High-intensity interval training in the form of isometric contraction improves fatigue resistance in dystrophin-deficient muscle.

Duchenne muscular dystrophy is a genetic muscle-wasting disorder characterized by progressive muscle weakness and easy fatigability. Here we examined whether high-intensity interval training (HIIT) in the form of isometric contraction improves fatigue resistance in skeletal muscle from dystrophin-deficient mdx52 mice. Isometric HIIT was performed on plantar flexor muscles in vivo with supramaximal electrical stimulation every other day for 4 weeks (a total of 15 sessions). In the non-trained contralateral gastrocnemius muscle from mdx52 mice, the decreased fatigue resistance was associated with a reduction in the amount of peroxisome proliferator-activated receptor γ coactivator 1-α, citrate synthase activity, mitochondrial respiratory complex II, LC3B-II/I ratio, and mitophagy-related gene expression (i.e. Pink1, parkin, Bnip3 and Bcl2l13) as well as an increase in the phosphorylation levels of Src Tyr416 and Akt Ser473, the amount of p62, and the percentage of Evans Blue dye-positive area. Isometric HIIT restored all these alterations and markedly improved fatigue resistance in mdx52 muscles. Moreover, an acute bout of HIIT increased the phosphorylation levels of AMP-activated protein kinase (AMPK) Thr172, acetyl CoA carboxylase Ser79, unc-51-like autophagy activating kinase 1 (Ulk1) Ser555, and dynamin-related protein 1 (Drp1) Ser616 in mdx52 muscles. Thus, our data show that HIIT with isometric contractions significantly mitigates histological signs of pathology and improves fatigue resistance in dystrophin-deficient muscles. These beneficial effects can be explained by the restoration of mitochondrial function via AMPK-dependent induction of the mitophagy programme and de novo mitochondrial biogenesis. KEY POINTS: Skeletal muscle fatigue is often associated with Duchenne muscular dystrophy (DMD) and leads to an inability to perform daily tasks, profoundly decreasing quality of life. We examined the effect of high-intensity interval training (HIIT) in the form of isometric contraction on fatigue resistance in skeletal muscle from the mdx52 mouse model of DMD. Isometric HIIT counteracted the reduced fatigue resistance as well as dystrophic changes in skeletal muscle of mdx52 mice. This beneficial effect could be explained by the restoration of mitochondrial function via AMP-activated protein kinase-dependent mitochondrial biogenesis and the induction of the mitophagy programme in the dystrophic muscles.

Improved skeletal muscle fatigue resistance in experimental autoimmune myositis mice following high-intensity interval training.

BACKGROUND: Muscle weakness and decreased fatigue resistance are key manifestations of systemic autoimmune myopathies (SAMs). We here examined whether high-intensity interval training (HIIT) improves fatigue resistance in the skeletal muscle of experimental autoimmune myositis (EAM) mice, a widely used animal model for SAM. METHODS: Female BALB/c mice were randomly assigned to control (CNT) or EAM groups (n = 28 in each group). EAM was induced by immunization with three injections of myosin emulsified in complete Freund's adjuvant. The plantar flexor (PF) muscles of mice with EAM were exposed to either an acute bout or 4 weeks of HIIT (a total of 14 sessions). RESULTS: The fatigue resistance of PF muscles was lower in the EAM than in the CNT group (P < 0.05). These changes were associated with decreased activities of citrate synthase and cytochrome c oxidase and increased expression levels of the endoplasmic reticulum stress proteins (glucose-regulated protein 78 and 94, and PKR-like ER kinase) (P < 0.05). HIIT restored all these alterations and increased the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and the mitochondrial electron transport chain complexes (I, III, and IV) in the muscles of EAM mice (P < 0.05). CONCLUSIONS: HIIT improves fatigue resistance in a SAM mouse model, and this can be explained by the restoration of mitochondria oxidative capacity via inhibition of the ER stress pathway and PGC-1α-mediated mitochondrial biogenesis.

Larger improvements in fatigue resistance and mitochondrial function with high- than with low-intensity contractions during interval training of mouse skeletal muscle.

