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Results for “CDC2-CDC28 Kinases”

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At least 307 records · Page 17Linked to original sources
PubMed1994

tsJT16, a cell cycle ts mutant of rat fibroblast defective in early G0/G1 transition, fails to induce G1-cyclin and cdk2 genes after serum stimulation at the nonpermissive temperature.

tsJT16 is a cell-cycle temperature-sensitive (ts) mutant derived from rat fibroblasts whose functional defect appears soon after the growth stimulation from G0 phase. In addition to c-fos, c-myc and ornithine decarboxylase gene, 7 primarily inducible genes, c-jun, KC, JE, 2F1, 2A9, egr-1, and egr-2, were further shown to be expressed after serum stimulation at both permissive and nonpermissive temperatures. However, expression of secondarily inducible genes, cyclin D1 and D3 and cdk2, was ts and was cycloheximide sensitive. Expression of cyclin C was not inhibited by cycloheximide but it was ts. Failure in expression of G1 cyclins and Cdk2 is suggested to be a causal event for inability of growth induction of tsJT16 at the nonpermissive temperature.

Animals↗
PubMed1994

Selective repression of growth-regulating cdk2, cyclin E and E2F1 genes in human cell senescence.

Normal human diploid TIG-1 fibroblasts underwent replicative senescence around 64-68 population doubling levels (PDL) by the irreversible serum-unresponsive G1-growth arrest. Repression of growth-promoting genes was searched in this study. The RT-PCR and Western blot analyses have shown that in senescent TIG-1 cells at PDL64-67, cdk2 and cyclin E were selectively repressed at the mRNA and protein levels even after serum stimulation, and cdc2 and cyclin A were less repressed than cdk2 and cyclin E. Such a specific lack of cdk2 and cyclin E proteins correlated with unphosphorylation of the retinoblastoma gene product (pRB) in senescent cells. Transcription factor E2F1 was also completely repressed at the mRNA and protein levels in senescent TIG-1 cells. Middle-passage cells exhibited active expressions of all the above genes and pRB phosphorylation. Therefore, the present results have indicated the selective repressions of cdk2, cyclin E and E2F1 in senescent cells.

Base Sequence↗
PubMed1995

A model of the G1 phase of the cell cycle incorporating cyclin E/cdk2 complex and retinoblastoma protein.

A mathematical model of cyclin E, cdk2 and retinoblastoma protein control of the G1 phase of the human cell cycle is proposed. The model includes retinoblastoma (Rb) protein phosphorylation by a cyclin E/cdk2 complex and its subsequent dephosphorylation at the end of the cell cycle. The numerical solutions to this model demonstrates the cyclic behavior of the cyclin E/cdk2 complex, with and without Rb function, cell cycle. This model suggests an inhibition of cyclin E/cdk2 complex formation (or its activation) by hypophosphorylated retinoblastoma protein. The experimental results of cell cycle arrest upon injection of transforming growth factor-beta, alpha-interferon or D-erythro-sphingosine during G1 phase are reproduced. Cell cycle behavior predicted by this model for increasing the concentration of hypophosphorylated retinoblastoma protein during the G1 phase is discussed. Additional results are obtained by numerical simulation.

CDC2-CDC28 Kinases↗
PubMed1995

Stabilization of cyclin E and cdk2 mRNAs at G1/S transition in Rat-1A cells emerging from the G0 state.

mRNAs for cyclin E and Cdk2 have a role in the commitment to DNA replication in the cell cycle, and are induced in Rat-1A cells by serum stimulation. Cyclin E and cdk2 genes are transcribed in quiescent cells, but their transcripts rapidly turn over and levels are kept low. The rate of transcription of the cdk2 gene is slightly increased after serum stimulation, while that of cyclin E is fairly constant. At the G1/S transition of serum-stimulated cells, transient stabilization of the two types of mRNAs occurs, an event which may lead to induction of each mRNA. Artificial expression of an immediate-early protein delta FosB results in proliferation of quiescent Rat-1A cells, and this is accompanied by an efficient induction of cyclin E and cdk2 mRNAs. In delta FosB-expressing cells, two types of mRNAs are stabilized to the same extent seen in serum-stimulated cells. The expression of cyclin E and cdk2 genes is upregulated by stabilization of their transcripts, at least in part. We propose that delta FosB may have a role in regulation of progression of the cell cycle in serum-stimulated Rat-1A cells by triggering stabilization of mRNAs for cyclin E and Cdk2.

Animals↗
PubMed1995

A complex between E2F and the pRb-related protein p130 is specifically targeted by the simian virus 40 large T antigen during cell transformation.

