Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 19 de 19
Filter
Add more filters










Publication year range
2.
J Neurosci Res ; 100(1): 149-164, 2022 01.
Article in English | MEDLINE | ID: mdl-34520585

ABSTRACT

Opioids are commonly used for the treatment of postoperative and post-traumatic pain; however, their therapeutic effectiveness is limited by undesirable and life-threatening side effects. Researchers have long attempted to develop opioid co-administration therapies that enhance analgesia, but the complexity of opioid analgesia and our incomplete mechanistic understanding has made this a daunting task. We discovered that subanalgesic morphine doses (100 ng/kg-10 µg/kg) augmented the acute analgesic effect of fentanyl (20 µg/kg) following subcutaneous drug co-administration to male rats. In addition, administration of equivalent drug ratios to naïve rat spinal cord membranes induced a twofold increase in G protein activation. The rate of GTP hydrolysis remained unchanged. We demonstrated that these behavioral and biochemical effects were mediated by the delta opioid receptor (DOP). Subanalgesic doses of the DOP-selective agonist SNC80 also augmented the acute analgesic effect of fentanyl. Furthermore, co-administration of the DOP antagonist naltrindole with both fentanyl-morphine and fentanyl-SNC80 combinations prevented augmentation of both analgesia and G protein activation. The mu opioid receptor (MOP) antagonist cyprodime did not block augmentation. Confocal microscopy of the substantia gelatinosa of rats treated with fentanyl, subanalgesic morphine, or this combination showed that changes in MOP internalization did not account for augmentation effects. Together, these findings suggest that augmentation of fentanyl analgesia by subanalgesic morphine is mediated by increased G protein activation resulting from a synergistic interaction between or heterodimerization of MOPs and DOPs. This finding is of great therapeutic significance because it suggests a strategy for the development of DOP-selective ligands that can enhance the therapeutic index of clinically used MOP drugs.


Subject(s)
Analgesia , Morphine , Analgesics, Opioid/pharmacology , Animals , Fentanyl/pharmacology , Fentanyl/therapeutic use , Male , Morphine/pharmacology , Pain , Rats , Receptors, Opioid, delta , Receptors, Opioid, mu
3.
Hepatology ; 57(5): 2004-13, 2013 May.
Article in English | MEDLINE | ID: mdl-23300120

ABSTRACT

UNLABELLED: Functions of p53 during mitosis reportedly include prevention of polyploidy and transmission of aberrant chromosomes. However, whether p53 plays these roles during genomic surveillance in vivo and, if so, whether this is done via direct or indirect means remain unknown. The ability of normal, mature hepatocytes to respond to stimuli, reenter the cell cycle, and regenerate liver mass offers an ideal setting to assess mitosis in vivo. In quiescent liver, normally high ploidy levels in adult mice increased with loss of p53. Following partial hepatectomy, p53(-/-) hepatocytes exhibited early entry into the cell cycle and prolonged proliferation with an increased number of polyploid mitoses. Ploidy levels increased during regeneration of both wild-type (WT) and p53(-/-) hepatocytes, but only WT hepatocytes were able to dynamically resolve ploidy levels and return to normal by the end of regeneration. We identified multiple cell cycle and mitotic regulators, including Foxm1, Aurka, Lats2, Plk2, and Plk4, as directly regulated by chromatin interactions of p53 in vivo. Over a time course of regeneration, direct and indirect regulation of expression by p53 is mediated in a gene-specific manner. CONCLUSION: Our results show that p53 plays a role in mitotic fidelity and ploidy resolution in hepatocytes of normal and regenerative liver.


Subject(s)
Liver/pathology , Mitosis/physiology , Ploidies , Transcription, Genetic/physiology , Tumor Suppressor Protein p53/physiology , Animals , Cell Cycle/physiology , Cell Proliferation , Hepatectomy , Liver/physiology , Liver/surgery , Liver Regeneration/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics
4.
J Cell Sci ; 124(Pt 17): 2903-13, 2011 Sep 01.
Article in English | MEDLINE | ID: mdl-21878498

