scholarly journals Inhibition of Cyclin-dependent Kinase 1 Induces Cytokinesis without Chromosome Segregation in an ECT2 and MgcRacGAP-dependent Manner

2005 ◽  
Vol 280 (43) ◽  
pp. 36502-36509 ◽  
Author(s):  
Fumihiko Niiya ◽  
Xiaozhen Xie ◽  
Kyung S. Lee ◽  
Hiroki Inoue ◽  
Toru Miki
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Cleavage Furrow ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Mitotic Spindles ◽  
Initiation Mechanism

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.

Phosphatidylinositol 3-kinase mediates integrin-dependent NF-(κ)B and MAPK activation through separate signaling pathways

2001 ◽  
Vol 114 (8) ◽  
pp. 1579-1589 ◽  
Author(s):  
M. Reyes-Reyes ◽  
N. Mora ◽  
A. Zentella ◽  
C. Rosales
Keyword(s):  
Signaling Pathways ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Monocytic Leukemia ◽  
Monocytic Cells ◽  
Mapk Activation

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, (β)1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via (β)1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor (κ)B (NF-(κ)B) activation was then analyzed. We found that integrins activated both NF-(κ)B and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-(κ)B activation. In contrast, a dominant negative mutant of Rac completely prevented NF-(κ)B activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-(κ)B is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Ρ augmented NF-(κ)B activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-(κ)B, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.

Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway

2004 ◽  
Vol 287 (6) ◽  
pp. L1127-L1133 ◽  
Author(s):  
Jun Tamaoki ◽  
Kazuo Isono ◽  
Kiyoshi Takeyama ◽  
Etsuko Tagaya ◽  
Junko Nakata ◽  
...  
Keyword(s):  
Carbon Black ◽  
Airway Epithelium ◽  
Ultrafine Particles ◽  
Egf Receptor ◽  
Kinase Inhibitor ◽  
Specific Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Dependent Manner

Exposure to ambient ultrafine particles induces airway inflammatory reactions and tissue remodeling. In this experiment, to determine whether ultrafine carbon black (ufCB) affects proliferation of airway epithelium and, if so, what the mechanism of action is, we studied human primary bronchial epithelial cell cultures. Incubation of cells in the serum-free medium with ufCB increased incorporations of [3H]thymidine and [3H]leucine into cells in a time- and dose-dependent manner. This effect was attenuated by Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD) and apocynin, an inhibitor of NADPH oxidase, and completely inhibited by pretreatment with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors AG-1478 and BIBX-1382, and the mitogen-activated protein kinase kinase inhibitor PD-98059. Transfection of a dominant-negative mutant of H-Ras likewise abolished the effect ufCB. Stimulation with ufCB also induced processing of membrane-anchored proheparin-binding (HB)-EGF, release of soluble HB-EGF into the medium, association of phosphorylated EGF-R and Shc with glutathione- S-transferase-Grb2 fusion protein, and phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment with AG-1478, [Glu52] Diphtheria toxin, a specific inhibitor of HB-EGF, neutralizing HB-EGF antibody, Cu/Zn SOD, and apocynin each inhibited ufCB-induced ERK activation. These results suggest that ufCB causes oxidative stress-mediated proliferation of airway epithelium, involving processing of HB-EGF and the concomitant activation of EGF-R and ERK cascade.

scholarly journals Targeted Comparative RNA Interference Analysis Reveals Differential Requirement of Genes Essential for Cell Proliferation

2006 ◽  
Vol 17 (11) ◽  
pp. 4837-4845 ◽  
Author(s):  
Yuichi J. Machida ◽  
Yuefeng Chen ◽  
Yuka Machida ◽  
Ankit Malhotra ◽  
Sukumar Sarkar ◽  
...  
Keyword(s):  
Rna Interference ◽  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Ribosome Biogenesis ◽  
Cancer Cell Line ◽  
Prostate Cancer Cell Line ◽  
Breast Epithelial Cell ◽  
Epithelial Cell Line ◽  
Dependent Manner

Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 growth-regulated genes in parallel in a breast epithelial cell line, MCF10A, and a prostate cancer cell line, PC3. Depletion of an unexpectedly high number of genes (25%) differentially affected proliferation of the two cell lines. Knockdown of many genes that spare PC3 (p53−) but inhibit MCF10A (p53+) proliferation induces p53 in MCF10A cells. EBNA1BP2, involved in ribosome biogenesis, is an example of such a gene, with its depletion arresting MCF10A at G1/S in a p53-dependent manner. TARCOR is thus useful for identifying cell type–specific genes and pathways involved in proliferation and also for exploring the heterogeneity of cell lines. In particular, our data emphasize the importance of considering the genetic status, when performing siRNA screens in mammalian cells.

scholarly journals A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

1997 ◽  
Vol 17 (12) ◽  
pp. 7362-7374 ◽  
Author(s):  
J A Diehl ◽  
C J Sherr
Keyword(s):  
Cyclin D1 ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Cyclin Dependent Kinase ◽  
Linker Peptide ◽  
Cdk Activating Kinase

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.

scholarly journals The Ca2+-binding Protein ALG-2 Is Recruited to Endoplasmic Reticulum Exit Sites by Sec31A and Stabilizes the Localization of Sec31A

2006 ◽  
Vol 17 (11) ◽  
pp. 4876-4887 ◽  
Author(s):  
Akinori Yamasaki ◽  
Katsuko Tani ◽  
Akitsugu Yamamoto ◽  
Naomi Kitamura ◽  
Masayuki Komada
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Rich Region ◽  
Linked Gene ◽  
Transport Vesicles ◽  
Er Exit Sites ◽  
A Cell

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca2+-dependent manner and colocalized with Sec31A at ER exit sites. A Ca2+binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca2+chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca2+-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.