Interval training (IT) results in improved fatigue resistance in skeletal muscle mainly due to an increased aerobic capacity, which involves increased muscle mitochondrial content and/or improved mitochondrial function. We hypothesized that IT with high-intensity contractions is more effective in increasing mitochondrial function, and hence fatigue resistance, than low-intensity contractions. To study this hypothesis without interference from differences in muscle fiber recruitment obliged to occur during voluntary contractions, IT was performed with in situ supramaximal electrical stimulation where all muscle fibers are recruited. We compared the effect of IT with repeated low-intensity (20 Hz stimulation, IT20) and high-intensity (100 Hz stimulation, IT100) contractions on fatigue resistance and mitochondrial content and function in mouse plantar flexor muscles. Muscles were stimulated every other day for 4 weeks. The averaged peak torque during IT bouts was 4.2-fold higher with IT100 than with IT20. Both stimulation protocols markedly improved in situ fatigue resistance, although the improvement was larger with IT100. The citrate synthase activity, a biomarker of mitochondrial content, was similarly increased with IT20 and IT100. Conversely, increased expression of mitochondrial respiratory chain (MRC) complexes I, III, and IV was only observed with IT100 and this was accompanied by increases in MRC supercomplex formation and pyruvate-malate-driven state 3 respiration in isolated mitochondria. In conclusion, the IT-induced increase in fatigue resistance is larger with high-intensity than with low-intensity contractions and this is linked to improved mitochondrial function due to increased expression of MRC complexes and assembly of MRC supercomplexes.

Preconditioning contractions prevent prolonged force depression and Ca2+-dependent proteolysis of STAC3 after damaging eccentric contractions.

Preconditioning contractions (PCs) have been shown to markedly improve recovery from eccentric contractions (ECCs)-induced force depression. We here examined the mechanism behind the effects of PCs with focusing on the SH3 and cysteine-rich domain 3 (STAC3) that is essential for coupling membrane depolarization to Ca2+ release from the sarcoplasmic reticulum. Rat medial gastrocnemius (MG) muscles were excised immediately (REC0), 1 day (REC1), and 4 days (REC4) after exposure to 100 repeated damaging ECCs in vivo. PCs with 10 repeated nondamaging ECCs were applied 2 days before the damaging ECCs. Damaging ECCs induced in vivo isometric torque depression at 50 and 100 Hz stimulation frequencies, which was accompanied by a significant decrease in the amount of full-length STAC3, an activation of calpain 1, and an increased number of Evans Blue dye-positive fibers in MG muscles at REC1 and REC4. Interestingly, PCs attenuated all these deleterious alterations induced by damaging ECCs. Moreover, mechanistic experiments performed on normal muscle samples exposed to various concentration of Ca2+ showed a Ca2+-dependent proteolysis of STAC3, which was prevented by calpain inhibitor MDL-28170. In conclusion, PCs may improve recovery from force depression after damaging ECCs, in part by inhibiting the loss of STAC3 due to the increased permeability of cell membrane and subsequent activation of calpain 1.NEW & NOTEWORTHY The SH3 and cysteine-rich domain 3 (STAC3) is a skeletal muscle-specific protein that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release. No studies, however, examined the role of STAC3 in protective effects of preconditioning contractions (PCs) against damaging eccentric contractions (ECCs). Here, we demonstrate that PCs may improve recovery from damaging ECCs-induced force depression, in part by an inhibition of Ca2+-dependent proteolysis of STAC3 due to increased membrane permeability and subsequent calpain 1 activation.

High-but not moderate-intensity exercise acutely attenuates hypercapnia-induced vasodilation of the internal carotid artery in young men.

