Dephosphorylation and subcellular compartment change of the mitotic Bloom's syndrome DNA helicase in response to ionizing radiation.

Bloom's syndrome is a rare human autosomal recessive disorder that combines a marked genetic instability and an increased risk of developing all types of cancers and which results from mutations in both copies of the BLM gene encoding a RecQ 3'-5' DNA helicase. We recently showed that BLM is phosphorylated and excluded from the nuclear matrix during mitosis. We now show that the phosphorylated mitotic BLM protein is associated with a 3'-5' DNA helicase activity and interacts with topoisomerase III alpha. We demonstrate that in mitosis-arrested cells, ionizing radiation and roscovitine treatment both result in the reversion of BLM phosphorylation, suggesting that BLM could be dephosphorylated through the inhibition of cdc2 kinase. This was supported further by our data showing that cdc2 kinase activity is inhibited in gamma-irradiated mitotic cells. Finally we show that after ionizing radiation, BLM is not involved in the establishment of the mitotic DNA damage checkpoint but is subjected to a subcellular compartment change. These findings lead us to propose that BLM may be phosphorylated during mitosis, probably through the cdc2 pathway, to form a pool of rapidly available active protein. Inhibition of cdc2 kinase after ionizing radiation would lead to BLM dephosphorylation and possibly to BLM recruitment to some specific sites for repair.     

Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase

Following the identification of the cdc2 kinase as a major element controlling entry of cells into mitosis, it is important to define the physiological target range of this enzyme. Here, we demonstrate that two major nucleolar proteins, nucleolin and NO38, are highly phosphorylated during mitosis. Importantly, the two nucleolar proteins are also phosphorylated by highly purified starfish cdc2 kinase in vitro, on sites that correspond to those observed specifically during mitosis in vivo. A repeated motif (TPXKK) is identified as the likely mitotic phosphoacceptor site in nucleolin, in that a synthetic peptide mimicking this site functions as both a substrate and a competitive inhibitor of cdc2 kinase. These results identify two novel candidate substrates for cdc2 kinase, and they implicate protein phosphorylation in controlling mitotic changes in nucleolar structure and activity.     

Phosphorylation of p97(VCP) and p47 in vitro by p34cdc2 kinase.

The hexameric ATPase p97/yeast Cdc48p has been implicated in a number of cellular events that are regulated during mitosis, including homotypic membrane fusion, spindle pole body function, and ubiquitin-dependent protein degradation. p97/Cdc48p contains two conserved consensus p34cdc2 kinase phosphorylation sites within its second ATP binding domain. This domain is likely to play a role in stabilising the hexameric form of the protein. We therefore investigated whether p97 could be phosphorylated by p34cdc2 kinase in vitro, and whether phosphorylation might influence the oligomeric status of p97. Monomeric, but not hexameric, p97 was phosphorylated by p34cdc2 kinase, as was the p97-associated protein p47. However, phosphorylation by p34cdc2 kinase did not impair subsequent re-hexamerisation of p97, implying that the phosphorylated residue(s) are not critical for interaction between p97 monomers. Moreover, p97 within both interphase and mitotic cytosols was almost exclusively hexameric, suggesting that the activity of p97 is not regulated during mitosis by influencing the extent of oligomerisation.     

Raptor is Phosphorylated by cdc2 during Mitosis

Background

The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK) modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR) signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1). As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.

Methodology/Principal Findings

We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.

Conclusions/Significance

This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct modulation of the mTORC1 complex during mitosis.     

A role for the p34cdc2 kinase and phosphatases in the regulation of phosphorylation and disassembly of lamin B2 during the cell cycle.

While the p34cdc2 kinase is considered to be a critical regulator of mitosis, its function has not yet been directly linked to one of the key events during the onset of mitosis: nuclear envelope breakdown. Here we show that a major structural protein of the nuclear envelope, lamin B2, is phosphorylated by p34cdc2. Results from two-dimensional phosphopeptide mapping experiments demonstrate that the p34cdc2-specific phosphopeptides represent both mitotic and interphase specific phosphorylations of lamin B2 and include the major interphase phosphorylation site. In mitotic cells we detected two distinct forms of lamin B2 which differ in electrophoretic mobility and in degree of phosphorylation. The phosphorylation pattern of lamin B2 generated in vitro by p34cdc2 was more closely related to the less phosphorylated mitotic lamin B2, suggesting that another kinase(s) in addition to p34cdc2 is involved in generating the mitotic phosphorylation pattern. In addition, we show that treatment of interphase cells with okadaic acid, a potent phosphatase inhibitor, leads to the acquisition of mitosis-specific phosphopeptides and can reversibly increase the detergent-solubility of lamin B2. However, the M-phase-like phosphorylation of lamin B2 in itself is not sufficient to induce its disassembly from the nuclear lamina suggesting that an additional event(s) besides phosphorylation is required.     

Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase

As cells enter mitosis, the protein-tyrosine kinase, p60c-src, is known to be extensively phosphorylated on threonine in its amino-terminal region. In the present work, extracts of mitotic cells were searched for the protein kinase responsible for this phosphorylation. HeLa cells and Xenopus eggs were found to contain a mitosis-specific protein kinase activity capable of phosphorylating highly purified p60c-src in vitro on threonine residues. Tryptic phosphopeptide maps indicate that the mitotic HeLa kinase phosphorylates the same sites in vitro as those used during mitosis in vivo. In addition, this mitotic HeLa kinase comigrates on gel filtration with p34cdc2-associated histone H1 kinase, a well known regulator of mitotic events. Finally, antibodies to the C-terminal peptide of human p34cdc2 specifically deplete p60c-src-phosphorylating activity from mitotic extracts. These results suggest that p60c-src may act as an effector of p34cdc2 in certain mitotic processes.     

Phosphorylation of the Bloom's syndrome helicase and its role in recovery from S-phase arrest

Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3(+) related) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G(2)/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.     

Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leads to loss of Mcm4,6,7 helicase activity.

Mcm proteins that play an essential role in eukaryotic DNA replication are phosphorylated in vivo, and cyclin-dependent protein kinase is at least in part responsible for the phosphorylation of Mcm4. Our group reported that the DNA helicase activity of Mcm4,6,7 complex, which may be involved in initiation of DNA replication, is inhibited following phosphorylation by Cdk2/cyclin A in vitro. Here, we further examined the interplay between mouse Mcm4,6,7 complex and cyclin-dependent kinases and determined the sites required for the phosphorylation of Mcm4. Six Ser and Thr residues, in all, were required for the phosphorylation. Inhibition of Mcm4,6,7 helicase activity by Cdk2/cyclin A was largely relieved by introducing mutations in these residues of Mcm4. Anti-phosphothreonine antibodies raised against one of these sites reacted with Mcm4 prepared from HeLa cells at mitotic phase but did not bind to those at G(1) and G(1)/S, suggesting that this site is mainly phosphorylated in the mitotic phase. Mcm4,6,7 complex purified from HeLa cells at the mitotic phase exhibited a low level of DNA helicase activity, compared with the complexes prepared from cells at other phases. These results suggest that phosphorylation of Mcm4 at specific sites leads to loss of Mcm4,6,7 DNA helicase activity.     

A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells.     

Two different protein kinases act on a different time schedule as glial filament kinases during mitosis.

Glial fibrillary acidic protein (GFAP) is a component of glial filaments specific to astroglia. We now report the spatial and temporal distributions of four phosphorylated sites in the GFAP molecule during mitosis of astroglial cells, determined by antibodies which can distinguish phosphorylated epitopes from non-phosphorylated-epitopes. Immunofluorescence microscopy showed that the Ser8 residues in the entire cytoplasmic glial filament system are initially phosphorylated when the cells enter mitosis. In cytokinesis, the phosphoSer8 residues become dephosphorylated, whereas Thr7, Ser13 and Ser34 in glial filaments at the cleavage furrow become the preferred sites of phosphorylation. The cdc2 kinase purified from mitotic cells can phosphorylate GFAP at Ser8 but not at Thr7, Ser13 or Ser34, in vitro. These results suggest that cdc2 kinase acts as a glial filament kinase only at the G2-M phase transition while other glial filament kinases are probably activated at the cleavage furrow before final separation of the daughter cells.     

Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis

Tyrosine phosphorylation of cdc2 is regulated in the cell cycle of mouse 3T3 fibroblasts. Phosphotyrosine in cdc2 is detectable at the onset of DNA synthesis and becomes maximal in the G2 phase of the cell cycle. Quantitative tyrosine dephosphorylation of cdc2 occurs during entry into mitosis and no phosphotyrosine is detected during the G1 phase of the cell cycle. While increasing tyrosine phosphorylation of cdc2 correlates with the formation of a cdc2/p62 complex, the tyrosine phosphorylated cdc2 is inactive as a histone H1 kinase. cdc2 is fully dephosphorylated in its most active mitotic form, yet specific tyrosine dephosphorylation of interphase cdc2 in vitro is insufficient to activate the kinase. In vivo inhibition of tyrosine dephosphorylation by exposure of cells to a phosphatase inhibitor is associated with G2 arrest, which is reversible upon the removal of the phosphatase inhibitor. Tyrosine dephosphorylation of cdc2 may be one of a number of obligatory steps in the mitotic activation of the kinase.     

Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis

We have previously shown that overexpressed chicken pp60c-src has retarded mobility, novel serine/threonine phosphorylation, and enhanced kinase activity during NIH 3T3 cell mitosis. Here we show that novel mitotic phosphorylations occur at Thr 34, Thr 46, and Ser 72. The possibility, previously raised, that Ser 17 is dephosphorylated during mitosis is excluded. The phosphorylated sites lie in consensus sequences for phosphorylation by p34cdc2, the catalytic component of maturation promoting factor (MPF). Furthermore, highly purified MPF from metaphase-arrested Xenopus eggs phosphorylated both wild-type and kinase-defective pp60c-src at these sites. Altered phosphorylation alone is sufficient to account for the large retardation in mitotic pp60c-src electrophoretic mobility: phosphorylation of normal pp60c-src by MPF retarded mobility and dephosphorylation of mitotic pp60c-src restored normal mobility. These results suggest that pp60c-src is one of the targets for MPF action, which may account in part for the pleiotropic changes in protein phosphorylation and cellular architecture that occur during mitosis.     

Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks

The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.     

Mitotic phosphorylation of the dynein light intermediate chain is mediated by cdc2 kinase

Cytoplasmic dynein, a large minus-end-directed microtubule motor, performs multiple functions during the cell cycle. In interphase, dynein moves membrane organelles, while in mitosis it moves chromosomes and helps to form the mitotic spindle. The cell-cycle regulation of dynein activity may be controlled, at least in part, by the phosphorylation of its light intermediate chains (DLIC), since a 10-fold increase in light intermediate chain phosphorylation correlates with a decrease in dynein-based membrane transport of similar magnitude in mitosis. In this study, we sought to identify the kinase responsible for this potentially important phosphorylation event. We show that bacterially-expressed chicken light intermediate chain (chDLIC) will undergo mitosis-specific phosphorylation when added to Xenopus egg extracts. Mutation of a conserved cdc2 kinase consensus site (Ser197) abolishes this phosphorylation event, and mass spectroscopy analysis confirms that the wild-type DLIC is stoichiometrically phosphorylated at this site when incubated with metaphase but not interphase extracts. We also show that purified cdc2 kinase phosphorylates purified DLICs at Ser197 in vitro and that Ser197 phosphorylation is dramatically reduced in metaphase extracts depleted of cdc2 kinase. These results indicate that cdc2 kinase directly phosphorylates dynein and thus may be an important regulator of dynein activity in the cell cycle.     

Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin

As cells enter mitosis, the intermediate filament (IF) networks of interphase BHK-21 cells are depolymerized to form cytoplasmic aggregates of disassembled IFs, and the constituent IF proteins, vimentin and desmin are hyperphosphorylated at several specific sites. We have characterized one of two endogenous vimentin kinases from a particulate fraction of mitotic cell lysates. Through several purification steps, vimentin kinase activity copurifies with histone H1 kinase and both activities bind to p13suc1-Sepharose. The final enriched kinase preparation consists primarily of p34cdc2 and polypeptides of 65 and 110 kd. The purified kinase complex phosphorylates vimentin in vitro at a subset of sites phosphorylated in vivo during mitosis. Furthermore, phosphorylation of in vitro polymerized vimentin IFs by the purified kinase causes their disassembly. Therefore, vimentin is a substrate of p34cdc2 and phosphorylation of vimentin contributes to M phase reorganization of the IF network.     

Changes in regulatory phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during normal cell cycle progression and checkpoint arrests

Entry into mitosis is catalyzed by cdc2 kinase. Previous work identified the cdc2-activating phosphatase cdc25C and the cdc2-inhibitory kinase wee1 as targets of the incomplete replication-induced kinase Chk1. Further work led to the model that checkpoint kinases block mitotic entry by inhibiting cdc25C through phosphorylation on Ser287 and activating wee1 through phosphorylation on Ser549. However, almost all conclusions underlying this idea were drawn from work using recombinant proteins. Here, we report that in the early Xenopus egg cell cycles, phosphorylation of endogenous cdc25C Ser287 is normally high during interphase and shows no obvious increase after checkpoint activation. By contrast, endogenous wee1 Ser549 phosphorylation is low during interphase and increases after activation of either the DNA damage or replication checkpoints; this is accompanied by a slight increase in wee1 kinase activity. Blocking mitotic entry by adding the catalytic subunit of PKA also results in increased wee1 Ser549 phosphorylation and maintenance of cdc25C Ser287 phosphorylation. These results argue that in response to checkpoint activation, endogenous wee1 is indeed a critical responder that functions by repressing the cdc2-cdc25C positive feedback loop. Surprisingly, endogenous wee1 Ser549 phosphorylation is highest during mitosis just after the peak of cdc2 activity. Treatments that block inactivation of cdc2 result in further increases in wee1 Ser549 phosphorylation, suggesting a previously unsuspected role for wee1 in mitosis.     

BLM, the Bloom's syndrome protein, varies during the cell cycle in its amount, distribution, and co-localization with other nuclear proteins

BLM, the protein encoded by the gene mutated in Bloom's syndrome (BS), is a phylogenetically highly conserved DNA helicase that varies in amount and distribution in the nucleus during the cell-division cycle. It is undetectable in many cells as they emerge from mitosis but becomes abundant during G(1) and remains so throughout S, G(2), and mitosis. BLM is widely distributed throughout the nucleus but at certain times also becomes concentrated in foci that vary in number and size. It co-localizes transitorily with replication protein A (RPA) and promyelocytic leukemia protein (PML) nuclear bodies, and at times it enters the nucleolus. The observations support the hypothesis that BLM is distributed variously about the nucleus to manipulate DNA in some, very possibly several, nucleic acid transactions, when and where they take place. The specific transaction(s) remain to be identified. Although absence from the nucleus of functional BLM - the situation in BS - obviously is not lethal in the human, other helicases would appear to be unable to substitute for it completely, witness the hypermutability and hyperrecombinability of BS cells.