The kinase VRK1 is required for normal meiotic progression in mammalian oogenesis

The kinase VRK1 has been implicated in mitotic and meiotic progression in invertebrate species, but whether it mediates these events during mammalian gametogenesis is not completely understood. Previous work has demonstrated a role for mammalian VRK1 in proliferation of male spermatogonia, yet whether VRK1 plays a role in meiotic progression, as seen in Drosophila, has not been determined. Here, we have established a mouse strain bearing a gene trap insertion in the VRK1 locus that disrupts Vrk1 expression. In addition to the male proliferation defects, we find that reduction of VRK1 activity causes a delay in meiotic progression during oogenesis, results in the presence of lagging chromosomes during formation of the metaphase plate, and ultimately leads to the failure of oocytes to be fertilized. The activity of at least one phosphorylation substrate of VRK1, p53, is not required for these defects. These results are consistent with previously defined functions of VRK1 in meiotic progression in Drosophila oogenesis, and indicate a conserved role for VRK1 in coordinating proper chromosomal configuration in female meiosis.     

The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair

The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.     

The C terminus of mammalian phospholipase D is required for catalytic activity

The activity of phospholipase D (PLD) is regulated by a variety of hormonal stimuli and provides a mechanistic pathway for response of cells to extracellular stimuli. The two identified mammalian PLD enzymes possess highly homologous C termini, which are required for catalytic activity. Mutational analysis of PLD1 and PLD2 reveals that modification of as little as the C-terminal threonine or the addition of a single alanine attenuates activity of the enzyme. Protein folding appears to be intact because mutant enzymes express to similar levels in Sf9 cells and addition of peptides representing the C-terminal amino acids, including the simple hexamer PMEVWT, restores partial activity to several of the mutants. Analysis of several mutants suggests a requirement for the hydrophobic reside at the -2-position but not an absolute requirement for the hydroxyl side chain of threonine at the C terminus. The inability of peptides amidated at their C termini to effect restoration of activity indicates the involvement of the C-terminal alpha carboxyl group in functional activity of these enzymes. The ability of peptides to restore activity to PLD enzymes mutated at the C terminus suggests a flexible interaction of this portion of the molecule with a catalytic core constructed on conserved HKD motifs. Participation of these C termini residues in either stabilization of the catalytic site or the enzymatic reaction itself remains to be determined. This requirement for the C terminus provides an excellent potential site for interaction with regulatory proteins that may either enhance or down-regulate the activity of these enzymes in vitro.     

The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity

Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoyl-bearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoyl-bearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes.     

Protein kinase Cepsilon activates protein kinase B/Akt via DNA-PK to protect against tumor necrosis factor-alpha-induced cell death

We have previously shown that protein kinase Cepsilon (PKCepsilon) protects breast cancer cells from tumor necrosis factor-alpha (TNF)-induced cell death. In the present study, we have investigated if the antiapoptotic function of PKCepsilon is mediated via Akt and the mechanism by which PKCepsilon regulates Akt activity. TNF caused a transient increase in Akt phosphorylation at Ser473 in MCF-7 cells. Overexpression of PKCepsilon in MCF-7 cells increased TNF-induced Akt phosphorylation at Ser473 resulting in its activation. Knockdown of PKCepsilon by small interfering RNA (siRNA) decreased TNF-induced Akt phosphorylation/activation and increased cell death. Introduction of constitutively active Akt protected breast cancer MCF-7 cells from TNF-mediated cell death and partially restored cell survival in PKCepsilon-depleted cells. Depletion of Akt in MCF-7 cells abolished the antiapoptotic effect of PKCepsilon on TNF-mediated cell death. Akt was constitutively associated with PKCepsilon and DNA-dependent protein kinase (DNA-PK), and this association was increased by TNF treatment. Overexpression of PKCepsilon enhanced the interaction between Akt and DNA-PK. Knockdown of DNA-PK by siRNA inhibited TNF-induced Akt phosphorylation and the antiapoptotic effect of Akt and PKCepsilon. These results suggest that PKCepsilon activates Akt via DNA-PK to mediate its antiapoptotic function. Furthermore, we report for the first time that DNA-PK can regulate receptor-initiated apoptosis via Akt.     

