Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient

Adachi, Jun; Hashiguchi, Kazunari; Nagano, Maiko; Sato, Misako; Sato, Ayako; Fukamizu, Kazuna; Ishihama, Yasushi; Tomonaga, Takeshi

doi:10.1021/acs.analchem.6b01232

Cited by 66 publications

(68 citation statements)

References 13 publications

(22 reference statements)

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“…Digestion was stopped by adding trifluoroacetic acid (final concentration, 1%). The samples were subjected to a desalting and washing step using C 18 /SCX-StageTips (58). Digested peptides were eluted with elution buffer (0.5 M ammonium acetate, 30% acetonitrile) and evaporated.…”

Section: Methodsmentioning

confidence: 99%

S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

Sato

Watanabe

Suzuki

et al. 2019

J Virol

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Temporally controlled gene expression is necessary for the propagation of herpesviruses. To achieve this, herpesviruses encode several transcriptional regulators. In Epstein-Barr virus, BcRF1 associates with five viral proteins (BDLF4, BGLF3, BFRF2, BVLF1, and BDLF3.5) to form the viral late (L) gene regulatory complex, which is called the viral preinitiation complex (vPIC), on TATT-containing promoters. However, regulation of the vPIC has been largely unexplored. In this study, we performed two screens using a kinase inhibitor library and identified a series of cyclin-dependent kinase (CDK) inhibitors that downregulated the expression of L genes without any impact on viral DNA replication through destabilization of the BDLF4 protein. Knockdown of CDK2 by short hairpin RNA (shRNA) and proteasome inhibitor treatment showed that phosphorylation of the BDLF4 protein prevented ubiquitin-mediated degradation. Moreover, we demonstrated that cyclin A-and E-associated CDK2 complexes phosphorylated BDLF4 in vitro, and we identified several serine/threonine phosphorylation sites in BDLF4. Phosphoinactive and phosphomimic mutants revealed that phosphorylation at threonine 91 plays a role in stabilizing BDLF4. Therefore, our findings indicate that S-like-phase CDKs mediate the regulation of L gene expression through stabilization of the BDLF4 protein, which makes the temporal L gene expression system more robust. IMPORTANCE Late (L) genes represent more than one-third of the herpesvirus genome, suggesting that many of these genes are indispensable for the life cycle of the virus. With the exception of BCRF1, BDLF2, and BDLF3, Epstein-Barr virus L genes are transcribed by viral regulators, which are known as the viral preinitiation complex (vPIC) and the host RNA polymerase II complex. Because the vPIC is conserved in beta-and gammaherpesviruses, studying the control of viral L gene expression by the vPIC contributes to the development of drugs that specifically inhibit these processes in beta-and gammaherpesvirus infections/diseases. In this study, we demonstrated that CDK inhibitors induced destabilization of the vPIC component BDLF4, leading to a reduction in L gene expression and subsequent progeny production. Our findings suggest that CDK inhibitors may be a therapeutic option against betaand gammaherpesviruses in combination with existing inhibitors of herpesvirus lytic replication, such as ganciclovir.

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Section: Methodsmentioning

confidence: 99%

S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

Sato

Watanabe

Suzuki

et al. 2019

J Virol

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“…Protein extraction from cell pellets, trypsin digestion and enrichment of phosphopeptides were performed as described previously 55 . Enriched phosphopeptides for each sample were labelled with Tandem Mass Tag (TMT, Thermo Fischer) according to the manufacturer’s protocol at one-tenth scale.…”

Section: Methodsmentioning

confidence: 99%

“…The combined sample was desalted and trapped by octadecyl (C18)-strong cation exchange using StageTip 56, 57 , then fractionated into 7 fractions by elution with each of the buffers as described previously 55 . These 7 fractions of TMT-labelled samples were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS).…”

Section: Methodsmentioning

confidence: 99%

TKI-addicted ROS1-rearranged cells are destined to survival or death by the intensity of ROS1 kinase activity

Ogura

Nagatake-Kobayashi

Adachi

et al. 2017

Sci Rep

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ROS1 rearrangement is observed in 1–2% of non-small cell lung cancers (NSCLC). The ROS1 tyrosine kinase inhibitor (TKI) crizotinib has induced marked tumour shrinkage in ROS1-rearranged cancers. However, emergence of acquired resistance to TKI is inevitable within a few years. Previous findings indicate that cabozantinib overcomes secondary mutation–mediated crizotinib-resistance in ROS1-fusion-positive cells. Here we attempted to establish cabozantinib-resistant cells by N-ethyl-N-nitrosourea mutagenesis screening using CD74-ROS1–expressing Ba/F3 cells. Two resistant cell lines with CD74-ROS1 F2004V or F2075C mutations, which are homologous to ALK F1174 or F1245 mutations, survived in the presence of a low dose of ROS1-TKI. Removal of ROS1-TKI from these TKI-addicted cells induced excessive activation of ROS1 tyrosine kinase followed by apoptosis. We succeeded in recapturing the TKI-addicted phenotype using doxycycline-inducible CD74-ROS1 mutant over-expression in Ba/F3 cells, suggesting that excessive ROS1 oncogenic signaling itself induced apoptosis instead of cell growth. Phosphoproteomic analysis and high-throughput inhibitor screening revealed that excessive ROS1 signaling in the TKI-addicted cells phosphorylated or activated apoptosis-related molecules such as FAF1 or p38. Collectively, our findings partly clarify molecular mechanisms of excessive ROS1 oncogenic signaling that mediates paradoxical induction of apoptosis.

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“…improved the performance of SCX StageTip fractionation by using acid-based elution instead of salt-based elution, and the results showed that more than 20,000 phosphopeptides from seven fractions were identified with high reproducibility. 79 Due to the high selectivity of phosphopeptide enrichment, fractionation of phosphopeptides followed by phosphopeptide enrichment, often called the “enrichment-fractionation” approach, is prevalent in a variety of recent reports. 57, 79, 80 …”

Section: Peptide Chromatographic Separationsmentioning

confidence: 99%

“…79 Due to the high selectivity of phosphopeptide enrichment, fractionation of phosphopeptides followed by phosphopeptide enrichment, often called the “enrichment-fractionation” approach, is prevalent in a variety of recent reports. 57, 79, 80 …”

Section: Peptide Chromatographic Separationsmentioning

confidence: 99%

Recent advances in phosphoproteomics and application to neurological diseases

Arrington

Hsu

Elder

et al. 2017

Analyst

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Phosphorylation has an incredible impact on the biological behavior of proteins, altering everything from intrinsic activity to cellular localization and complex formation. It is no surprise then that this post-translational modification has been the subject of intense study and that, with the advent of faster, more accurate instrumentation, the number of large-scale mass spectrometry-based phosphoproteomic studies has swelled over the past decade. Recent developments in sample preparation, phosphorylation enrichment, quantification, and data analysis strategies permit both targeted and ultra-deep phosphoproteome profiling, but challenges remain in pinpointing biologically relevant phosphorylation events. We describe here technological advances that have facilitated phosphoproteomic analysis of cells, tissues, and biofluids and note applications to neuropathologies in which the phosphorylation machinery may be dysregulated, much as it is in cancer.

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Improved Proteome and Phosphoproteome Analysis on a Cation Exchanger by a Combined Acid and Salt Gradient

Cited by 66 publications

References 13 publications

S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

TKI-addicted ROS1-rearranged cells are destined to survival or death by the intensity of ROS1 kinase activity

Recent advances in phosphoproteomics and application to neurological diseases

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