The p130 protein is a recently cloned member of the retinoblastoma protein family. We show here that transformation of NIH3T3-L1 fibroblasts (L1 cells) by the simian virus 40 large T antigen (LTAg) depends on the disruption of DNA binding complexes between transcription factor E2F and p130. LTAg binds to the pocket region of p130 in vivo and disrupts the E2F-p130 complexes. E2F-p130 complexes are present only in quiescent L1 cells and disappear at the G1/S phase boundary concomitantly to induction of DNA synthesis and expression of the E2F-regulated cdc2 gene. p130 is a substrate of cyclin-dependent kinase 2 (Cdk2) in vitro and associates with a Cdk in vivo which is activated upon serum stimulation in late G1. Overexpression of p130 inhibits cdc2 promoter activity and entry of quiescent L1 cells into S phase. The results demonstrate that p130 is negative regulator of cell cycle progression which is specifically targeted by LTAg during cell transformation.

3T3 Cells↗
PubMed1996

Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells.

Human mammary epithelial cells (HMEC) isolated from reduction mammoplasty tissue are proliferative for several passages but then enter a period termed ¿selection' or M0 during which the majority of the cells become larger, flattened, and less proliferative. Early passage HMEC (prior to M0) were transduced with human papillomavirus type-16 E6, E7, or E6/E7 by using recombinant retroviral vectors. E7 alone or E6/E7, but not E6 alone, alleviated the M0 proliferation block, suggesting a possible role for Rb or Rb-related proteins, but not p53, in M0. In addition, cells in M0 did not have increased levels of p53 or p21 proteins, further indicating that induction of these proteins is not required for the M0 proliferation block. Early passage cells contained Rb which was primarily hyperphosphorylated while cells in M0 contained Rb protein which was predominantly underphosphorylated. Cells in M0 accumulated in G1 or B0 and expressed reduced levels of cyclin A and CDK2 proteins.

Animals↗
PubMed1996

CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus.

Cytoplasmic polyadenylation is a key mechanism controlling maternal mRNA translation in early development. In most cases, mRNAs that undergo poly(A) elongation are translationally activated; those that undergo poly(A) shortening are deactivated. Poly(A) elongation is regulated by two cis-acting sequences in the 3'-untranslated region (UTR) of responding mRNAs, the polyadenylation hexanucleotide AAUAAA and the U-rich cytoplasmic polyadenylation element (CPE). Previously, we cloned and characterized the Xenopus oocyte CPE binding protein (CPEB), showing that it was essential for the cytoplasmic polyadenylation of B4 RNA. Here, we show that CPEB also binds the CPEs of G10, c-mos, cdk2, cyclins A1, B1 and B2 mRNAs. We find that CPEB is necessary for polyadenylation of these RNAs in egg extracts, suggesting that this protein is required for polyadenylation of most RNAs during oocyte maturation. Our data demonstrate that the complex timing and extent of polyadenylation are partially controlled by CPEB binding to multiple target sites in the 3' UTRs of responsive mRNAs. Finally, injection of CPEB antibody into oocytes not only inhibits polyadenylation in vivo, but also blocks progesterone-induced maturation. This is due to inhibition of polyadenylation and translation of c-mos mRNA, suggesting that CPEB is critical for early development.

Animals↗
PubMed1996

Neu differentiation factor (Heregulin) activates a p53-dependent pathway in cancer cells.

Previously we reported that neu differentiation factor (NDF)/heregulin (HRG) elevates tyrosine phosphorylation of its receptors erbB-3, erbB-4, and erbB-2 (through heterodimer formation). We also showed that both NDF/HRG and antibodies to erbB-2 can arrest growth and induce differentiation in breast cancer cells. In this study, we report on the mechanism of NDF/HRG-induced cellular effects. We show that NDF/HRG and antibodies to erbB-2 receptors up-regulate expression of p53 by stabilizing the protein. This is accompanied by up-regulation of the p53 inducible gene, p21CIP1/WAF1, in a variety of cell lines: MCF7 and their derivatives (MCF7/HER2, MN1 and MCF-7-puro), ZR75T and LnCap cells. The induction of p21 is further enhanced when cells are treated with both NDF/HRG and DNA-damaging chemotherapeutic agents (i.e. doxorubicin). The NDF/HRG mediated induction of p21 is dependent on wildtype p53, as it fails to occur in cells expressing dominant negative p53 (MDD2). Furthermore, p21 induction is capable of inactivating cdk2 complexes as measured by Histone H1 phosphorylation assays. Finally, we show that in primary cultures of breast and other cancers, p21 is significantly induced in response to NDF/HRG treatment. Collectively, these observations suggest that the mechanism of breast cancer cell growth inhibition and differentiation via erbB receptors activation is through a p53-mediated pathway.

