Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27(kip1) with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2-Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad-Max complexes and de-repressed by Myc via a single highly conserved E-box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase-dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc-dependent dissociation of p27 from Cdk2 and Myc-induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2-deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.
- MeSH
- Cell Line MeSH
- Cell Cycle physiology MeSH
- 3T3 Cells MeSH
- Cyclin D2 MeSH
- Cyclins genetics MeSH
- DNA Primers MeSH
- Cyclin-Dependent Kinase Inhibitor p27 MeSH
- Rats MeSH
- Humans MeSH
- Mice MeSH
- Tumor Suppressor Proteins * MeSH
- Microtubule-Associated Proteins metabolism MeSH
- Cell Cycle Proteins * MeSH
- Proto-Oncogene Proteins c-myc physiology MeSH
- Gene Expression Regulation physiology MeSH
- Base Sequence MeSH
- Sequence Homology, Nucleic Acid MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2.
- MeSH
- Cell Line MeSH
- Cell Cycle MeSH
- COS Cells MeSH
- Cyclin A metabolism MeSH
- Cyclin-Dependent Kinase 2 MeSH
- Cyclin-Dependent Kinases metabolism MeSH
- DNA Primers genetics MeSH
- Phosphorylation MeSH
- Nuclear Proteins chemistry genetics metabolism MeSH
- CDC2-CDC28 Kinases * MeSH
- Rats MeSH
- Humans MeSH
- Protein Serine-Threonine Kinases metabolism MeSH
- Protein Kinases metabolism MeSH
- Cell Cycle Proteins chemistry genetics metabolism MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- S Phase MeSH
- Base Sequence MeSH
- Subcellular Fractions metabolism MeSH
- In Vitro Techniques MeSH
- Transfection MeSH
- Binding Sites MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cellular signal transduction cascades triggered by mitogenic or antiproliferative cues eventually converge on a biochemical mechanism centered around the retinoblastoma tumor suppressor (pRb), the so-called RB pathway that governs G1-phase progression and guards the commitment to enter S phase. pRb, together with its immediate upstream regulators, the D-type cyclins, their partner cyclin-dependent kinases Cdk4 and Cdk6, and the Cdk inhibitors, form a functional unit that is involved in major decisions about cellular fate, and whose components, including the proto-oncogenic cyclin D-dependent kinases, are commonly deregulated in many types of cancer. We report here the production and characterization of a series of 12 monoclonal antibodies (MAbs) that specifically recognize either Cdk4 or Cdk6. These antibodies are proving to be invaluable molecular probes for defining abundance, subcellular localization, binding partners, and ultimately the function(s) of these cell cycle-regulatory kinases. Localization of the target epitopes was mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and two antibodies recognizing sequences adjacent to N-terminus of Cdk4 can discriminate between the wild-type protein and the oncogenic, melanoma-associated R24C mutant of this kinase. Individual antibodies of our panel recognize distinct pools of Cdk4/6, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, kinase assays, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Collectively, the antibodies described in this study provide the means for immunochemical analyses of the cyclin D-dependent kinases in human and animal cells, and represent useful molecular tools that should help better understand the biological roles of Cdk4 and Cdk6 in normal cell-cycle control, and their oncogenic activity in tumor cells.
- MeSH
- Cell Line MeSH
- Cyclin-Dependent Kinase 4 MeSH
- Cyclin-Dependent Kinase 6 MeSH
- Cyclin-Dependent Kinases chemistry genetics immunology MeSH
- DNA Primers genetics MeSH
- Hybridomas immunology MeSH
- Immunochemistry MeSH
- Immunohistochemistry MeSH
- Rats MeSH
- Humans MeSH
- Epitope Mapping MeSH
- Antibodies, Monoclonal * MeSH
- Mutation MeSH
- Mice MeSH
- Neoplasms enzymology genetics MeSH
- Protein Serine-Threonine Kinases chemistry immunology MeSH
- Proto-Oncogene Proteins * MeSH
- Retinoblastoma Protein metabolism MeSH
- Base Sequence MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Novel mRNA isoforms encoding the enamel matrix proteins amelin-1, amelin-2 and ameloblastin have been recently described. We have applied detailed immunohistochemical as well as non-radioactive in situ hybridization analyses to follow amelin-1 expression in developing rat incisors and molars. We constructed an expression vector, overproduced recombinant amelin in Escherichia coli and prepared an antibody. In addition to the previously reported amelin mRNA expression patterns in ameloblasts, the amelin message was also detected in pulpal mesenchymal cells including preodontoblasts and young odontoblasts. The signal in these cells persisted until deposition of mantle dentin became evident. The immunolocalization of amelin-1 in preodontoblasts and ameloblasts essentially followed the pattern of mRNA expression. The most intense staining was found in the enamel matrix adjacent to secretory ameloblasts. Focal accumulations of immunoreactive material were found at the dentinoenamel junction during the maturation stage. Also, using 5'-RACE (Rapid Amplification of cDNA Ends) we could confirm only amelin-1 and ameloblastin messages in the total RNA pool from rat molars and conclude that amelin-2 is a truncated form of ameloblastin. The sequential expression of amelin in mesenchymal and epithelial cells suggests it plays a role in cell differentiation during early tooth development.
- MeSH
- Ameloblasts metabolism MeSH
- DNA Primers genetics MeSH
- Epithelial Cells metabolism MeSH
- In Situ Hybridization MeSH
- Immunohistochemistry MeSH
- DNA, Complementary genetics MeSH
- Rats MeSH
- RNA, Messenger genetics MeSH
- Mesoderm metabolism MeSH
- Odontoblasts metabolism MeSH
- Odontogenesis * genetics MeSH
- Rats, Sprague-Dawley MeSH
- Dental Enamel Proteins * genetics metabolism MeSH
- Recombinant Fusion Proteins genetics MeSH
- Base Sequence MeSH
- Gene Expression Regulation, Developmental MeSH
- Dental Enamel cytology metabolism growth & development MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected-K166E, L568X, and 3121-2 A-->G (in homozygosity)-accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations.
- MeSH
- Alleles MeSH
- Asian People * genetics MeSH
- Cystic Fibrosis ethnology genetics MeSH
- DNA Primers MeSH
- Electrophoresis, Polyacrylamide Gel * methods MeSH
- Exons MeSH
- Humans MeSH
- Models, Genetic MeSH
- Mutation * genetics MeSH
- DNA Mutational Analysis * methods MeSH
- Polymerase Chain Reaction MeSH
- Cystic Fibrosis Transmembrane Conductance Regulator * genetics MeSH
- Retrospective Studies MeSH
- Sensitivity and Specificity MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