PURPOSE: Exercise-induced increases in shear rate (SR) across different exercise intensities may differentially affect hypercapnia-induced vasodilation of the internal carotid artery (ICA), a potential index of cerebrovascular function. We aimed to elucidate the effects of exercise intensity on ICA SR during exercise and post-exercise hypercapnia-induced vasodilation of the ICA in young men. METHODS: Twelve healthy men completed 30 min of cycling at moderate [MIE; 65 ± 5% of age-predicted maximal heart rate (HRmax)] and high (HIE; 85 ± 5% HRmax) intensities. Hypercapnia-induced vasodilation was induced by 3 min of hypercapnia (target end-tidal partial pressure of CO2 + 10 mmHg) and was assessed at pre-exercise, 5 min and 60 min after exercise. Doppler ultrasound was used to measure ICA diameter and blood velocity during exercise and hypercapnia tests. RESULTS: SR was not altered during either exercise (interaction and main effects of time; both P > 0.05). ICA conductance decreased during HIE from resting values (5.1 ± 1.3 to 3.2 ± 1.0 mL·min-1·mmHg-1; P < 0.01) but not during MIE (5.0 ± 1.3 to 4.0 ± 0.8 mL·min-1·mmHg-1; P = 0.11). Consequently, hypercapnia-induced vasodilation declined immediately after HIE (6.9 ± 1.7% to 4.0 ± 1.4%; P < 0.01), but not after MIE (7.2 ± 2.1% to 7.3 ± 1.8%; P > 0.05). Sixty minutes after exercise, hypercapnia-induced vasodilation returned to baseline values in both trials (MIE 8.0 ± 3.1%; HIE 6.4 ± 2.9%; both P > 0.05). CONCLUSION: The present study showed blunted hypercapnia-induced vasodilation of the ICA immediately after high-intensity exercise, but not a moderate-intensity exercise in young men. Given that the acute response is partly linked to the adaptive response in the peripheral endothelial function, the effects of aerobic training on cerebrovascular health may vary depending on exercise intensity.

Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

VAB-10 spectraplakin acts in cell and nuclear migration in Caenorhabditis elegans.

Cytoskeletal regulation is important in cell migration. The Caenorhabditis elegans gonadal distal tip cells (DTCs) offer a simple model with which to investigate the mechanism of cell migration in organogenesis. Here, we report that one of the spectraplakin isoforms, VAB-10B1, plays an essential role in cell and nuclear migration of DTCs by regulating the actin and microtubule (MT) cytoskeleton. In the vab-10(tk27) mutant, which lacks VAB-10B1, alignment of filamentous (F)-actin and MTs was weakly and severely disorganized, respectively, which resulted in a failure to translocate the DTC nucleus and a premature termination of DTC migration. An MT growing-tip marker, EBP-2-GFP, revealed that polarized outgrowth of MTs towards the nuclei of migrating DTCs was strikingly impaired in tk27 animals. A vab-10 mini-gene encoding only the actin- and MT-binding domains significantly rescued the gonadal defects, suggesting that VAB-10B1 has a role in linking actin and MT filaments. These results suggest that VAB-10B1/spectraplakin regulates the polarized alignment of MTs, possibly by linking F-actin and MTs, which enables normal nuclear translocation and cell migration of DTCs.

C. elegans mig-6 encodes papilin isoforms that affect distinct aspects of DTC migration, and interacts genetically with mig-17 and collagen IV.

The gonad arms of C. elegans hermaphrodites acquire invariant shapes by guided migrations of distal tip cells (DTCs), which occur in three phases that differ in the direction and basement membrane substrata used for movement. We found that mig-6 encodes long (MIG-6L) and short (MIG-6S) isoforms of the extracellular matrix protein papilin, each required for distinct aspects of DTC migration. Both MIG-6 isoforms have a predicted N-terminal papilin cassette, lagrin repeats and C-terminal Kunitz-type serine proteinase inhibitory domains. We show that mutations affecting MIG-6L specifically and cell-autonomously decrease the rate of post-embryonic DTC migration, mimicking a post-embryonic collagen IV deficit. We also show that MIG-6S has two separable functions - one in embryogenesis and one in the second phase of DTC migration. Genetic data suggest that MIG-6S functions in the same pathway as the MIG-17/ADAMTS metalloproteinase for guiding phase 2 DTC migrations, and MIG-17 is abnormally localized in mig-6 class-s mutants. Genetic data also suggest that MIG-6S and non-fibrillar network collagen IV play antagonistic roles to ensure normal phase 2 DTC guidance.

MIG-17/ADAMTS controls cell migration by recruiting nidogen to the basement membrane in C. elegans.

