Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chromatin condensation and nuclear envelope breakdown are characteristic features of apoptotic cell death, but the mechanisms underlying these phenomena have not been identified. Solubilization of nuclear lamin is responsible for both events in mitosis. In this work, we report that glucocorticoids stimulate rapid degradation of lamin B1 that occurs before oligonucleosomal DNA fragmentation in apoptotic thymocytes. Protease inhibitors and the Ca2+ buffering agent BAPTA-AM block lamin degradation and DNA fragmentation, indicating that the processes are regulated by similar or identical mechanisms. Incubation of isolated thymocyte nuclei with Ca2+ stimulates lamin degradation before the detection of oligonucleosomal DNA fragments. However, in contrast to lamin dissolution during mitosis and some other forms of apoptosis, glucocorticoid-induced degradation of lamin B1 in thymocytes is not accompanied by dephosphorylation-mediated activation of cdc2. Our results demonstrate that lamin degradation is an early feature of apoptosis in thymocytes and suggest that chromatin condensation and breakdown of the nuclear envelope may occur as a result of disruption of nuclear lamina architecture.
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PMID:Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei. 753 14

Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane, which binds directly to both lamin B1 and chromosomes in a mitotic phosphorylation-regulated manner. The biochemical and physiological properties of LAP2 suggest an important role in nuclear envelope re-assembly at the end of mitosis and/or anchoring of the nuclear lamina and interphase chromosomes to the nuclear envelope. We describe the cDNA cloning of LAP2 and characterization of its membrane topology and targeting to the nuclear envelope. The LAP2 cDNA sequence predicts a protein of 452 amino acids, containing a large hydrophilic domain with several potential cdc2 kinase phosphorylation sites and a single putative membrane-spanning sequence at residues 410-433. Immunogold localization of an LAP2 epitope in isolated nuclear envelopes indicates that the large amino-terminal hydrophilic domain (residues 1-409) is exposed to the nucleoplasm. By expressing deletion mutants of LAP2 in cultured cells, we have identified multiple regions in its nucleoplasmic domain that promote localization at the nuclear envelope. These data suggest that targeting of LAP2 to the nuclear envelope is mediated by cooperative interactions with multiple binding sites at the inner nuclear membrane.
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PMID:Cloning of a cDNA for lamina-associated polypeptide 2 (LAP2) and identification of regions that specify targeting to the nuclear envelope. 773 15

Protein kinase C (PKC) is activated at the nuclear membrane in response to a variety of mitogenic stimuli. In human leukemic cells, the beta II PKC isotype is selectively translocated and activated at the nucleus. We recently identified the nuclear envelope component lamin B1 as a major substrate for nuclear PKC both in whole cells and in vitro. Using highly purified human beta II PKC and isolated nuclear envelopes from the human promyelocytic (HL60) leukemia cell line, we have now determined the major sites for beta II PKC-mediated lamin B phosphorylation. Using a combination of cyanogen bromide cleavage, direct microsequencing, tryptic phosphopeptide, and phosphate release analyses, two major sites of PKC-mediated phosphorylation, Ser395 and Ser405, have been identified. These sites lie within the carboxyl-terminal domain of lamin B immediately adjacent to the central alpha-helical rod domain. Functionally, beta II PKC-mediated phosphorylation of these sites leads to the time-dependent solubilization of lamin B indicative of mitotic nuclear envelope breakdown in vitro. beta II PKC-mediated lamin B phosphorylation is inhibited by 1) a monoclonal antibody directed against the active site of PKC, 2) a PKC pseudosubstrate inhibitor peptide, and 3) a PKC peptide substrate. Two observations indicate that PKC-mediated lamin B phosphorylation and solubilization is due to direct phosphorylation of lamin B by PKC rather than indirect activation of a cdc2 kinase. Neither immunodepletion with p13suc1 Sepharose beads nor the presence of a p34cdc2 kinase peptide substrate had any effect on PKC-mediated lamin B phosphorylation. Therefore, we conclude that beta II PKC represents a physiologically relevant lamin kinase that can directly modulate nuclear lamina structure in vitro. Nuclear beta II PKC, like p34cdc2 kinase, may function to regulate nuclear lamina structural stability during cell cycle.
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PMID:Identification of protein kinase C (PKC) phosphorylation sites on human lamin B. Potential role of PKC in nuclear lamina structural dynamics. 846 84

