ABSTRACT
Cdc25A is a tyrosine phosphatase that is involved in the regulation of the G1/S phase transition by activating cyclin E/Cdk2 and cyclin A/Cdk2 complexes through removal of inhibitory phosphorylations. The E6 and E7 oncoproteins of the high-risk human papillomaviruses (HPV) interact with and functionally abrogate the p53 and pRB proteins, respectively. In the present study we have investigated the regulation of the Cdc25A promoter during G1 and S-phases of the cell cycle and by the HPV-16 E7 oncoprotein. Serum induction leads to a derepression of the Cdc25A promoter and can be mediated through two E2F binding sites, E2F-A and E2F-C. While E2F-A is by both E2F1 and E2F4, E2F-C is regulated only by E2F1. The Cdc25A promoter is transactivated by the E7 oncogene of HPV-16. Furthermore, Cdc25A levels are highly increased in E7-expressing cell lines. Inducible expression of E7 leads to an immediate increase in Cdc25A protein levels. These data suggest that Cdc25A may be a critical target of HPV-16 E7 in the disruption of the G1/S phase transition.
Subject(s)
Carrier Proteins , Cell Cycle Proteins , Gene Expression Regulation, Enzymologic , Oncogene Proteins, Viral/genetics , cdc25 Phosphatases/genetics , 3T3 Cells , Animals , Base Sequence , Binding Sites , Cell Cycle/genetics , Consensus Sequence , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , E2F Transcription Factors , E2F1 Transcription Factor , E2F4 Transcription Factor , Humans , Mice , Molecular Sequence Data , Papillomaviridae/genetics , Papillomavirus E7 Proteins , Promoter Regions, Genetic , Retinoblastoma-Binding Protein 1 , Transcription Factor DP1 , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , cdc25 Phosphatases/biosynthesisABSTRACT
The cell adhesion molecule L1, a 200-220-kDa type I membrane glycoprotein of the Ig superfamily, mediates many neuronal processes. Originally studied in the nervous system, L1 is expressed by hematopoietic and many epithelial cells, suggesting a more expanded role. L1 supports homophilic L1-L1 and integrin-mediated cell binding and can also bind with high affinity to the neural proteoglycan neurocan; however, the binding site is unknown. We have dissected the L1 molecule and investigated the cell binding ability of Ig domains 1 and 6. We report that RGD sites in domain 6 support alpha5beta1- or alphavbeta3-mediated integrin binding and that both RGD sites are essential. Cooperation of RGD sites with neighboring domains are necessary for alpha(5)beta(1). A T cell hybridoma and activated T cells could bind to L1 in the absence of RGDs. This binding was supported by Ig domain 1 and mediated by cell surface-exposed neurocan. Lymphoid and brain-derived neurocan were structurally similar. We also present evidence that a fusion protein of the Ig 1-like domain of L1 can bind to recombinant neurocan. Our results support the notion that L1 provides distinct cell binding sites that may serve in cell-cell or cell-matrix interactions.
Subject(s)
Chondroitin Sulfate Proteoglycans/metabolism , Integrins/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Neural Cell Adhesion Molecules/metabolism , Animals , Binding Sites , Cations/metabolism , Fluorescent Antibody Technique , Glycoside Hydrolases/pharmacology , Heparin/pharmacology , Hybridomas/metabolism , Immunoglobulins/chemistry , Lectins, C-Type , Leukocyte L1 Antigen Complex , Membrane Glycoproteins/genetics , Mice , Mutagenesis, Site-Directed , Neural Cell Adhesion Molecules/genetics , Neurocan , Oligopeptides/chemistry , Oligopeptides/genetics , Protein Binding , Recombinant Proteins/metabolism , T-Lymphocytes/metabolismABSTRACT
We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.
Subject(s)
Cell Cycle Proteins/metabolism , Mitosis , Nuclear Proteins , Phosphoprotein Phosphatases/metabolism , cdc25 Phosphatases , Biological Transport , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/genetics , Cell Nucleus/enzymology , Cell Nucleus/genetics , Chromosomes/genetics , Chromosomes/metabolism , Cyclin B/genetics , Cyclin B/metabolism , Cyclin B1 , Cytoplasm/enzymology , DNA/biosynthesis , Dose-Response Relationship, Drug , Gene Expression , HeLa Cells , Humans , Interphase , Microinjections , Microscopy, Fluorescence , Mutation , Phosphoprotein Phosphatases/genetics , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Time FactorsABSTRACT
The L1 adhesion molecule is a member of the immunoglobulin superfamily which is expressed by neural and hematopoietic cells. L1 is primarily a cell surface molecule but in its released form it becomes embedded in the extracellular matrix. In addition to the established L1-L1 homotypic interaction, L1 can bind to alpha v beta 3 in the human. The 6th Ig-like domain is critical for this function. We now demonstrate that a fusion protein containing the 6th Ig-like domain of L1 (6.L1-Fc) can support the migration of human MED-B1 (alpha v beta 3+) but not of Nalm-6 cells (alpha 5 beta 1+). The migration was blocked in the presence of a mab to alpha v beta 3 and was not seen on a 6.L1-Fc in which the RGD site was mutated. Activation of human T lymphocytes in the presence of PHA and PMA led to the induction of alpha v beta 3 and alpha v beta 5 expression and concomitantly induced migration of the cells on 6.L1-Fc. The migration was blocked by mabs to alpha v beta 3 but not to alpha v beta 5. Our results suggest that L1 exposed at the cell surface or as a matrix constituent can serve as a potent substrate for alpha v beta 3 mediated cell migration.
