitination maintains sumoylation pathway homeostasis by promoting target protein desumoylation and/or degradation. Thus, STUbLs establish a novel mode of communication between the sumoylation and ubiquitination pathways. STUbLs are evolutionarily conserved and include: Schizosaccharomyces pombe Slx8‐Rfp (founding member), Homo sapiens RNF4, Dictyostelium discoideum MIP1 and Saccharomyces cerevisiae Slx5–Slx8. Cells lacking Slx8‐Rfp accumulate sumoylated proteins, display genomic instability, and are hypersensitive to genotoxic stress. These phenotypes are suppressed by deletion of the major SUMO ligase Pli1, demonstrating the specificity of STUbLs as regulators of sumoylated proteins. Notably, human RNF4 expression restores SUMO pathway homeostasis in fission yeast lacking Slx8‐Rfp, underscoring the evolutionary functional conservation of STUbLs. The DNA repair factor Rad60 and its human homolog NIP45, which contain SLDs, are candidate STUbL targets. Consistently, Rad60 and Slx8‐Rfp mutants have similar DNA repair defects.

p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast.

We have investigated the mechanism by which fission yeast p80cdc25 induces mitosis. The in vivo active domain was localized to the C‐terminal 23 kDa of p80cdc25. This domain produced as a bacterial fusion protein (GST‐cdc25) caused tyrosyl dephosphorylation and activation of immunoprecipitated p34cdc2. Furthermore, GST‐cdc25 dephosphorylated both para‐nitrophenyl‐phosphate (pNPP) and casein phosphorylated on serine in vitro. Reaction requirements and inhibitor sensitivities were the same as those of phosphotyrosine phosphatases (PTPases). Analysis of cdc25 C‐terminal domains from a variety of species revealed a conserved motif having critical residues present at the active site of PTPases. Mutation of the cdc25 Cys480 codon, corresponding to an essential cysteine in the active site of PTPases, abolished the phosphatase activity of GST‐cdc25. These data indicate that cdc25 proteins define a novel subclass of eukaryotic PTPases, and strongly argue that cdc25 proteins directly dephosphorylate and activate p34cdc2 kinase to induce M‐phase.

Cell cycle regulation of human WEE1.

WEE1 kinase negatively regulates entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of CDC2/cyclin B kinase. We report here an investigation of human WEE1. Endogenous WEE1 migrates as an approximately 94 kDa protein in SDS‐PAGE, substantially larger than the 49 kDa protein encoded by the original human WEE1 cDNA clone that was truncated at the 5′‐end. Antibody depletion experiments demonstrate that WEE1 accounts for most of the activity that phosphorylates CDC2 on Tyr15 in an in vitro assay of HeLa cell lysates, hence it is likely to have an important role in the mitotic control of human cells. WEE1 activity was not found to be elevated in HeLa cells arrested in S phase, suggesting that unreplicated DNA does not delay M phase by hyperactivating WEE1. WEE1 activity is strongly suppressed during M phase, suggesting that negative regulation of WEE1 could be part of the mechanism by which activation of CDC2/cyclin B kinase is promoted during the G2/M transition. M phase WEE1 is re‐activated in samples prepared in the absence of protein phosphatase inhibitors, demonstrating that WEE1 is inhibited by a mechanism that requires protein phosphorylation.

Human Mus81-Associated Endonuclease Cleaves Holliday Junctions In Vitro

Mus81, a protein with homology to the XPF subunit of the ERCC1-XPF endonuclease, is important for replicational stress tolerance in both budding and fission yeast. Human Mus81 has associated endonuclease activity against structure-specific oligonucleotide substrates, including synthetic Holliday junctions. Mus81-associated endonuclease resolves Holliday junctions into linear duplexes by cutting across the junction exclusively on strands of like polarity. In addition, Mus81 protein abundance increases in cells following exposure to agents that block DNA replication. Taken together, these findings suggest a role for Mus81 in resolving Holliday junctions that arise when DNA replication is blocked by damage or by nucleotide depletion. Mus81 is not related by sequence to previously characterized Holliday junction resolving enzymes, and it has distinct enzymatic properties that suggest it uses a novel enzymatic strategy to cleave Holliday junctions.

Involvement of the MKK6-p38gamma cascade in gamma-radiation-induced cell cycle arrest.

ABSTRACT The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38γ (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for γ-irradiation-induced G2arrest. Activation of the MKK6-p38γ cascade is sufficient to induce G2 arrest in cells, and expression of dominant negative alleles of MKK6 or p38γ allows cells to escape the DNA damage-induce G2 delay. Activation of p38γ is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by γ irradiation leads to the activation of MKK6, p38γ, and Cds1 and that activation of both MKK6 and p38γ is essential for the proper regulation of the G2checkpoint in mammalian cells.

Holliday junction resolution in human cells: two junction endonucleases with distinct substrate specificities

Enzymatic activities that cleave Holliday junctions are required for the resolution of recombination intermediates and for the restart of stalled replication forks. Here we show that human cell‐free extracts possess two distinct endonucleases that can cleave Holliday junctions. The first cleaves Holliday junctions in a structure‐ and sequence‐specific manner, and associates with an ATP‐dependent branch migration activity. Together, these activities promote branch migration/resolution reactions similar to those catalysed by the Escherichia coli RuvABC resolvasome. Like RuvC‐mediated resolution, the products can be religated. The second, containing Mus81 protein, cuts Holliday junctions but the products are mostly non‐ligatable. Each nuclease has a defined substrate specificity: the branch migration‐associated resolvase is highly specific for Holliday junctions, whereas the Mus81‐associated endonuclease is one order of magnitude more active upon replication fork and 3′‐flap structures. Thus, both nucleases are capable of cutting Holliday junctions formed during recombination or through the regression of stalled replication forks. However, the Mus81‐associated endonuclease may play a more direct role in replication fork collapse by catalysing the cleavage of stalled fork structures.