ABSTRACT

Mutations in the Caenorhabditis elegans separase gene, sep-1, are embryonic lethal. Newly fertilized mutant embryos have defects in polar body extrusion, fail to undergo cortical granule exocytosis, and subsequently fail to complete cytokinesis. Chromosome nondisjunction during the meiotic divisions is readily apparent after depletion of sep-1 by RNAi treatment, but much less so in hypomorphic mutant embryos. To identify factors that influence the activity of separase in cortical granule exocytosis and cytokinesis, we carried out a genetic suppressor screen. A mutation in the protein phosphatase 5 (pph-5) gene was identified as an extragenic suppressor of sep-1. This mutation suppressed the phenotypes of hypomorphic separase mutants but not RNAi depleted animals. Depletion of pph-5 caused no phenotypes on its own, but was effective in restoring localization of mutant separase to vesicles and suppressing cortical granule exocytosis and cytokinesis phenotypes. The identification of PPH-5 as a suppressor of separase suggests that a new phospho-regulatory pathway plays an important role in regulating anaphase functions of separase.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Cell Cycle Proteins/metabolism , Endopeptidases/metabolism , Nuclear Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Alleles , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Cell Cycle Proteins/genetics , Cytokinesis/genetics , Cytoplasmic Granules/genetics , Cytoplasmic Granules/metabolism , Endopeptidases/genetics , Exocytosis/physiology , Mutation , Nuclear Proteins/biosynthesis , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Phosphoprotein Phosphatases/biosynthesis , Phosphoprotein Phosphatases/deficiency , Phosphoprotein Phosphatases/genetics , Separase
5.
PLoS Genet ; 6(11): e1001218, 2010 Nov 24.
Article in English | MEDLINE | ID: mdl-21124864

ABSTRACT

The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3-dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.


Subject(s)
Anaphase , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/cytology , Caenorhabditis elegans/metabolism , Chromosome Segregation , Cyclin B/metabolism , Spindle Apparatus/metabolism , Animals , Caenorhabditis elegans/embryology , Cell Nucleus/metabolism , Cytoplasmic Dyneins/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Gene Deletion , Kinetochores/metabolism , Metaphase , RNA Interference , Time Factors
6.
PLoS One ; 4(10): e7450, 2009 Oct 14.
Article in English | MEDLINE | ID: mdl-19826475

ABSTRACT

The germinal center kinases (GCK) constitute a large, highly conserved family of proteins that has been implicated in a wide variety of cellular processes including cell growth and proliferation, polarity, migration, and stress responses. Although diverse, these functions have been attributed to an evolutionarily conserved role for GCKs in the activation of ERK, JNK, and p38 MAP kinase pathways. In addition, multiple GCKs from different species promote apoptotic cell death. In contrast to these paradigms, we found that a C. elegans GCK, GCK-1, functions to inhibit MAP kinase activation and apoptosis in the C. elegans germline. In the absence of GCK-1, a specific MAP kinase isoform is ectopically activated and oocytes undergo abnormal development. Moreover, GCK-1- deficient animals display a significant increase in germ cell death. Our results suggest that individual germinal center kinases act in mechanistically distinct ways and that these functions are likely to depend on organ- and developmental-specific contexts.


Subject(s)
Apoptosis , Caenorhabditis elegans Proteins/metabolism , Gene Expression Regulation, Enzymologic , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Caenorhabditis elegans , Cell Proliferation , Cytoplasm/metabolism , Enzyme Activation , Female , Germinal Center Kinases , Male , Models, Biological , Oocytes/metabolism , Protein Structure, Tertiary , RNA Interference
7.
Curr Biol ; 19(3): R127-9, 2009 Feb 10.
Article in English | MEDLINE | ID: mdl-19211053

ABSTRACT

Condensins are conserved protein complexes that play integral roles in chromosome dynamics during mitosis and meiosis. Caenorhabditis elegans has been thought to be unusual in that it appeared to lack a typical condensin I complex. However, recent biochemical excavating in the nematode has unearthed the 'missing' condensin I complex as well as the worm homologs of long-lost canonical condensin subunits.