Antibodies, Monoclonal↗
PubMed1995

Transforming growth factor beta down-regulation of CKShs1 transcripts in growth-inhibited epithelial cells.

CKShs1 is a mammalian homologue of the yeast suc1 and CKS1 genes, for which the null mutation leads to arrest in both the G1 and G2 phases of the cell cycle in Saccharomyces cerevisiae. Northern blot analysis revealed that transcript levels of CKShs1 are strongly down-regulated in mink lung cells and moderately down-regulated in BALB keratinocytes within 10 h of exposure to transforming growth factor beta (TGF-beta), whereas growth arrest of both cell lines requires at least 15 h of TGF-beta treatment. As a genetic test for the potential role of CKShs1 in TGF-beta growth regulation, we analyzed a stably transfected derivative of mink lung cells that constitutively overexpresses a truncated form of the type 2 TGF-beta receptor and is resistant to TGF-beta growth inhibition; CKShs1 transcripts are not down-regulated by TGF-beta in this mutant cell line. TGF-beta down-regulation of CKShs1 transcripts is specific, since mRNA levels of mammalian G1 cyclins D1, D2, and D3 do not change in response to TGF-beta in either cell line. Cyclin D1 and cyclin D2 transcripts are strongly induced by epidermal growth factor, and beta 2-microglobulin transcripts are strongly induced by TGF-beta in BALB keratinocytes released from quiescence by addition of epidermal growth factor. Our results suggest a role for CKShs1 gene products in TGF-beta growth arrest of epithelial cells.

Animals↗
PubMed1995

Expression and activity of the retinoblastoma protein (pRB)-family proteins, p107 and p130, during L6 myoblast differentiation.

The activity of the E2 F-family of transcription factors is tightly linked to control of the cell cycle. p107 and p130, two closely related members of the retinoblastoma protein-family of negative cell cycle regulators, modulate the activity of the E2f-family proteins by direct interaction with these factors. To understand the role of p107 and p130 in progression through or exit from the cell cycle, we have characterized the expression, phosphorylation state, cyclin-binding, and E2f-binding activity of p107 and p130 during terminal differentiation of rat myoblast cells into immature skeletal muscle (myotubes). In exponentially growing L6 myoblasts, p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 can be observed throughout the cell cycle. During differentiation of L6 cells, p107 levels are reduced, while p130 protein levels are increased 8-fold. Despite both p107 and p130 becoming hypophosphorylated during myogenesis, the E2F-site DNA-binding complexes containing p107 observed in exponentially growing myoblasts are quantitatively replaced in myotubes with complexes containing only p130. In myotubes, p107 is not associated with E2f-family proteins that are capable of binding DNA. The failure to observe p107-containing complexes in myotubes appears to be due to the differentiation-specific induction of both p130 and cyclin D3, p107 is found in complexes with cyclin D3 in myotubes, and the addition of exogenous cyclin D3 or p130 to lysates from undifferentiated L6 cells was able to disrupt p107-containing E2F-site binding complexes. In myotubes, p130 also forms complexes with cyclin D3 as well as cyclin E, cdk2, and cdk4. We are able to copurify cyclin D3 with cyclin E from myotubes, indicating the presence of a macromolecular complex containing both cyclin E and cyclin D3 simultaneously bound to p130. Thus, in myoblasts, p107 is normally involved in regulation of E2f-family proteins during cell cycle progression, while p130 is a differentiation-specific regulator of E2f activity. Our results also provide evidence that the apparent positive regulator of cell cycle progression, cyclin D3, has a function in terminally differentiated muscle cells.

Animals↗
PubMed1996

Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin-dependent degradation of cyclin E.

A yeast screen was developed to identify mutations in human cyclin E that lead to stabilization of the protein in order to identify determinants important for cyclin E turnover. Both C-terminal truncations and missense mutations near the C-terminus of cyclin E conferred hyperstability in vivo, suggesting that sequences in this region were critical for turnover. The following observations indicate that autophosphorylation of CDK2/cyclin E on Thr380 of the cyclin regulates cyclin E destruction: (i) mutation of Thr380 to Ala stabilizes cyclin E in yeast and mammalian cells; (ii) cyclin E/CDK2 autophosphorylates on cyclin E in vitro and cyclin E is a phosphoprotein in vivo in mammalian cells; (iii) the T380A mutation eliminates phosphorylation on the same site in mammalian cells and in vitro; (iv) inhibiting CDK2 activity in vivo stabilizes cyclin E; (v) the T380A mutation prevents ubiquitination of cyclin E. These results suggest a model where activation of cyclinE/CDK2 is coupled to cyclin E turnover via site-specific phosphorylation, which acts as a signal for ubiquitination and proteasome processing.

Animals↗