James R. A. Hutchins

Inhibition of the G2 DNA Damage Checkpoint and of Protein Kinases Chk1 and Chk2 by the Marine Sponge Alkaloid Debromohymenialdisine

Cells can respond to DNA damage by activating checkpoints that delay cell cycle progression and allow time for DNA repair. Chemical inhibitors of the G2 phase DNA damage checkpoint may be used as tools to understand better how the checkpoint is regulated and may be used to sensitize cancer cells to DNA-damaging therapies. However, few inhibitors are known. We used a cell-based assay to screen natural extracts for G2checkpoint inhibitors and identified debromohymenialdisine (DBH) from a marine sponge. DBH is distinct structurally from previously known G2 checkpoint inhibitors. It inhibited the G2checkpoint with an IC50 of 8 μm and showed moderate cytotoxicity (IC50 = 25 μm) toward MCF-7 cells. DBH inhibited the checkpoint kinases Chk1 (IC50 = 3 μm) and Chk2 (IC50 = 3.5 μm) but not ataxia-telangiectasia mutated (ATM), ATM-Rad3-related protein, or DNA-dependent protein kinase in vitro, indicating that it blocks two major branches of the checkpoint pathway downstream of ATM. It did not cause the activation or inhibition of different signal transduction proteins, as determined by mobility shift analysis in Western blots, suggesting that it inhibits a narrow range of protein kinases in vivo.

Role of Importin-β in the Control of Nuclear Envelope Assembly by Ran

Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin. Here, we show that importin-beta, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-beta on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-beta in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-alpha is functional. Thus, importin-beta functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-alpha with karyophilic proteins carrying classical nuclear localization signals. Importin-beta links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division.

Many fingers on the mitotic trigger: post-translational regulation of the Cdc25C phosphatase.

AbstractThe mitotic inducer Cdc25 phosphatase controls the activation of Cdc2/cyclin B protein kinase and entry into mitosis in eukaryotic cells. Cdc25C is highly regulated by multiple post-translational modifications within its N-terminal regulatory domain and site-specific protein interactions. Phosphorylation of one inhibitory site targeted by multiple kinases determines the timing of Cdc25C activation and arrests cells in G2 in response to checkpoint, stress, developmental and extracellular signals. In mitosis, phosphorylation of several Ser/Thr residues and Pin1-catalysed peptidyl-proline isomerisation produces activation. Phosphorylation of one activating site is antagonistic to the proximal inhibitory site and maintains Cdc25C activity during mitosis. Phosphorylation and interacting proteins also modulate the nuclear import and export signals on Cdc25C, inducing dramatic changes in its localisation within the cell. Thus, the regulation of Cdc25C activity and localisation integrates multiple signals that govern the decision to enter mitosis.

Phosphorylation Regulates the Dynamic Interaction of RCC1 with Chromosomes during Mitosis

The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin alpha/beta and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.

Substrate specificity determinants of the checkpoint protein kinase Chk1

The Chk1 protein kinase plays a critical role in a DNA damage checkpoint pathway conserved between fission yeast and animals. We have developed a quantitative assay for Chk1 activity, using a peptide derived from a region of Xenopus Cdc25C containing Ser‐287, a known target of Chk1. Variants of this peptide were used to determine the residues involved in substrate recognition by Chk1, revealing the phosphorylation motif Φ‐X‐β‐X‐X‐(S/T)*, where * indicates the phosphorylated residue, Φ is a hydrophobic residue (M>I>L>V), β is a basic residue (R>K) and X is any amino acid. This motif suggests that Chk1 is a member of a group of stress‐response protein kinases which phosphorylate target proteins with related specificities.

Dephosphorylation of the inhibitory phosphorylation site S287 in Xenopus Cdc25C by protein phosphatase-2A is inhibited by 14-3-3 binding.

Cdc25C phosphatase induces mitosis by dephosphorylating and activating Cdc2/cyclin B protein kinase. Phosphorylation of Xenopus Cdc25C at serine 287 creates a binding site for a 14‐3‐3 protein and restrains activation during interphase. Here, we show that dephosphorylation of S287 is catalysed by protein phosphatase‐2A in Xenopus egg extracts. 14‐3‐3 protein binding to Cdc25C inhibits dephosphorylation of S287, providing a mechanism to maintain phosphorylation of that site during interphase. The rate of dephosphorylation of S287 is not increased in mitotic extracts, indicating that the phosphorylation status of the site is likely to be controlled through modulation of kinases or 14‐3‐3 binding activity.

Microtubule assembly by the Apc protein is regulated by importin-β—RanGTP

Mutations in the tumour suppressor Adenomatous polyposis coli (Apc) initiate most sporadic colorectal cancers. Apc is implicated in regulating microtubule (MT) dynamics in interphase and mitosis. However, little is known about the underlying mechanism or regulation of this Apc function. We identified importin-β as a binding partner of Apc that regulates its effect on MTs. Apc binds importin-β in vitro and in Xenopus egg extracts, and RanGTP inhibits this interaction. The armadillo-like repeat domain of importin-β binds to the middle of Apc, where it can compete with β-catenin. In addition, two independent sites in the C terminus of Apc bind the N-terminal region of importin-β. Binding to importin-β reduces the ability of Apc to assemble and bundle MTs in vitro and to promote assembly of microtubule asters in Xenopus egg extracts, but does not affect the binding of Apc to MTs or to EB1. Depletion of Apc decreases the formation of cold-stable spindles in Xenopus egg extracts. Importantly, the ability of purified Apc to rescue this phenotype was reduced when it was constitutively bound to importin-β. Thus, importin-β binds to Apc and negatively regulates the MT-assembly and spindle-promoting activity of Apc in a Ran-regulatable manner.

Dynamic localisation of Ran GTPase during the cell cycle

BackgroundRan GTPase has multiple functions during the cell division cycle, including nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. The activity of Ran is determined by both its guanine nucleotide-bound state and its subcellular localization.ResultsHere, we have characterised the localisation and mobility of Ran coupled to green fluorescent protein (GFP) during the cell cycle in live human cells. Ran-GFP is nuclear during interphase and is dispersed throughout the cell during mitosis. GFP-RanQ69L, a mutant locked in the GTP-bound state, is less highly concentrated in the nucleus and associates with nuclear pore complexes within the nuclear envelope. During mitosis, GFP-RanQ69L is excluded from chromosomes and localizes to the spindle. By contrast, GFP-RanT24N, a mutant with low affinity for nucleotides, interacts relatively stably with chromatin throughout the cell cycle and is highly concentrated on mitotic chromosomes.ConclusionThese results show that Ran interacts dynamically with chromatin, nuclear pore complexes and the mitotic spindle during the cell cycle. These interactions are dependent on the nucleotide-bound state of the protein. Our data indicate that Ran-GTP generated at chromatin is highly mobile and interacts dynamically with distal structures that are involved in nuclear transport and mitotic spindle assembly.