Subject(s)
Adenosine Triphosphatases/metabolism , Caenorhabditis elegans/physiology , Cell Nucleus Division/physiology , Chromosomes/physiology , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Adenosine Triphosphatases/genetics , Animals , Caenorhabditis elegans/genetics , DNA-Binding Proteins/genetics , Models, Molecular , Multiprotein Complexes/genetics
8.
Genes Dev ; 22(20): 2869-85, 2008 Oct 15.
Article in English | MEDLINE | ID: mdl-18923084

ABSTRACT

The Shugoshin/Aurora circuitry that controls the timely release of cohesins from sister chromatids in meiosis and mitosis is widely conserved among eukaryotes, although little is known about its function in organisms whose chromosomes lack a localized centromere. Here we show that Caenorhabditis elegans chromosomes rely on an alternative mechanism to protect meiotic cohesin that is shugoshin-independent and instead involves the activity of a new chromosome-associated protein named LAB-1 (Long Arm of the Bivalent). LAB-1 preserves meiotic sister chromatid cohesion by restricting the localization of the C. elegans Aurora B kinase, AIR-2, to the interface between homologs via the activity of the PP1/Glc7 phosphatase GSP-2. The localization of LAB-1 to chromosomes of dividing embryos and the suppression of mitotic-specific defects in air-2 mutant embryos with reduced LAB-1 activity support a global role of LAB-1 in antagonizing AIR-2 in both meiosis and mitosis. Although the localization of a GFP fusion and the analysis of mutants and RNAi-mediated knockdowns downplay a role for the C. elegans shugoshin protein in cohesin protection, shugoshin nevertheless helps to ensure the high fidelity of chromosome segregation at metaphase I. We propose that, in C. elegans, a LAB-1-mediated mechanism evolved to offset the challenges of providing protection against separase activity throughout a larger chromosome area.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Gene Expression Regulation, Developmental , Protein Serine-Threonine Kinases/antagonists & inhibitors , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Aurora Kinase B , Aurora Kinases , Caenorhabditis elegans , Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans Proteins/genetics , Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation , DNA-Binding Proteins/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Fluorescent Antibody Technique , Immunoglobulin G/immunology , Meiosis/physiology , Meiotic Prophase I/physiology , Mitosis/physiology , Molecular Sequence Data , Multiprotein Complexes/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Helminth/genetics , RNA, Helminth/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Sister Chromatid Exchange , Cohesins
9.
Dev Cell ; 15(4): 603-16, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18854144

ABSTRACT

The Aurora B kinase is the enzymatic core of the chromosomal passenger complex, which is a critical regulator of mitosis. To identify novel regulators of Aurora B, we performed a genome-wide screen for suppressors of a temperature-sensitive lethal allele of the C. elegans Aurora B kinase AIR-2. This screen uncovered a member of the Afg2/Spaf subfamily of Cdc48-like AAA ATPases as an essential inhibitor of AIR-2 stability and activity. Depletion of CDC-48.3 restores viability to air-2 mutant embryos and leads to abnormally high AIR-2 levels at the late telophase/G1 transition. Furthermore, CDC-48.3 binds directly to AIR-2 and inhibits its kinase activity from metaphase through telophase. While canonical p97/Cdc48 proteins have been assigned contradictory roles in the regulation of Aurora B, our results identify a member of the Afg2/Spaf AAA ATPases as a critical in vivo inhibitor of this kinase during embryonic development.


Subject(s)
Adenosine Triphosphatases/metabolism , Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans/physiology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Adenosine Triphosphatases/genetics , Alleles , Amino Acid Substitution , Animals , Aurora Kinase B , Aurora Kinases , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Glutathione Transferase/metabolism , Lysine/metabolism , Mitosis , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , RNA Interference , Recombinant Proteins/metabolism , Temperature , Valosin Containing Protein
10.
J Biol Chem ; 283(19): 12763-8, 2008 May 09.
Article in English | MEDLINE | ID: mdl-18334486

ABSTRACT

The Aurora kinases comprise an evolutionarily conserved protein family that is required for a variety of cell division events, including spindle assembly, chromosome segregation, and cytokinesis. Emerging evidence suggests that once phosphorylated, a subset of Aurora substrates can enhance Aurora kinase activity. Our previous work revealed that the Caenorhabditis elegans Tousled-like kinase TLK-1 is a substrate and activator of the AIR-2 Aurora B kinase in vitro and that partial loss of TLK-1 enhances the mitotic defects of an air-2 mutant. However, given that these experiments were performed in vitro and with partial loss of function alleles in vivo, a necessary step forward in our understanding of the relationship between the Aurora B and Tousled kinases is to prove that TLK-1 expression is sufficient for Aurora B activation in vivo. Here, we report that heterologous expression of wild-type and kinase-inactive forms of TLK-1 suppresses the lethality of temperature-sensitive mutants of the yeast Aurora B kinase Ipl1. Moreover, kinase-dead TLK-1 associates with and augments the activity of Ipl1 in vivo. Together, these results provide critical and compelling evidence that Tousled has a bona fide kinase-independent role in the activation of Aurora B kinases in vivo.


