Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells

Rines, Daniel R.; Gómez-Ferrerı́a, Marı́a Ana; Zhou, Yingyao; DeJesus, Paul; Grob, Seanna; Batalov, Serge; Labow, M; Huesken, Dieter; Mickanin, Craig; Hall, Jonathan; Reinhardt, Mischa; Natt, François; Lange, Joerg; Sharp, David J.; Chanda, Sumit K.; Caldwell, Jeremy S.

doi:10.1186/gb-2008-9-2-r44

Cited by 58 publications

(55 citation statements)

References 33 publications

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“…Genome-wide siRNA libraries were screened for regulation of TNF-induced IL-8 scretion in HeLa cells (33)(34)(35), and several siRNAs targeting BMX were found to be potent inhibitors (data not shown). A selection of siRNAs designed to target different sequences in the human BMX mRNA were transfected in HeLa cells according to the HiPerfect protocol, and TNF-induced IL-8 was measured (Fig.…”

Section: Depletion Of Bmx Protein Results In Decreased Secretion Of Il-8mentioning

confidence: 99%

“…All small interfering RNAs (siRNAs) were synthesized at Novartis with the target sequences listed in Supplemental Table 2. The siRNA libraries have been described earlier and comprise siRNA targeting 24,373 predicted human genes (33), as well as 5000 "druggable" genes, predominately enzymes and receptors (34,35). Synthesis of siRNA was performed with standard phosphoramidite chemistry.…”

Section: Methodsmentioning

confidence: 99%

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The Tyrosine Kinase BMX Is an Essential Mediator of Inflammatory Arthritis in a Kinase-Independent Manner

Gottar-Guillier

Dodeller

Huesken

et al. 2011

The Journal of Immunology

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Inflammatory cytokines like TNF play a central role in autoimmune disorders such as rheumatoid arthritis. We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of a shared inflammatory signaling pathway. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1β, or TLR agonists. BMX was required for phosphorylation of p38 MAPK and JNK, as well as activation of NF-κB. The following epistasis analysis indicated that BMX acts downstream of or at the same level as the complex TGF-β activated kinase 1 (TAK1)–TAK1 binding protein. At the cellular level, regulation of the IL-8 promoter required the pleckstrin homology domain of BMX, which could be replaced by an ectopic myristylation signal, indicating a requirement for BMX membrane association. In addition, activation of the IL-8 promoter by in vitro BMX overexpression required its catalytic activity. Genetic ablation of BMX conferred protection in the mouse arthritis model of passive K/BxN serum transfer, confirming that BMX is an essential mediator of inflammation in vivo. However, genetic replacement with a catalytically inactive BMX allele was not protective in the same arthritis animal model. We conclude that BMX is an essential component of inflammatory cytokine signaling and that catalytic, as well as noncatalytic functions of BMX are involved.

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Section: Depletion Of Bmx Protein Results In Decreased Secretion Of Il-8mentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Tyrosine Kinase BMX Is an Essential Mediator of Inflammatory Arthritis in a Kinase-Independent Manner

Gottar-Guillier

Dodeller

Huesken

et al. 2011

The Journal of Immunology

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“…The kinase siRNA SMARTpool library (Dharmacon, catalog no. H-003500) and the four siRNAs targeting 5,000 druggable genes (Human Druggable Genome siRNA Set V1.0 [Qiagen]) were transfected into the HCV replicon cells as previously described (21,23,48). The total number of siRNAs screened was 20,779, of which 20,000 were 4 individual siRNAs targeting 5,000 druggable genes and 779 SMARTpool kinases.…”

Section: Methodsmentioning

confidence: 99%

Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

Borawski¹,

Troke²,

Puyang³

et al. 2009

J Virol

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178

174

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Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.Hepatitis C virus (HCV) causes liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (52). The HCV genome is a single-stranded RNA molecule where both the 5Ј and the 3Ј untranslated region (UTR) contain highly conserved RNA structures necessary for polyprotein translation and genome replication (43). The processed polyprotein yields at least three structural proteins and six nonstructural proteins. The structural proteins include the core, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The viral proteins processed by signal peptidases form viral particles that assemble at the endoplasmic reticulum (ER) and/or Golgi bodies and are released from the host cell by viral budding. The structural protein coding regions are separated from nonstructural proteins by the short membrane peptide p7, thought to function as an ion channel (43, 53). The nonstructural proteins NS2, NS3/4A, NS5A, and NS5B are involved in coordinating the intracellular processes of the virus life cycle, including polyprotein processing and viral RNA replication (34).The Luc-1b cell is a human hepatoma cell line (Huh7) that contains a genotype 1b HCV subgenomic replicon, a luciferase reporter, and a neomycin selection marker, allowing HCV replication to be studied both in vitro and in vivo (8,36). This subgenomic replicon lacks the coding regions for NS2 and the structural proteins but contains the nonstructural proteins in cis, which are required for replicat...

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“…Although the specific role of AKT3 in colorectal cancer has not been established, previous studies have suggested AKT3 as a possible mitotic regulator (43,44). Because the inhibition of AURKB causes cytokinetic failure and aneuploidy, and a possible role of AKT in microtubule stabilization has been suggested (45), we suspected that AKT3 may have non-apoptotic activity associated with the cytokinetic machinery, which might counteract aneuploidy induction by AURKis and modulate the chemosensitivity of cancer cells to AURKi.…”

Section: Discussionmentioning

confidence: 94%

Functional Effects of AKT3 on Aurora Kinase Inhibitor-induced Aneuploidy

Noguchi

Hongama

Hariki

et al. 2017

Journal of Biological Chemistry

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Edited by Eric R. FearonThe suppression of mitotic Aurora kinases (AURKs) by AURK inhibitors frequently causes cytokinetic failure, leading to polyploidy or aneuploidy, indicating the critical role of AURKmediated phosphorylation during cytokinesis. We demonstrate the deregulated expression of AKT3 in Aurora kinase inhibitor (AURKi)-resistant cells, which we established from human colorectal cancer HCT 116 cells. The AKT family, which includes AKT1, -2, and -3, plays multiple roles in antiapoptotic functions and drug resistance and is involved in cell growth and survival pathways. We found that an AKT inhibitor, AZD5363, showed synergistic effect with an AURKi, VX-680, on two AKT3-expressing AURKi-resistant cell lines, and AKT3 knockdown sensitized cells to VX-680. Consistent with these activities, AKT3 expression suppressed AURKi-induced apoptosis and conferred resistance to AURKi. Thus, AKT3 expression affects cell sensitivity to AURKi. Moreover, we found that AKT3 expression suppressed AURKi-induced aneuploidy, and inversely AKT3 knockdown enhanced it. In addition, partial co-localization of AKT3 with AURKB was observed during anaphase. Overall, this study suggests that AKT3 could repress the antiproliferative effects of AURKi, with a novel activity particularly suppressing the aneuploidy induction.

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Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells

Abstract: A loss-of-function screen for siRNAs that arrest human cells in metaphase reveals genes involved in mitotic spindle integrity.

Cited by 58 publications

References 33 publications

The Tyrosine Kinase BMX Is an Essential Mediator of Inflammatory Arthritis in a Kinase-Independent Manner

The Tyrosine Kinase BMX Is an Essential Mediator of Inflammatory Arthritis in a Kinase-Independent Manner

Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

Functional Effects of AKT3 on Aurora Kinase Inhibitor-induced Aneuploidy

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