USP7 deubiquitinase controls HIV-1 production by stabilizing Tat protein

2017 ◽  
Vol 474 (10) ◽  
pp. 1653-1668 ◽  
Author(s):  
Amjad Ali ◽  
Rameez Raja ◽  
Sabihur Rahman Farooqui ◽  
Shaista Ahmad ◽  
Akhil C. Banerjea
Keyword(s):  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Virus Production ◽  
Dependent Manner ◽  
Tat Protein ◽  
Viral Production ◽  
Cellular Processes ◽  
Latently Infected ◽  
Dub Inhibitor ◽  
Hiv 1

Deubiquitinases (DUBs) are key regulators of complex cellular processes. HIV-1 Tat is synthesized early after infection and is mainly responsible for enhancing viral production. Here, we report that one of the DUBs, USP7, stabilized the HIV-1 Tat protein through its deubiquitination. Treatment with either a general DUB inhibitor (PR-619) or USP7-specific inhibitor (P5091) resulted in Tat protein degradation. The USP7-specific inhibitor reduced virus production in a latently infected T-lymphocytic cell line J1.1, which produces large amounts of HIV-1 upon stimulation. A potent increase in Tat-mediated HIV-1 production was observed with USP7 in a dose-dependent manner. As expected, deletion of the USP7 gene using the CRISPR-Cas9 method reduced the Tat protein and supported less virus production. Interestingly, the levels of endogenous USP7 increased after HIV-1 infection in human T-cells (MOLT-3) and in mammalian cells transfected with HIV-1 proviral DNA. Thus, HIV-1 Tat is stabilized by the host cell deubiquitinase USP7, leading to enhanced viral production, and HIV-1 in turn up-regulates the USP7 protein level.

scholarly journals Rho and Anillin-dependent Control of mDia2 Localization and Function in Cytokinesis

2010 ◽  
Vol 21 (18) ◽  
pp. 3193-3204 ◽  
Author(s):  
Sadanori Watanabe ◽  
Katsuya Okawa ◽  
Takashi Miki ◽  
Satoko Sakamoto ◽  
Tomoko Morinaga ◽  
...  
Keyword(s):  
Rna Interference ◽  
Rho Gtpase ◽  
Small Gtpase ◽  
Cleavage Furrow ◽  
3T3 Cells ◽  
Actin Nucleation ◽  
Inhibitory Domain ◽  
And Function ◽  
Rho Effectors ◽  
Nih 3T3

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.

scholarly journals G1/S Cyclin-dependent Kinase Regulates Small GTPase Rho1p through Phosphorylation of RhoGEF Tus1p inSaccharomyces cerevisiae

2008 ◽  
Vol 19 (4) ◽  
pp. 1763-1771 ◽  
Author(s):  
Keiko Kono ◽  
Satoru Nogami ◽  
Mitsuhiro Abe ◽  
Masafumi Nishizawa ◽  
Shinichi Morishita ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Cyclin Dependent Kinase ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Temporal And Spatial ◽  
Cdk Phosphorylation

Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.

scholarly journals Cyclin-dependent Kinase 9 Links RNA Polymerase II Transcription to Processing of Ribosomal RNA

2013 ◽  
Vol 288 (29) ◽  
pp. 21173-21183 ◽  
Author(s):  
Kaspar Burger ◽  
Bastian Mühl ◽  
Michaela Rohrmoser ◽  
Britta Coordes ◽  
Martin Heidemann ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Rna ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Rrna Processing ◽  
Cyclin Dependent Kinase ◽  
Small Nucleolar Rna ◽  
Nucleolar Rna ◽  
Rnapii Transcription

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3′ extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3′ processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3′ rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

scholarly journals Tuba activates Cdc42 during neuronal polarization downstream of the small GTPase Rab8a

2019 ◽  
Author(s):  
Pamela J. Urrutia ◽  
Felipe Bodaleo ◽  
Daniel A. Bórquez ◽  
Victoria Rozes-Salvador ◽  
Cristopher Villablanca ◽  
...  
Keyword(s):  
Small Gtpases ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Neuronal Polarity ◽  
Dependent Manner ◽  
Gain Of Function ◽  
Molecular Determinants ◽  
Axon Specification ◽  
Neuronal Polarization

ABSTRACTThe acquisition of neuronal polarity is a complex molecular process that involves several different cellular mechanisms that need to be finely coordinated to define the somatodendritic and axonal compartments. Amongst such mechanisms, cytoskeleton and membrane dynamics control both the morphological transitions that define neuronal polarity acquisition as well as provide molecular determinants to specific sites in neurons at a defined time point. Small GTPases from the Rab and Rho families are well known molecular determinants of neuronal differentiation. However, during axon specification, a molecular link that couples proteins from these two families has yet to be identified. In this paper, we describe the role of Tuba, a Cdc42-specific guanine nucleotide-exchange factor (GEF), in neuronal polarity through a Rab8a-dependent mechanism. Rab8a or Tuba gain-of-function generates neurons with supernumerary axons whereas Rab8a or Tuba loss-of-function abrogated axon specification, phenocopying the well-established effect of Cdc42 on neuronal polarity. Neuronal polarization associated to Rab8a is also evidenced in vivo, since a dominant negative version of Rab8a severely impaired neuronal migration.Remarkably, Rab8a activates Cdc42 in a Tuba-dependent manner, and dominant negative mutants of both GTPases reciprocally prevent the effect over polarity acquisition in the gain-of-function scenarios. Our results strongly suggest that a positive feedback loop linking Rab8a and Cdc42 activities via Tuba, is a primary event in neuronal polarization. In addition, we identified the GEF responsible for Cdc42 activation that is essential to specify axons in cultured neurons.

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