The activity of the DNA-dependent protein kinase (DNA-PK) complex is determinant in the cellular response to nitrogen mustards

The DNA-dependent protein kinase plays a critical role in mammalian DNA double strand break (DSB) repair and in specialized recombination, such as lymphoid V(D)J recombination. Its regulatory subunit Ku (dimer of the Ku70 and Ku80 protein) binds to DNA and recruits the kinase catalytic sub-unit, DNA-PKcs. We show here that three different strains deficient in either the Ku80 (xrs-6) or DNA-PKcs (V-3, scid) component of DNA-PK are markedly sensitive (3.5- to 5-fold) to a group of DNA cross-linking agents, the nitrogen mustards (NMs) (melphalan and mechlorethamine) as compared to their parental cell line. Importantly, the level of hypersensitivity to these drugs was close to the level of hypersensitivity observed for radiomimetic agents that create DSBs in DNA (bleomycin and neocarzinostatin). In addition, sensitivity to NMs was restored to the parental level in the xrs-6 cell line stably transfected with the human Ku80 gene (xrs-6/Ku80), showing unequivocally that DNA-PK is involved in this phenotype. These results indicate that a function of the whole DNA-PK protein complex is involved in the cellular response to NMs and suggest that the repair of DNA interstrand cross-links induced in DNA by NMs involved a DNA-PK dependent pathway that shares common features with DNA DSBs repair.     

The meiosis-specific modification of mammalian telomeres

During meiosis, rapid chromosome movements within the nucleus enable homologous chromosomes to acquire physical juxtaposition. In most organisms, chromosome ends, telomeres, tethered to the transmembrane LINC-complex mediate this movement by transmitting cytoskeletal forces to the chromosomes. While the majority of molecular studies have been performed using lower eukaryotes as model systems, recent studies have identified mammalian meiotic telomere regulators, including the LINC-complex SUN1/KASH5 and the meiosis-specific telomere binding protein TERB1. This review highlights the molecular regulations of mammalian meiotic telomeres in comparison with other model systems and discusses some future perspectives.     

The epigenetic regulation of mammalian telomeres

Increasing evidence indicates that chromatin modifications are important regulators of mammalian telomeres. Telomeres provide well studied paradigms of heterochromatin formation in yeast and flies, and recent studies have shown that mammalian telomeres and subtelomeric regions are also enriched in epigenetic marks that are characteristic of heterochromatin. Furthermore, the abrogation of master epigenetic regulators, such as histone methyltransferases and DNA methyltransferases, correlates with loss of telomere-length control, and telomere shortening to a critical length affects the epigenetic status of telomeres and subtelomeres. These links between epigenetic status and telomere-length regulation provide important new avenues for understanding processes such as cancer development and ageing, which are characterized by telomere-length defects.     

The kinase activity of aurora B is required for kinetochore-microtubule interactions during mitosis

As a component of the "chromosomal passenger protein complex," the aurora B kinase is associated with centromeres during prometaphase and with midzone microtubules during anaphase and is required for both mitosis and cytokinesis. Ablation of aurora B causes defects in both prometaphase chromosomal congression and the spindle checkpoint; however, the mechanisms underlying these defects are unclear. To address this question, we have examined chromosomal movement, spindle organization, and microtubule motor distribution in NRK cells transfected with a kinase-inactive, dominant-negative mutant of aurora B, aurora B(K-R). In cells overexpressing aurora B(K-R) fused with GFP, centromeres moved in a synchronized and predominantly unidirectional manner, as opposed to the independent, bidirectional movement in control cells expressing a similar level of wild-type aurora B-GFP. In addition, most kinetochores became physically separated from spindle microtubules, which appeared as a striking bundle between the spindle poles. These defects were associated with a microtubule-dependent depletion of motor proteins dynein and CENP-E from kinetochores. Our observations suggest that aurora B regulates the association of motor proteins with kinetochores during prometaphase. Interactions of kinetochore motors with microtubules may in turn regulate the organization of microtubules, the movement of prometaphase chromosomes, and the release of the spindle checkpoint.     