We report the first RNAi-induced phenotypes in mammalian cultured cells using RNA interference mediated by duplexes of 21-nt RNAs. The 21 gene products studied have different functions and subcellular localizations. Knockdown experiments monitored by immunofluorescence and immunoblotting show that even major cellular proteins such as actin and vimentin can be silenced efficiently. Genes were classified as essential or nonessential depending on impaired cell growth after RNA silencing. Phenotypes also involved altered cell morphology and aberrant mitotic arrest. Among the essential genes identified by RNAi for which such information was previously not available are lamin B1, lamin B2, NUP153, GAS41, ARC21, cytoplasmic dynein, the protein kinase cdk1 and both beta- and gamma-actin. Newly defined nonessential genes are emerin and zyxin. Several genes previously characterized by other methods such as knockout of murine genes are included as internal controls and gave identical results when RNAi was used. In the case of two nonessential genes (lamin A/C and zyxin) RNAi provides a recognizable phenotype. Our results complete the characterization of the mammalian nuclear lamins. While lamins A/C appear as nonessential proteins in the mouse embryo and in RNAi treated cultured cells, the two other lamins, B1 and B2, are now identified as essential proteins. Interestingly the inner nuclear membrane protein emerin, thought to be a ligand of lamin A/C, is also a nonessential protein in tissue culture cells.
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PMID:Identification of essential genes in cultured mammalian cells using small interfering RNAs. 1179 20

The role of protein kinase C-beta(II) (PKC-beta(II)) in etoposide (VP-16)-induced apoptosis was studied using polyomavirus-transformed pyF111 rat fibroblasts in which PKC-beta(II) specific activity in the nuclear membrane (NM) doubled and the enzyme was cleaved into catalytic fragments. No PKC-beta(II) complexes with lamin B1 and/or active caspases were immunoprecipitable from the NM of proliferating untreated cells, but large complexes of PKC-beta(II) holoprotein and its catalytic fragments with lamin B1, active caspase-3 and -6, and inactive phospho-CDK-1, but not PKC-beta(I) or PKC-delta, could be immunoprecipitated from the NM of VP-16-treated cells, suggesting that PKC-beta(II) is an apoptotic lamin kinase. By 30 min after normal nuclei were mixed with cytoplasms from VP-16-treated, but not untreated, cells, PKC-beta(II) holoprotein had moved from the apoptotic cytoplasm to the normal NM, and lamin B1 was phosphorylated before cleavage by caspase-6. Lamin B1 phosphorylation was partly reduced, but its cleavage was completely prevented, despite the presence of active caspase-6, by adding a selective PKC-betas inhibitor, hispidin, to the apoptotic cytoplasms. Thus, a PKC-beta(II) response to VP-16 seems necessary for lamin B1 cleavage by caspase-6 and nuclear lamina dissolution in apoptosing pyF111 fibroblasts. The possibility of PKC-beta(II) being an apoptotic lamin kinase in these cells was further suggested by lamin B1-bound PKC-delta being inactive or only slightly active and by PKC-alpha not combining with the lamin.
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PMID:Protein kinase C-beta II Is an apoptotic lamin kinase in polyomavirus-transformed, etoposide-treated pyF111 rat fibroblasts. 1190 Nov 53

Most DNA synthesis in HeLa cell nucleus is concentrated in discrete foci. These synthetic sites can be identified by electron microscopy after allowing permeabilized cells to elongate nascent DNA in the presence of biotin-dUTP. Biotin incorporated into nascent DNA can be then immunolabeled with gold particles. Two types of DNA synthetic sites/replication factories can be distinguished at ultrastructural level: (1) electron-dense structures--replication bodies (RB), and (2) focal replication sites with no distinct underlying structure--replication foci (RF). The protein composition of these synthetic sites was studied using double immunogold labeling. We have found that both structures contain (a) proteins involved in DNA replication (DNA polymerase alpha, PCNA), (b) regulators of the cell cycle (cyclin A, cdk2), and (c) RNA processing components like Sm and SS-B/La auto antigens, p80-coilin, hnRNPs A1 and C1/C2. However, at least four regulatory and structural proteins (Cdk1, cyclin B1, PML and lamin B1) differ in their presence in RB and RF. Moreover, in contrast to RF, RB have structural organization. For example, while DNA polymerase alpha, PCNA and hnRNP A1 were diffusely spread throughout RB, hnRNP C1/C2 was found only at the very outside. Surprisingly, RB contained only small amounts of DNA. In conclusion, synthetic sites of both types contain similar but not the same sets of proteins. RB, however, have more developed microarchitecture, apparently with specific functional zones. This data suggest possible differences in genome regions replicated by these two types of replication factories.
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PMID:The microarchitecture of DNA replication domains. 1624 14