Subject(s)
Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Animals , Aurora Kinases , Caenorhabditis elegans , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Enzyme Activation , Genes, Lethal/genetics , Intracellular Signaling Peptides and Proteins , Mutation/genetics , Protein Binding , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
11.
Development ; 133(4): 697-709, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16421191

ABSTRACT

Maturation promoting factor (MPF), a complex of cyclin-dependent kinase 1 and cyclin B, drives oocyte maturation in all animals. Mechanisms to block MPF activation in developing oocytes must exist to prevent precocious cell cycle progression prior to oocyte maturation and fertilization. This study sought to determine the developmental consequences of precociously activating MPF in oocytes prior to fertilization. Whereas depletion of Myt1 in Xenopus oocytes causes nuclear envelope breakdown in vitro, we found that depletion of the Myt1 ortholog WEE-1.3 in C. elegans hermaphrodites causes precocious oocyte maturation in vivo. Although such oocytes are ovulated, they are fertilization incompetent. We have also observed novel phenotypes in these precociously maturing oocytes, such as chromosome coalescence, aberrant meiotic spindle organization, and the expression of a meiosis II post-fertilization marker. Furthermore, co-depletion studies of CDK-1 and WEE-1.3 demonstrate that WEE-1.3 is dispensable in the absence of CDK-1, suggesting that CDK-1 is a major target of WEE-1.3 in C. elegans oocytes.


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/physiology , Oocytes/physiology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/physiology , Xenopus Proteins/genetics , Animals , CDC2 Protein Kinase/physiology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Chromosome Aberrations , Cyclin B/metabolism , Disorders of Sex Development , Female , Fertilization , Germ Cells , Maturation-Promoting Factor/antagonists & inhibitors , Maturation-Promoting Factor/physiology , Meiosis , Phenotype , Phosphorylation , RNA Interference , Tubulin/metabolism
12.
Cell ; 122(5): 723-34, 2005 Sep 09.
Article in English | MEDLINE | ID: mdl-16143104

ABSTRACT

A balance in the activities of the Ipl Aurora kinase and the Glc7 phosphatase is essential for normal chromosome segregation in yeast. We report here that this balance is modulated by the Set1 methyltransferase. Deletion of SET1 suppresses chromosome loss in ipl1-2 cells. Conversely, combination of SET1 and GLC7 mutations is lethal. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. We find that Set1 is required for methylation of conserved lysines in a kinetochore protein, Dam1. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. Our studies demonstrate that Set1 has important, unexpected functions in mitosis. Moreover, our findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function.


Subject(s)
Chromosome Segregation/physiology , DNA-Binding Proteins/physiology , Methyltransferases/physiology , Protein Kinases/physiology , Saccharomyces cerevisiae Proteins/physiology , Transcription Factors/physiology , Amino Acid Sequence , Aurora Kinases , Cell Cycle Proteins/metabolism , Chromosome Deletion , Chromosome Segregation/genetics , DNA-Binding Proteins/genetics , Gene Deletion , Histone-Lysine N-Methyltransferase , Intracellular Signaling Peptides and Proteins , Methylation , Methyltransferases/genetics , Microtubule-Associated Proteins/metabolism , Mitosis/genetics , Mitosis/physiology , Molecular Sequence Data , Mutation , Phenotype , Phosphorylation , Protein Kinases/genetics , Protein Serine-Threonine Kinases , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Transcription Factors/genetics
13.
Curr Biol ; 15(10): 894-904, 2005 May 24.
Article in English | MEDLINE | ID: mdl-15916946

