scholarly journals Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry

2002 ◽  
Vol 157 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Ian Marc Bonapace ◽  
Lucia Latella ◽  
Roberto Papait ◽  
Francesco Nicassio ◽  
Alessandra Sacco ◽  
...  
Keyword(s):  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Gene Encoding ◽  
Adenovirus E1a ◽  
Differentiated Cells ◽  
Molecular Determinant ◽  
Tumor Viruses ◽  
Cdk2 Complex ◽  
Nih 3T3 ◽  
Phase Entry

Terminal differentiation exerts a remarkably tight control on cell proliferation. However, the oncogenic products of DNA tumor viruses, such as adenovirus E1A, can force postmitotic cells to proliferate, thus representing a powerful tool to study progression into S phase. In this study, we identified the gene encoding Np95, a murine nuclear phosphoprotein, as an early target of E1A-induced transcriptional events. In terminally differentiated (TD) cells, the activation of Np95 was specifically induced by E1A, but not by overexpression of E2F-1 or of the cyclin E (cycE)–cyclin-dependent kinase 2 (cdk2) complex. In addition, the concomitant expression of Np95 and of cycE–cdk2 was alone sufficient to induce S phase in TD cells. In NIH-3T3 cells, the expression of Np95 was tightly regulated during the cell cycle, and its functional ablation resulted in abrogation of DNA synthesis. Thus, expression of Np95 is essential for S phase entry. Previous evidence suggested that E1A, in addition to its well characterized effects on the pRb/E2F-1 pathway, activates a parallel and complementary pathway that is also required for the reentry in S phase of TD cells (Tiainen, M., D. Spitkousky, P. Jansen-Dürr, A. Sacchi, and M. Crescenzi. 1996. Mol. Cell. Biol. 16:5302–5312). From our results, Np95 appears to possess all the characteristics to represent the first molecular determinant identified in this pathway.

scholarly journals Cortactin Modulates RhoA Activation and Expression of Cip/Kip Cyclin-Dependent Kinase Inhibitors To Promote Cell Cycle Progression in 11q13-Amplified Head and Neck Squamous Cell Carcinoma Cells

2010 ◽  
Vol 30 (21) ◽  
pp. 5057-5070 ◽  
Author(s):  
David R. Croucher ◽  
Danny Rickwood ◽  
Carole M. Tactacan ◽  
Elizabeth A. Musgrove ◽  
Roger J. Daly
Keyword(s):  
Cell Cycle ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Cycle Progression ◽  
Kinase Inhibitors ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Fadu Cells ◽  
Phase Entry

ABSTRACT The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.

scholarly journals Requirement for Phospholipase C-γ1 Enzymatic Activity in Growth Factor-Induced Mitogenesis

1998 ◽  
Vol 18 (1) ◽  
pp. 590-597 ◽  
Author(s):  
Zhixiang Wang ◽  
Stefan Glück ◽  
Lianfeng Zhang ◽  
Michael F. Moran
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Phospholipase C ◽  
Cell Cycle Progression ◽  
Mdck Cells ◽  
Cell Receptor ◽  
S Phase ◽  
Dominant Negative ◽  
Nih 3T3 ◽  
Phase Entry

ABSTRACT The cytoplasmic regions of the receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) bind and activate phospholipase C-γ1 (PLC-γ1) and other signaling proteins in response to ligand binding outside the cell. Receptor binding by PLC-γ1 is a function of its SH2 domains and is required for growth factor-induced cell cycle progression into the S phase. Microinjection into MDCK epithelial cells and NIH 3T3 fibroblasts of a polypeptide corresponding to the noncatalytic SH2-SH2-SH3 domains of PLC-γ1 (PLC-γ1 SH2-SH2-SH3) blocked growth factor-induced S-phase entry. Treatment of cells with diacylglycerol (DAG) or DAG and microinjected inositol-1,4,5-triphosphate (IP3), the products of activated PLC-γ1, did not stimulate cellular DNA synthesis by themselves but did suppress the inhibitory effects of the PLC-γ1 SH2-SH2-SH3 polypeptide but not the cell cycle block imposed by inhibition of the adapter protein Grb2 or p21 Ras. Two c-fos serum response element (SRE)-chloramphenicol acetyltransferase (CAT) reporter plasmids, a wild-type version, wtSRE-CAT, and a mutant, pm18, were used to investigate the function of PLC-γ1 in EGF- and PDGF-induced mitogenesis. wtSRE-CAT responds to both protein kinase C (PKC)-dependent and -independent signals, while the mutant, pm18, responds only to PKC-independent signals. Microinjection of the dominant-negative PLC-γ1 SH2-SH2-SH3 polypeptide greatly reduced the responses of wtSRE-CAT to EGF stimulation in MDCK cells and to PDGF stimulation in NIH 3T3 cells but had no effect on the responses of mutant pm18. These results indicate that in addition to Grb2-mediated activation of Ras, PLC-γ1-mediated DAG production is required for EGF- and PDGF-induced S-phase entry and gene expression, possibly through activation of PKC.

scholarly journals The Rb family contains a conserved cyclin-dependent-kinase-regulated transcriptional repressor motif.