Mutations in the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) family of secreted proteases cause diseases linked to ECM abnormalities. However, the mechanisms by which these enzymes modulate the ECM during development are mostly unexplored. The Caenorhabditis elegans MIG-17/ADAMTS protein is secreted from body wall muscle cells and localizes to the basement membrane (BM) of the developing gonad where it controls directional migration of gonadal leader cells. Here we show that specific amino acid changes in the ECM proteins fibulin-1C (FBL-1C) and type IV collagen (LET-2) result in bypass of the requirement for MIG-17 activity in gonadal leader cell migration in a nidogen (NID-1)-dependent and -independent manner, respectively. The MIG-17, FBL-1C and LET-2 activities are required for proper accumulation of NID-1 at the gonadal BM. However, mutant FBL-1C or LET-2 in the absence of MIG-17 promotes NID-1 localization. Furthermore, overexpression of NID-1 in mig-17 mutants substantially rescues leader cell migration defects. These results suggest that functional interactions among BM molecules are important for MIG-17 control of gonadal leader cell migration. We propose that FBL-1C and LET-2 act downstream of MIG-17-dependent proteolysis to recruit NID-1 and that LET-2 also activates a NID-1-independent pathway, thereby inducing the remodeling of the BM required for directional control of leader cell migration.

bHLH transcription factors regulate organ morphogenesis via activation of an ADAMTS protease in C. elegans.

The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases plays important roles in animal development and pathogenesis. However, transcriptional regulation of ADAMTS proteins during development remains largely unexplored. Here we show that basic helix-loop-helix (bHLH) transcription factors regulate the expression of an ADAMTS protease that is required for gonad development in Caenorhabditis elegans. Mutations in the gene mig-24 cause shortened and swollen gonad arms due to a defect in gonadal leader cell migration, although leader cell specification appears to occur normally. The MIG-24 protein is a bHLH transcription factor of the Achaete-Scute family and is specifically expressed in gonadal leader cells. MIG-24 can physically interact with HLH-2, an E/Daughterless family bHLH transcription factor and bind the promoter region of gon-1, which encodes an ADAMTS protease required for gonadal leader cell migration. Mutations or RNA interference of mig-24 and hlh-2 severely impaired gon-1 expression and forced expression of GON-1 in leader cells in mig-24 mutants partially rescued the gonadal elongation defect. We propose that, unlike most previously characterized Achaete-Scute transcription factors that are involved in cell fate specification, MIG-24 acts with HLH-2 in specified cells to control cell migration by activating the expression of the GON-1 ADAMTS protease.

Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin.

Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.

Isolation and characterization of the TIGA genes, whose transcripts are induced by growth arrest.

We report here the isolation of 44 genes that are upregulated after serum starvation and/or contact inhibition. These genes have been termed TIGA, after Transcript Induced by Growth Arrest. We found that there are two kinds of G0 phases caused by serum starvation, namely, the shallow G0 (or G0/G1) and the deep G0 phases. The shallow G0 is induced by only a few hours of serum starvation, while deep G0 is generated after 3 days of serum starvation. We propose that mammalian cells enter deep G0 through a G0 gate, which is only opened on the third day of serum starvation. TIGA1, one of the uncharacterized TIGA genes, encodes a homolog of cyanate permease of bacteria and localizes in mitochondria. This suggests that Tiga1 is involved in the inorganic ion transport and metabolism needed to maintain the deep G0 phase. Ectopic expression of TIGA1 inhibited not only tumor cell proliferation but also anchorage-independent growth of cancer cell lines. A microsatellite marker, ENDL-1, allowed us to detect loss of heterozygosity around the TIGA1 gene region (5q21-22). Further analysis of the TIGA genes we have identified here may help us to better understand the mechanisms that regulate the G0 phase.

Different requirements for the association of ATR-ATRIP and 9-1-1 to the stalled replication forks.