ABSTRACT

BACKGROUND: The Aurora kinases control multiple aspects of mitosis, among them centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora activity is regulated in part by a subset of Aurora substrates that, once phosphorylated, can enhance Aurora kinase activity. Aurora A substrate activators include TPX2 and Ajuba, whereas the only known Aurora B substrate activator is the chromosomal passenger INCENP. RESULTS: We report that the C. elegans Tousled kinase TLK-1 is a second substrate activator of the Aurora B kinase AIR-2. Tousled kinase (Tlk) expression and activity have been linked to ongoing DNA replication, and Tlk can phosphorylate the chromatin assembly factor Asf. Here, we show that TLK-1 is phosphorylated by AIR-2 during prophase/prometaphase and that phosphorylation increases TLK-1 kinase activity in vitro. Phosphorylated TLK-1 increases AIR-2 kinase activity in a manner that is independent of TLK-1 kinase activity but depends on the presence of ICP-1/INCENP. In vivo, TLK-1 and AIR-2 cooperate to ensure proper mitotic chromosome segregation. CONCLUSIONS: The C. elegans Tousled kinase TLK-1 is a substrate and activator of the Aurora B kinase AIR-2. These results suggest that Tousled kinases have a previously unrecognized role in mitosis and that Aurora B associates with discrete regulatory complexes that may impart distinct substrate specificities and functions to the Aurora B kinase.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/physiology , Chromosome Segregation/physiology , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Animals , Aurora Kinase B , Blotting, Western , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA, Complementary/genetics , Immunohistochemistry , Immunoprecipitation , Phosphorylation , Plasmids/genetics , Protein Serine-Threonine Kinases/genetics , RNA Interference , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Two-Hybrid System Techniques
14.
Mol Biol Cell ; 16(2): 742-56, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15548597

ABSTRACT

BimC kinesins are required for mitotic spindle assembly in a variety of organisms. These proteins are localized to centrosomes, spindle microtubules, and the spindle midzone. We have previously shown that the Caenorhabditis elegans Aurora B kinase AIR-2 is required for the localization of the ZEN-4 kinesin protein to midzone microtubules. To determine whether the association of BimC kinesins with spindle microtubules is also dependent on AIR-2, we examined the expression pattern of BMK-1, a C. elegans BimC kinesin, in wild-type and AIR-2-deficient embryos. BMK-1 is highly expressed in the hermaphrodite gonad and is localized to meiotic spindle microtubules in the newly fertilized embryo. In mitotic embryos, BMK-1 is associated with spindle microtubules from prophase through anaphase and is concentrated at the spindle midzone during anaphase and telophase. In the absence of AIR-2, BMK-1 localization to meiotic and mitotic spindles is greatly reduced. This is not a consequence of loss of ZEN-4 localization because BMK-1 is appropriately localized in ZEN-4-deficient embryos. Furthermore, AIR-2 and BMK-1 directly interact with one another and the C-terminal tail domain of BMK-1 is specifically phosphorylated by AIR-2 in vitro. Together with our previous data, these results suggest that at least one function of the Aurora B kinases is to recruit spindle-associated motor proteins to their sites of action.


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/metabolism , Helminth Proteins/metabolism , Kinesins/physiology , Mitosis , Protein Serine-Threonine Kinases/physiology , Spindle Apparatus , Amino Acid Sequence , Animals , Aurora Kinase B , Aurora Kinases , Blotting, Western , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/metabolism , Conserved Sequence , Embryo, Nonmammalian , Glutathione Transferase/metabolism , Immunohistochemistry , Kinesins/metabolism , Mitogen-Activated Protein Kinase 7/biosynthesis , Mitogen-Activated Protein Kinase 7/chemistry , Mitogen-Activated Protein Kinase 7/genetics , Mitogen-Activated Protein Kinase 7/metabolism , Molecular Sequence Data , Phosphorylation , Point Mutation , Precipitin Tests , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , RNA Interference , Recombinant Proteins/biosynthesis , Sequence Homology, Amino Acid , Temperature , Threonine/chemistry , Two-Hybrid System Techniques
15.
Curr Biol ; 13(22): 1921-9, 2003 Nov 11.
Article in English | MEDLINE | ID: mdl-14614817