1996 ◽  
Vol 16 (12) ◽  
pp. 7173-7181 ◽  
Author(s):  
K N Chow ◽  
P Starostik ◽  
D C Dean
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Transcriptional Repressor ◽  
S Phase ◽  
Domain Swapping ◽  
Cyclin Dependent Kinase ◽  
Cyclin Dependent Kinases ◽  
Tumor Viruses ◽  
Cdk Phosphorylation ◽  
Repressor Activity

Progression through the cell cycle is dependent on the sequential expression of cyclins, which combine with cyclin-dependent kinases (cdks) to form active kinases. The transition from G1 to S phase is dependent on D cyclins in complex with cdk4 or cdk6 and cyclin E complexed with cdk2. One target of G1 cyclins is the retinoblastoma susceptibility protein (Rb). Rb is a transcriptional repressor that is selectively targeted to genes through interaction with the E2F family of cell cycle transcription factors. Rb is a member of a family of proteins that include p107 and p130. The three proteins share a region known as the pocket that is important for binding E2F and is also the binding site for oncoproteins from DNA tumor viruses that inactivate Rb. We have found that two conserved domains within the Rb pocket (A and B) interact to form a transcriptional repressor motif (K. N. B. Chow and D. C. Dean, Mol. Cell. Biol. 16:4862-4868, 1996). Here we demonstrate that p107 also has an A-B repressor motif, and using domain swapping and coimmunoprecipitation assays, we compare A and B from Rb and p107. Finally and most importantly, we demonstrate that the A-B interaction which forms the repressor motif is blocked by G1 cdk phosphorylation, thereby blocking repressor activity. This A-B repressor motif is then the first example of a cdk-regulated transcriptional repressor.

scholarly journals Phosphorylation of the Cyclin-Dependent Kinase Inhibitor p21Cip1 on Serine 130 Is Essential for Viral Cyclin-Mediated Bypass of a p21Cip1-Imposed G1 Arrest

2006 ◽  
Vol 26 (6) ◽  
pp. 2430-2440 ◽  
Author(s):  
Annika Järviluoma ◽  
Emma S. Child ◽  
Grzegorz Sarek ◽  
Papinya Sirimongkolkasem ◽  
Gordon Peters ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitor ◽  
Primary Effusion Lymphoma ◽  
S Phase ◽  
G1 Arrest ◽  
Cyclin Dependent Kinase ◽  
Partial Restoration ◽  
Viral Cyclin ◽  
Phase Entry

ABSTRACT K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.

scholarly journals Expression of E1A in terminally differentiated muscle cells reactivates the cell cycle and suppresses tissue-specific genes by separable mechanisms.

1996 ◽  
Vol 16 (10) ◽  
pp. 5302-5312 ◽  
Author(s):  
M Tiainen ◽  
D Spitkovsky ◽  
P Jansen-Dürr ◽  
A Sacchi ◽  
M Crescenzi
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitor ◽  
Cyclin E ◽  
Muscle Cells ◽  
S Phase ◽  
Specific Gene ◽  
Muscle Creatine Kinase ◽  
Tissue Specific ◽  
Adenovirus E1a ◽  
Differentiated Cells

Terminally differentiated cells are characterized by permanent withdrawal from the cell cycle; they do not enter S phase even when stimulated by growth factors or retroviral oncogenes. We have shown, however, that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. In this report, we describe the molecular events triggered by E1A in terminally differentiated skeletal muscle cells. We found that in myotubes infected with the adenovirus mutant dl520, 12S E1A bypasses the early G1 phase and activates the expression of late-G1 genes, such as the cyclin E and cyclin A genes, cdk2, PCNA, and B-myb. Of these, the cyclin E gene and cdk2 were significantly overexpressed in comparison with levels in proliferating, undifferentiated myoblasts. p130 and pRb were phosphorylated before the infected myotubes entered S phase, despite the high expression of the cyclin-dependent kinase inhibitor p21, and E2F was released. Our results suggest that one of the mechanisms that E1A uses to overcome the proliferative block of terminally differentiated cells involves coordinated overexpression of cyclin E and cdk2. Following E1A expression, the myogenic transcription factors MyoD and myogenin and the muscle-specific structural genes encoding muscle creatine kinase and myosin heavy chain were downregulated. The muscle regulatory factors were also silenced in myotubes infected with adenovirus E1A mutants incapable of reactivating the cell cycle in terminally differentiated muscle cells. Thus, the suppression of the differentiation program is not a consequence of cell cycle reactivation in myotubes, and it is induced by an independent mechanism. Our results show that E1A reactivates the cell cycle and suppresses tissue-specific gene expression in terminally differentiated muscle cells, thus causing dedifferentiation.