DNA replication checkpoint, a surveillance mechanism for S-phase progression, plays a crucial role for the maintenance of genome integrity. A variety of factors have been characterized to be involved in the checkpoint signal transduction. Rpa, a single strand DNA binding protein, was found to be responsible for forming a structure that is recognized by checkpoint sensors and then emits the initial signal for the activation of DNA damage checkpoint. Here we use a mutant of rpa1 gene, rfa1-t11, that has defects in recruiting checkpoint sensor proteins to the site of double strand break, to examine the mutant's effects on the activation of DNA replication checkpoint. We found that the mutant cells activated DNA replication checkpoint normally and showed no defects in recruiting ATR-ATRIP, a major sensor complex that is essential for DNA replication/damage checkpoint, to the site of stalled forks. In contrast, the mutant was defective in recruiting 9-1-1 complex, another sensor complex that functions in DNA damage checkpoint signal transduction, to the stalled forks. Moreover we found that sensitivity for HU obviously appeared in rfa1-t11 mutant when Mrc1 was deleted, while deletion of Rad9, an adaptor specific for damage checkpoint, had subtle effect. These data strongly suggest that rfa1-t11 mutant was mainly defective for activating DNA damage checkpoint and molecular requirement for the recruitment of ATR-ATRIP and 9-1-1 to the stressed forks may be different.

Microtubule bundle formation and cell death induced by the human CLASP/Orbit N-terminal fragment.

Previously we have identified the Drosophila orbit gene whose hypomorphic mutations cause abnormal chromosome segregation (Inoue et al., 2000). The orbit encodes Orbit/Mast, a 165-kDa microtubule-associated protein (MAP) with GTP-binding motifs. Two human homologues of the Orbit/Mast, CLASP1 (hOrbit1) and CLASP2 (hOrbit2) have been identified. Using an antibody to CLASP1/hOrbit1 polypeptide, we confirmed that the polypeptide of about 150 kDa associates with microtubule purified from the porcine brain. Thus, we conjectured that CLASP1 may be a human orthologue of the Drosophila Orbit/Mast, and therefore we named it h (human) Orbit1. We constructed the plasmid for expression of a fusion protein of the putative microtubule-binding domain (1-662 out of 1289 residues) of hOrbit1 and the green fluorescent protein (GFP), and then, transfected the plasmid into Tet off cells derived from HeLa cell. Confocal laser scanning microscopic observation revealed that the GFP-fluorescence associated with short and thin filaments in the perinuclear region during the short period after plasmid transfection, and colocalized with only part of the microtubules. GFP fluorescence was later detected on the abnormally longer and thick bundles of microtubule filaments. Finally the bundles formed networks in the perinuclear region. The results suggest that the GFP-hOrbit1 N-terminal fragment (GFP-hOrbit1 NF) binds to the newly formed microtubules rather than the pre-formed ones, and that displacement of the endogenous hOrbit by the fragment might cause abnormal bundling of microtubules. Interestingly, the expression of the GFP-hOrbit1 NF results in cell death associated with nuclear fragmentation.

DNA polymerase lambda directly binds to proliferating cell nuclear antigen through its confined C-terminal region.

DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases. Here, we show that Pol lambda directly binds to proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA replication and repair enzymes, both in vitro and in vivo. A pull-down assay using deletion mutants of Pol lambda showed that the confined C-terminal region of Pol lambda directly binds to PCNA. Furthermore, a synthetic peptide of 20-mers derived from the C-terminal region of Pol lambda competes with full-length Pol lambda for binding to PCNA. The residues between amino acids 518 and 537 of Pol lambda are required for binding to PCNA, and are different from the consensus PCNA interacting motif (PIM). Pol lambda associates with PCNA in vivo by immunoprecipitation analysis and EGFP-tagged Pol lambda co-localizes with PCNA as spots within a nucleus using fluorescent microscopy. Through direct binding, PCNA suppressed the distributive nucleotidyltransferase activity of Pol lambda. Pol micro, which also belongs to the family X of DNA polymerases, binds to PCNA by a pivotal amino acid residue.

Two functional coding single nucleotide polymorphisms in STK15 (Aurora-A) coordinately increase esophageal cancer risk.