ABSTRACT

BACKGROUND: The Tousled kinases comprise an evolutionarily conserved family of proteins that have been previously implicated in chromatin remodeling, DNA replication, and DNA repair. Here, we used RNA mediated interference (RNAi) to determine the function of the C. elegans Tousled kinase (TLK-1) during embryonic development. RESULTS: TLK-1-deficient embryos arrested with a phenotype reminiscent of embryos that are broadly defective in transcription, and the expression of several reporter genes was dramatically reduced in tlk-1(RNAi) embryos. Furthermore, posttranslational modifications of RNA polymerase II (RNAPII) and histone H3 that have been correlated with transcription elongation, phosphorylation of the RNAPII CTD at Serine 2, and methylation of histone H3 at Lysine 36 were found at significantly reduced levels in tlk-1(RNAi) embryos as compared to wild-type. CONCLUSIONS: These results reveal a surprising requirement for a Tousled-like kinase in transcriptional regulation during development, likely during the elongation phase. In addition, our results confirm that the link between RNAPII phosphorylation and histone H3 methylation previously observed in budding yeast is functionally conserved in metazoans.


Subject(s)
Caenorhabditis elegans/enzymology , Methyltransferases/genetics , Phosphotransferases/genetics , Protein Serine-Threonine Kinases/physiology , RNA Polymerase II/genetics , Transcription, Genetic/genetics , Animals , Base Sequence , Blotting, Western , Caenorhabditis elegans/embryology , Caenorhabditis elegans Proteins , Chromatin/enzymology , Genes, Regulator/genetics , Histones/genetics , Immunohistochemistry , Methylation , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/genetics , RNA Interference , Sequence Analysis, DNA
16.
Development ; 130(8): 1605-20, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12620985

ABSTRACT

In C. elegans, mutants in the anaphase-promoting complex or cyclosome (APC/C) exhibit defects in germline proliferation, the formation of the vulva and male tail, and the metaphase to anaphase transition of meiosis I. Oocytes lacking APC/C activity can be fertilized but arrest in metaphase of meiosis I and are blocked from further development. To examine the cell cycle and developmental consequences of reducing but not fully depleting APC/C activity, we analyzed defects in embryos and larvae of mat-1/cdc-27 mutants grown at semi-permissive temperatures. Hypomorphic embryos developed to the multicellular stage but were slow to complete meiosis I and displayed aberrant meiotic chromosome separation. More severely affected embryos skipped meiosis II altogether and exhibited striking defects in meiotic exit. These latter embryos failed to produce normal eggshells or establish normal asymmetries prior to the first mitotic division. In developing larvae, extended M-phase delays in late-dividing cell lineages were associated with defects in the morphogenesis of the male tail. This study reveals the importance of dosage-specific mutants in analyzing molecular functions of a ubiquitously functioning protein within different cell types and tissues, and striking correlations between specific abnormalities in cell cycle progression and particular developmental defects.


Subject(s)
Caenorhabditis elegans/growth & development , Cell Cycle/physiology , Embryo, Nonmammalian/physiology , Larva/physiology , Ligases/metabolism , Ubiquitin-Protein Ligase Complexes , Amino Acid Sequence , Anaphase-Promoting Complex-Cyclosome , Animals , Animals, Genetically Modified , Caenorhabditis elegans/anatomy & histology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Disorders of Sex Development , Embryo, Nonmammalian/anatomy & histology , Larva/anatomy & histology , Ligases/genetics , Male , Molecular Sequence Data , Morphogenesis , Phenotype , RNA Interference , Sequence Alignment , Temperature , Transgenes
17.
Genesis ; 34(4): 244-50, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12434334

ABSTRACT

Many kinases are required for progression through the eukaryotic cell cycle. The Aurora kinases comprise a highly conserved family of serine/threonine kinases that have been implicated in chromosome segregation and cytokinesis in several organisms. We have isolated a sterile Caenorhabditis elegans mutant in which the majority of the locus encoding the Aurora A kinase air-1 has been deleted. Complementation tests with previously isolated sterile mutations in the air-1 genetic interval demonstrate that the air-1 and let-412 loci are identical. Previous analysis of AIR-1 function by RNA-mediated interference (RNAi) has shown that AIR-1 is required for embryonic survival. The characterization of the three sterile air-1 mutant alleles described here extends these studies by revealing an allelic series that differentially affects postembryonic cell divisions and germline development.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/growth & development , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Aurora Kinase A , Aurora Kinases , Caenorhabditis elegans/genetics , Cell Division , Central Nervous System/cytology , Central Nervous System/growth & development , Central Nervous System/metabolism , Female , Gene Expression Regulation, Developmental , Germ Cells/cytology , Germ Cells/metabolism , Humans , Molecular Sequence Data , Mutation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , RNA Interference , Sequence Homology, Amino Acid , Vulva/growth & development , Vulva/metabolism
18.
J Biol Chem ; 277(31): 27577-80, 2002 Aug 02.
Article in English | MEDLINE | ID: mdl-12048181

