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V Baldin

Publications and source records attributed to V Baldin.

30 records · Page 2Linked to original sources
PubMed1995

Subcellular localisation of human wee1 kinase is regulated during the cell cycle.

Wee1 kinase-dependent phosphorylation of cdc2 maintains the cdc2/cyclin B complex in an inert form until it is activated by the cdc25 tyrosine phosphatase at the end of G2. As described for cdc25, cell cycle-linked changes in the intracellular localisation of wee1 may constitute an important aspect of the temporal regulation of cdc2 activity. Here we report that the subcellular distribution of human wee1 changes during the cell cycle in HeLa and IMR90 cells. During interphase, wee1 is found almost exclusively in the nucleus. When the cell enters mitosis, wee1 is relocalised into the cytoplasm. During cell division, wee1 becomes restricted to the mitotic equator and by the end of mitosis it is found exclusively in association with midbody bridges, a phenomenon that is dependent on microtubule assembly. The relocalisations of wee1 and its association with subcellular structures may play key regulatory roles at different stages of the cell cycle and during mitosis.

Biological Transport↗
PubMed1994

Characterization of an active GST-human Cdc2 fusion protein kinase expressed in the fission yeast Schizosaccharomyces pombe: a new approach to the study of cell cycle control proteins.

Characterization of cdk (cyclin dependent kinases) substrates and studies of their regulation require purified enzymatic complexes of cdc2-related catalytic and cyclin regulatory subunits. We produced human Cdc2 kinase in the fission yeast Schizosaccharomyces pombe as a fusion protein with glutathione S-transferase (GST). The GST-human Cdc2p fusion protein was active in vivo since it rescued a temperature-sensitive allele of cdc2. The fusion protein was purified using a one-step chromatography procedure with glutathione-Sepharose and exhibited a catalytic activity in vitro. Yeast cyclin B and suc1 were found in association with GST-Cdc2. A 17-fold stimulation of GST-Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with the S. pombe cellular extract prior to affinity purification. This indicates that cyclin concentration is limiting in this overexpression system. These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for biochemical studies.

CDC2 Protein Kinase↗
PubMed1994

Activation of rDNA transcription by FGF-2: key role of protein kinase CKII.

Basic Fibroblast Growth Factor-2 (FGF-2) promotes G1 to S transition of quiescent sparse adult bovine aortic endothelial cells. In addition to signal transduction through interaction with tyrosine kinase high affinity receptor, FGF-2 is translocated to the nucleus and accumulated into the nucleolus. These data suggest that FGF-2 functions directly in nuclear events. In vivo, correlations were established between the entrance of FGF-2 into the nucleus and an increase in rDNA transcription and in protein phosphorylation. In vitro, in experiments carried out with nuclei isolated from quiescent cells, addition of FGF-2 increases rDNA transcription by a factor of 5 and also increases protein phosphorylation. Nucleolin, a factor involved in control of rDNA transcription is preferentially phosphorylated. It has been shown that nucleolin and other factors implicated in rDNA transcription are substrates of protein kinase CKII. Using purified kinase CKII and nucleolin in an in vitro phosphorylation assay, we have shown that FGF-2 activates the protein kinase activity. These results suggest that FGF-2 could act as an activator of rDNA transcription through interactions with the protein kinase CKII.

Animals↗
PubMed1993

Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts.

In mammalian cells inhibition of the cdc2 function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of affinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis.

Amino Acid Sequence↗
PubMed1993

Cyclin D1 is a nuclear protein required for cell cycle progression in G1.

A cascade of events is triggered upon the addition of growth factor to quiescent mammalian cells, which ultimately restarts proliferation by inducing the transition from G0/G1 to S-phase. We have studied cyclin D1, a putative G1 cyclin, in normal diploid human fibroblasts. Cyclin D1 accumulated and reached a maximum level before S-phase upon the addition of serum to quiescent cells. The protein was localized to the nucleus, and it disappeared from the nucleus as cells proceeded into S-phase. Microinjection of anti-cyclin D1 antibodies or antisense plasmid prevented cells from entering S-phase, and the kinetics of inhibition showed that cyclin D1 is required at a point in the cell cycle earlier than cyclin A. These results demonstrate that cyclin D1 is a critical target of proliferative signals in G1.

Antisense Elements (Genetics)↗
PubMed1990

Glucocorticoids repress ribosome biosynthesis in lymphosarcoma cells by affecting gene expression at the level of transcription, posttranscription and translation.

Growth arrest of P1798 murine lymphosarcoma cells by glucocorticoids is accompanied by a remarkable decrease in transcription of rRNA and translation of mRNAs encoding basic ribosomal proteins (rps). Here we report that the expression of other genes involved in ribosome biogenesis is repressed in dexamethasone-treated P1798 cells. These include posttranscriptionally regulated decline in the abundance of the mRNA and primary transcript of nucleolin; abrupt drop in the transcription rate of U3 small nucleolar RNA; and inhibition of translation of mRNAs coding for P2 and L5, acidic and basic rps, respectively. Normal expression of these genes is resumed upon hormonal withdrawal.