STK15/Aurora-A is a serine/threonine kinase essential for chromosome segregation and cytokinesis, and is considered to be a cancer susceptibility gene in mice and humans. Two coding single nucleotide polymorphisms in Aurora-A, 91T>A [phenylalanine/isoleucine (F/I)] and 169G>A [valine/isoleucine (V/I)], create four haplotypes, 91T-169G, 91A-169G, 91T-169A, and 91A-169A. We evaluated the association between these coding single nucleotide polymorphisms and esophageal cancer risk by genotyping 197 esophageal cancer cases and 146 controls. Haplotype 91A-169A (I31/I57) was observed to be statistically more frequent in cancer cases (odds ratio, 3.1452; 95% confidence interval, 1.0258-9.6435). Functional differences among the four isoforms were then analyzed to reveal the source of the cancer risk. Kinase activity levels of I31/I57 and F31/I57 were reduced to 15% and 40% compared with I31/V57 in vivo and in vitro. We considered the differences between the kinase activities and divided individuals into four categories of Aurora-A haplotype combination. Category I had 57.5% or less kinase activity compared with the most common category, category III, and had a significantly higher estimated cancer risk (odds ratio, 5.5328; 95% confidence interval, 1.8149-16.8671). Abnormal nuclear morphology, a characteristic of genomic instability, was observed to be 30 to 40 times more frequent in human immortalized fibroblast cells overexpressing I31/I57 or F31/I57 compared with the others. Furthermore, significantly higher levels of chromosomal instability were observed in cancers in category I (homozygote 91T-169A) than those in category III (homozygous 91A-169G). These results indicate that the less kinase active Aurora-A haplotype combinations might induce genomic instability and increase esophageal cancer risk either in a recessive or a dominant manner.

Amino-terminal domain of ATRIP contributes to intranuclear relocation of the ATR-ATRIP complex following DNA damage.

ATM and rad3-related protein kinase (ATR), a member of the phosphoinositide kinase-like protein kinase family, plays a critical role in cellular responses to DNA structural abnormalities in conjunction with its interacting protein, ATRIP. Here, we show that the amino-terminal portion of ATRIP is relocalized to DNA damage-induced nuclear foci in an RPA-dependent manner, despite its lack of ability to associate with ATR. In addition, ATR-free ATRIP protein can be recruited to the nuclear foci. Our results suggest that the N-terminal domain of the ATRIP protein contributes to the cell cycle checkpoint by regulating the intranuclear localization of ATR.

The centrosomal protein Lats2 is a phosphorylation target of Aurora-A kinase.

Human Lats2, a novel serine/threonine kinase, is a member of the Lats kinase family that includes the Drosophila tumour suppressor lats/warts. Lats1, a counterpart of Lats2, is phosphorylated in mitosis and localized to the mitotic apparatus. However, the regulation, function and intracellular distribution of Lats2 remain unclear. Here, we show that Lats2 is a novel phosphorylation target of Aurora-A kinase. We first showed that the phosphorylated residue of Lats2 is S83 in vitro. Antibody that recognizes this phosphorylated S83 indicated that the phosphorylation also occurs in vivo. We found that Lats2 transiently interacts with Aurora-A, and that Lats2 and Aurora-A co-localize at the centrosomes during the cell cycle. Furthermore, we showed that the inhibition of Aurora-A-induced phosphorylation of S83 on Lats2 partially perturbed its centrosomal localization. On the basis of these observations, we conclude that S83 of Lats2 is a phosphorylation target of Aurora-A and this phosphorylation plays a role of the centrosomal localization of Lats2.

Regulation of checkpoint kinases through dynamic interaction with Crb2.

ATR/Rad3-like kinases promote the DNA damage checkpoint through regulating Chk1 that restrains the activation of cyclin-dependent kinases. In fission yeast, Crb2, a BRCT-domain protein that is similar to vertebrate 53BP1, plays a crucial role in establishing this checkpoint. We report here that Crb2 regulates DNA damage checkpoint through temporal and dynamic interactions with Rad3, Chk1 and replication factor Cut5. The active complex formation between Chk1 and Crb2 is regulated by Rad3 and became maximal during the checkpoint arrest. Chk1 activation seems to need two steps of interaction changes: the loss of Rad3-Chk1 and Rad3-Crb2 interactions, and the association between hyperphosphorylated forms of Chk1 and Crb2. Chk1 is the major checkpoint kinase for the arrest of DNA polymerase mutants. The in vitro assay of Chk1 showed that its activation requires the presence of Crb2 BRCT. Hyperphosphorylation of Crb2 is also dependent on its intact BRCT. Finally, we show direct interaction between Rad3 and Crb2, which is inhibitory to Rad3 activity. Hence, Crb2 is the first to interact with both Rad3 and Chk1 kinases.