ABSTRACT

How the events of mitosis are coordinated is not well understood. Intriguing mitotic regulators include the chromosomal passenger proteins. Loss of either of the passengers inner centromere protein (INCENP) or the Aurora B kinase results in chromosome segregation defects and failures in cytokinesis. Furthermore, INCENP and Aurora B have identical localization patterns during mitosis and directly bind each other in vitro. These results led to the hypothesis that INCENP is a direct substrate of Aurora B. Here we show that the Caenorhabditis elegans Aurora B kinase AIR-2 specifically phosphorylated the C. elegans INCENP ICP-1 at two adjacent serines within the carboxyl terminus. Furthermore, the full length and a carboxyl-terminal fragment of ICP-1 stimulated AIR-2 kinase activity. This increase in AIR-2 activity required that AIR-2 phosphorylate ICP-1 because mutation of both serines in the AIR-2 phosphorylation site of ICP-1 abolished the potentiation of AIR-2 kinase activity by ICP-1. Thus, ICP-1 is directly phosphorylated by AIR-2 and functions in a positive feedback loop that regulates AIR-2 kinase activity. Since the Aurora B phosphorylation site within INCENP and the functions of INCENP and Aurora B have been conserved among eukaryotes, the feedback loop we have identified is also likely to be evolutionarily conserved.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Aurora Kinases , Caenorhabditis elegans/enzymology , Conserved Sequence , Cytoskeletal Proteins/metabolism , Mitosis , Molecular Sequence Data , Phosphorylation , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid
19.
J Cell Biol ; 157(2): 219-29, 2002 Apr 15.
Article in English | MEDLINE | ID: mdl-11940606

ABSTRACT

Accurate chromosome segregation during cell division requires not only the establishment, but also the precise, regulated release of chromosome cohesion. Chromosome dynamics during meiosis are more complicated, because homologues separate at anaphase I whereas sister chromatids remain attached until anaphase II. How the selective release of chromosome cohesion is regulated during meiosis remains unclear. We show that the aurora-B kinase AIR-2 regulates the selective release of chromosome cohesion during Caenorhabditis elegans meiosis. AIR-2 localizes to subchromosomal regions corresponding to last points of contact between homologues in metaphase I and between sister chromatids in metaphase II. Depletion of AIR-2 by RNA interference (RNAi) prevents chromosome separation at both anaphases, with concomitant prevention of meiotic cohesin REC-8 release from meiotic chromosomes. We show that AIR-2 phosphorylates REC-8 at a major amino acid in vitro. Interestingly, depletion of two PP1 phosphatases, CeGLC-7alpha and CeGLC-7beta, abolishes the restricted localization pattern of AIR-2. In Ceglc-7alpha/beta(RNAi) embryos, AIR-2 is detected on the entire bivalent. Concurrently, chromosomal REC-8 is dramatically reduced and sister chromatids are separated precociously at anaphase I in Ceglc-7alpha/beta(RNAi) embryos. We propose that AIR-2 promotes the release of chromosome cohesion via phosphorylation of REC-8 at specific chromosomal locations and that CeGLC-7alpha/beta, directly or indirectly, antagonize AIR-2 activity.


Subject(s)
Caenorhabditis elegans/cytology , Caenorhabditis elegans/enzymology , Chromosome Pairing , Chromosome Segregation , Chromosomes/metabolism , Meiosis , Protein Serine-Threonine Kinases/metabolism , Anaphase , Animals , Aurora Kinase B , Aurora Kinases , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/metabolism , Chromosomes/genetics , Helminth Proteins/metabolism , Metaphase , Microscopy, Fluorescence , Mutation , Oocytes , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Transport , RNA, Double-Stranded/metabolism , Sequence Homology, Nucleic Acid , Time Factors
SELECTION OF CITATIONS
SEARCH DETAIL
...