Gradient of increasing Aurora B kinase activity is required for cells to execute mitosis

INCENP, Borealin, Survivin, and Aurora B kinase comprise the chromosomal passenger complex, an essential regulator of mitotic events. INCENP (inner centromere protein) binds and activates Aurora B through a feedback loop involving phosphorylation of a Thr-Ser-Ser (TSS) motif near the INCENP C terminus. Here, we have examined the role of the TSS motif in vertebrate cells using an DT40 INCENP(ON/OFF) conditional knock-out cell line in which mutants are expressed in the absence of wild-type INCENP. Our analysis confirms that regulated phosphorylation of the two serine residues (presumably by Aurora B) is critical for full activation of the kinase and is essential for cell viability. Cells expressing INCENP mutants bearing either phospho-null (TAA) or phospho-mimetic (TEE) mutations exhibit significant levels of Aurora B kinase activity but fail to undergo normal spindle elongation or complete cytokinesis. This work confirms previous suggestions that INCENP can act as a rheostat, with different INCENP mutants promoting differing degrees of kinase activation. Our results also reveal that mitotic progression is accompanied by a requirement for progressively higher levels of Aurora B kinase activity.     

Casein kinase II activity is required for transferrin receptor endocytosis.

The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis.     

Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment

Fray is a serine/threonine kinase expressed by the peripheral glia of Drosophila, whose function is required for normal axonal ensheathment. Null fray mutants die early in larval development and have nerves with severe swelling and axonal defasciculation. The phenotype is associated with a failure of the ensheathing glia to correctly wrap peripheral axons. When the fray cDNA is expressed in the ensheathing glia of fray mutants, normal nerve morphology is restored. Fray belongs to a novel family of Ser/Thr kinases, the PF kinases, whose closest relatives are the PAK kinases. Rescue of the Drosophila mutant phenotype with PASK, the rat homolog of Fray, demonstrates a functional homology among these proteins and suggests that the Fray signaling pathway is widely conserved.     

Cyclin-dependent kinase 2 (Cdk2) is required for centrosome duplication in mammalian cells

Centrosome duplication is indispensable for the formation of the bipolar mitotic spindle. Surprisingly, even if DNA replication or mitosis is inhibited, centrosome duplication can still occur [1] [2] [3] [4] [5]. Thus, it remains unknown how centrosome duplication is coordinated with the cell cycle. Here, we show that centrosome duplication requires cyclin-dependent kinase 2 (Cdk2) in mammalian cells. We have found that in Chinese hamster ovary (CHO) cells, whereas centrosome duplication is not inhibited by hydroxyurea (HU) treatment, which arrests the cells in S phase, it is inhibited by mimosine treatment, which arrests the cells in late G1 phase. Cdk2 activity was higher in HU-treated cells than in mimosine-treated cells. Remarkably, inhibition of the Cdk2 activity in HU-treated cells with butyrolactone I or roscovitine [6], or by expression of the Cdk inhibitor p21(Waf1/Cip1), blocked the continued centrosome duplication. Moreover, overexpression of Cdk2 reversed the inhibition of centrosome duplication by mimosine treatment. These results indicate a requirement of Cdk2 activity for centrosome duplication and therefore suggest an underlying mechanism for the coordination of centrosome duplication with the cell cycle.     

A novel ubiquitin-like domain in IkappaB kinase beta is required for functional activity of the kinase

Activation of NF-kappaB requires two highly related kinases named IKKalpha and IKKbeta that share identity in the nature and positioning of their structural domains. Despite their similarity, the kinases are functionally divergent, and we therefore sought to identify any structural features specific for IKKalpha or IKKbeta. We performed bioinformatics analysis, and we identified a region resembling a ubiquitin-like domain (UBL) that exists only in IKKbeta and that we named the UBL-like domain (ULD). Deletion of the ULD rendered IKKbeta catalytically inactive and unable to induce NF-kappaB activity, and overexpression of only the ULD dose-dependently inhibited tumor necrosis factor-alpha-induced NF-kappaB activity. The ULD could not be functionally replaced within IKKbeta by ubiquitin or the corresponding region of IKKalpha, whereas deletion of the equivalent section of IKKalpha did not affect its catalytic activity against IkappaBalpha or its activation by NF-kappaB-inducing kinase. We identified five residues conserved among the larger family of UBL-containing proteins and IKKbeta, and alanine scanning revealed that the leucine at position 353 (Leu(353)) is absolutely critical for IKKbeta-induced NF-kappaB activation. Most intriguingly, the L353A mutant was catalytically active but, unlike wild-type IKKbeta, formed a stable complex with the NF-kappaB p65 subunit. Our findings therefore establish the ULD as a critical functional domain specific for IKKbeta that might play a role in dissociating IKKbeta from p65.     

Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a "myosheet," was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.