scholarly journals Uncovering the role of APC-Cdh1 in generating the dynamics of S-phase onset

2014 ◽  
Vol 25 (4) ◽  
pp. 441-456 ◽  
Author(s):  
Xi Yuan ◽  
Jeyaraman Srividhya ◽  
Thomas De Luca ◽  
Ju-hyong E. Lee ◽  
Joseph R. Pomerening
Keyword(s):  
Kinase Inhibitor ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Cyclin E1 ◽  
Stimulus Response ◽  
Cyclin Dependent Kinase Inhibitor ◽  
E1 Protein ◽  
Reduced Frequency ◽  
Phase Entry

Cdh1, a coactivator of the anaphase-promoting complex (APC), is a potential tumor suppressor. Cdh1 ablation promotes precocious S-phase entry, but it was unclear how this affects DNA replication dynamics while contributing to genomic instability and tumorigenesis. We find that Cdh1 depletion causes early S-phase onset in conjunction with increase in Rb/E2F1-mediated cyclin E1 expression, but reduced levels of cyclin E1 protein promote this transition. We hypothesize that this is due to a weakened cyclin-dependent kinase inhibitor (CKI)–cyclin-dependent kinase 2 positive-feedback loop, normally generated by APC-Cdh1–mediated proteolysis of Skp2. Indeed, Cdh1 depletion increases Skp2 abundance while diminishing levels of the CKI p27. This lowers the level of cyclin E1 needed for S-phase entry and delays cyclin E1 proteolysis during S-phase progression while corresponding to slowed replication fork movement and reduced frequency of termination events. In summary, using both experimental and computational approaches, we show that APC-Cdh1 establishes a stimulus–response relationship that promotes S phase by ensuring that proper levels of p27 accumulate during G1 phase, and defects in its activation accelerate the timing of S-phase onset while prolonging its progression.

scholarly journals Glypican 1 Stimulates S Phase Entry and DNA Replication in Human Glioma Cells and Normal Astrocytes

2013 ◽  
Vol 33 (22) ◽  
pp. 4408-4421 ◽  
Author(s):  
Dianhua Qiao ◽  
Kristy Meyer ◽  
Andreas Friedl
Keyword(s):  
Dna Replication ◽  
Cyclin E ◽  
Glioma Cells ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Human Glioma ◽  
Human Glioma Cells ◽  
Human Gliomas ◽  
Phase Entry ◽  
Dna Rereplication

Malignant gliomas are highly lethal neoplasms with limited treatment options. We previously found that the heparan sulfate proteoglycan glypican 1 (GPC1) is universally and highly expressed in human gliomas. In this study, we investigated the biological activity of GPC1 expression in both human glioma cells and normal astrocytesin vitro. Expression of GPC1 inactivates the G1/S checkpoint and strongly stimulates DNA replication. Constitutive expression of GPC1 causes DNA rereplication and DNA damage, suggesting a mutagenic activity for GPC1. GPC1 expression leads to a significant downregulation of the tumor suppressors pRb, Cip/Kip cyclin-dependent kinase inhibitors (CKIs), and CDH1, and upregulation of the pro-oncogenic proteins cyclin E, cyclin-dependent kinase 2 (CDK2), Skp2, and Cdt1. These GPC1-induced changes are accompanied by a significant reduction in all types of D cyclins, which is independent of serum supplementation. It is likely that GPC1 stimulates the so-called Skp2 autoinduction loop, independent of cyclin D-CDK4/6. Knockdown of Skp2, CDK2, or cyclin E, three key elements within the network modulated by GPC1, results in a reduction of the S phase and aneuploid fractions, implying a functional role for these regulators in GPC1-induced S phase entry and DNA rereplication. In addition, a significant activation of both the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways by GPC1 is seen in normal human astrocytes even in the presence of growth factor supplement. Both pathways are constitutively activated in human gliomas. The surprising magnitude and the mitogenic and mutagenic nature of the effect exerted by GPC1 on the cell cycle imply that GPC1 may play an important role in both glioma tumorigenesis and growth.

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