Animals↗
PubMed1990

Translocation of bFGF to the nucleus is G1 phase cell cycle specific in bovine aortic endothelial cells.

Primary cultures of adult bovine aortic endothelial (ABAE) cells require bFGF to grow. G1-arrested cells, obtained after 48 h without serum and bFGF, were found to enter S phase and grow synchronously for at least two generations on addition of bFGF. In growing cells bFGF was detected both in the cytoplasm (90%) and in the nucleus (10%) where it accumulates in the nucleolus. It was not detected in the nucleus of confluent cells. bFGF uptake was continuous in the cytoplasm throughout the cell cycle with a maximum in G2, while nuclear uptake occurred only in late G1. Cytoplasmic bFGF (18.4 kd) is cleaved into a 16.5 kd peptide in G1 (t1/2 = 30 min). In the nucleus the 18.4 kd form was the only one detected 2 h following bFGF addition and was then cleaved into the 16.5 kd in early S phase. These results are consistent with the possibility that in addition to the classical pathway of signal transduction, bFGF is directly translocated to the nucleus in late G1, and could play a role in replication and/or in transcription of rDNA.

Animals↗
PubMed1989

Protein kinase NII and the regulation of rDNA transcription in mammalian cells.

Transcription of ribosomal RNA genes is generally accepted to correlate with cell growth. Using primary cultures of adult bovine aortic endothelial (ABAE) cells, we have shown that transcription of rDNA in confluent cells falls to 5% of the transcription level in growing cells. Protein kinase NII appears to be a limiting factor to promote rDNA transcription in isolated nuclei of confluent cells. Protein kinase NII was detected by immunocytochemistry in the cytoplasm, nuclei and nucleoli of growing cells while it was no longer present in nucleoli of confluent cells. The kinase activity, in isolated nuclei, was estimated by endogenous phosphorylation of a specific substrate, nucleolin. A 10% residual activity was present in confluent cell nuclei compared to growing cell nuclei. Concomitantly, the transcription 'in vitro' of rDNA in the corresponding nuclei was also highly reduced (by 85%). Addition of exogenous protein kinase NII to confluent cell nuclei induced a strong increase in the phosphorylation of specific proteins including nucleolin. In parallel, the transcription of rDNA was increased by a factor of 5, to nearly the level observed in nuclei prepared from growing cells. These data suggest that, in confluent cells, factors necessary for rDNA transcription machinery are present but inactive in the nucleolus and that the phosphorylation of one or several of these factors (nucleolin, topoisomerase I,...) by protein kinase NII is a key event in the regulation of rDNA transcription.

Alkaline Phosphatase↗
PubMed1987

Basic fibroblast growth factor enters the nucleolus and stimulates the transcription of ribosomal genes in ABAE cells undergoing G0----G1 transition.

The cellular action of growth factors, among them basic fibroblast growth factor (bFGF), is mediated by their interaction with a cell surface receptor, but the mechanism of transfer of mitogenic (or other) signals to the nucleus has not been identified. In this work, we show that bFGF is translocated to and accumulated in the nucleolus. Furthermore, the nucleolar localization of bFGF is correlated with a stimulation of transcription of ribosomal genes during G0----G1 transition induced by bFGF alone in adult bovine aortic endothelial cells (ABAE cells). Stimulation of ribosomal gene transcription is preceded by a significant increase of the major nonhistone nucleolar protein, nucleolin. In vitro, the growth factor has a direct effect on the enhancement of RNA polymerase I activity in isolated nuclei from quiescent sparse (G0) ABAE cells. The direct action of bFGF on the level of ribosomal gene transcription could correspond to an additional growth-signaling pathway, mediated by this growth factor.

Animals↗
PubMedDate not supplied

Use of CDC2 from etoposide-treated cells as substrate to assay CDC25 phosphatase activity.

Cyclin-dependent kinases (CDKs) regulate the key transition of the cell cycle in all organisms. In response to Etoposide (VP-16) induced DNA damage, cells undergo a G2-phase arrest resulting in the accumulation of inactive CDK1 (CDC2) kinase complexes. Here we report that upon Etoposide treatment CDC2 is phosphorylated on tyrosine 15 and is dephosphorylated and activated in vitro by recombinant CDC25 phosphatase. We also show that inactive CDC2 kinase from Etoposide-treated cells can be used as a substrate in a sensitive two-step assay of CDC25 phosphatase. This assay, which is very simple to set-up, is based on the monitoring of CDC2 kinase activity after CDC25-dependent dephosphorylation. It provides the possibility to use a highly physiological substrate in antimitotic drugs screening.

CDC2 Protein Kinase↗