Phosphoproteomic Analysis of Aurora Kinase Inhibition in Monopolar Cytokinesis

Polat, Ayşe Nur; Karayel, Özge; Giese, Sven H.; Harmanda, Büşra; Sanal, Erdem; Hu, Chi-Kuo; Renard, Bernhard Y.; Özlü, Nurhan

doi:10.1021/acs.jproteome.5b00645

Cited by 15 publications

(14 citation statements)

References 45 publications

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“…In-solution digestion was performed as described previously 19 . On-column dimethyl labeling was performed as described in 22 .…”

Section: Methodsmentioning

confidence: 99%

“…Statistical analysis was performed on the resulting PD peptide tables as described in 19 . The ratio normalization of the phosphopeptides was based on the regular peptides.…”

Section: Methodsmentioning

confidence: 99%

“…For the evaluation of the analysis of monopolar versus bipolar cytokinesis with label swaps, we treated the technical replicates as independent trials to assess the significance of the regulation by a binomial probability function. As described previously 19 , to derive a p-value based on the binomial, the number of trials was defined as the number of times, a peptide was quantified; the number of successes was defined as the number of times, a peptide passed the significance threshold; the probability of success was adjusted to the chosen alpha of 5%. Derived p-values were adjusted for multiple testing by Benjamini/Hochberg correction 64 .…”

Section: Methodsmentioning

confidence: 99%

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Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis

Karayel

Sanal

Giese

et al. 2018

Sci Rep

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The successful completion of cytokinesis requires the coordinated activities of diverse cellular components including membranes, cytoskeletal elements and chromosomes that together form partly redundant pathways, depending on the cell type. The biochemical analysis of this process is challenging due to its dynamic and rapid nature. Here, we systematically compared monopolar and bipolar cytokinesis and demonstrated that monopolar cytokinesis is a good surrogate for cytokinesis and it is a well-suited system for global biochemical analysis in mammalian cells. Based on this, we established a phosphoproteomic signature of cytokinesis. More than 10,000 phosphorylation sites were systematically monitored; around 800 of those were up-regulated during cytokinesis. Reconstructing the kinase-substrate interaction network revealed 31 potentially active kinases during cytokinesis. The kinase-substrate network connects proteins between cytoskeleton, membrane and cell cycle machinery. We also found consensus motifs of phosphorylation sites that can serve as biochemical markers specific to cytokinesis. Beyond the kinase-substrate network, our reconstructed signaling network suggests that combination of sumoylation and phosphorylation may regulate monopolar cytokinesis specific signaling pathways. Our analysis provides a systematic approach to the comparison of different cytokinesis types to reveal alternative ways and a global overview, in which conserved genes work together and organize chromatin and cytoplasm during cytokinesis.

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“…In-solution digestion was performed as described previously 19 . On-column dimethyl labeling was performed as described in 22 .…”

Section: Methodsmentioning

confidence: 99%

“…Statistical analysis was performed on the resulting PD peptide tables as described in 19 . The ratio normalization of the phosphopeptides was based on the regular peptides.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis

Karayel

Sanal

Giese

et al. 2018

Sci Rep

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“…In-solution digestion was performed as described previously (22). Briefly, cell lysate was incubated for 4 hours using Lys-C (enzyme:protein ratio of 1:100) in 4 M urea, followed by overnight trypsin (enzyme:protein ratio of 1:100) incubation in 1 M urea at 37°C.…”

Section: In-solution Digestion and Stable Isotope Dimethyl Labelingmentioning

confidence: 99%

Systems-level Analysis Reveals Multiple Modulators of Epithelial-mesenchymal Transition and Identifies DNAJB4 and CD81 as Novel Metastasis Inducers in Breast Cancer

Kagiali

Sanal

Karayel

et al. 2019

Molecular & Cellular Proteomics

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Epithelial-mesenchymal transition (EMT) is driven by complex signaling events that induce dramatic biochemical and morphological changes whereby epithelial cells are converted into cancer cells. However, the underlying molecular mechanisms remain elusive. Here, we used mass spectrometry based quantitative proteomics approach to systematically analyze the post-translational biochemical changes that drive differentiation of human mammary epithelial (HMLE) cells into mesenchymal. We identified 314 proteins out of more than 6,000 unique proteins and 871 phosphopeptides out of more than 7,000 unique phosphopeptides as differentially regulated. We found that phosphoproteome is more unstable and prone to changes during EMT compared to the proteome and multiple alterations at proteome level are not thoroughly represented by transcriptional data highlighting the necessity of proteome level analysis. We discovered cell state specific signaling pathways, such as Hippo, sphingolipid signaling, and unfolded protein response (UPR) by modeling the networks of regulated proteins and potential kinase-substrate groups. We identified two novel factors for EMT whose expression increased upon EMT induction: DnaJ heat shock protein family (Hsp40) member B4 (DNAJB4) and cluster of differentiation 81 (CD81). Suppression of DNAJB4 or CD81 in mesenchymal breast cancer cells resulted in decreased cell migration in vitro and led to reduced primary tumor growth, extravasation, and lung metastasis in vivo. Overall, we performed the global proteomic and phosphoproteomic analyses of EMT, identified and validated new mRNA and/or protein level modulators of EMT. This work also provides a unique platform and resource for future studies focusing on metastasis and drug resistance.

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“…Of 562 identified proteins, 64 mitotic spindle associated proteins were assigned as candidate substrates of AurA, AurB and PLK1. Our group focused on the potential targets of AurB kinase, specifically during cytokinesis by combining a monopolar cytokinesis approach with SILAC and small molecule inhibitors of Aurora kinases, VX680 and AZD1152 . More than 20 000 phosphopeptides were identified upon Aurora inhibition; 246 of them showing significant downregulation; intermediate filaments, microtubules and the actin cytoskeleton‐related proteins were among the potential Aurora kinase substrates during cytokinesis .…”

Section: Posttranslational Modification Analysis Of Cell Divisionmentioning

confidence: 99%

Proteomics in Cell Division

et al. 2017

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Cell division requires a coordinated action of the cell cycle machinery, cytoskeletal elements, chromosomes, and membranes. Cell division studies have greatly benefitted from the mass spectrometry (MS)-based proteomic approaches for probing the biochemistry of highly dynamic complexes and their coordination with each other as a cell progresses into division. In this review, the authors first summarize a wide-range of proteomic studies that focus on the identification of sub-cellular components/protein complexes of the cell division machinery including kinetochores, mitotic spindle, midzone, and centrosomes. The authors also highlight MS-based large-scale analyses of the cellular components that are largely understudied during cell division such as the cell surface and lipids. Then, the authors focus on posttranslational modification analyses, especially phosphorylation and the resulting crosstalk with other modifications as a cell undergoes cell division. Combining proteomic approaches that probe the biochemistry of cell division components with functional genomic assays will lead to breakthroughs toward a systems-level understanding of cell division.

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Phosphoproteomic Analysis of Aurora Kinase Inhibition in Monopolar Cytokinesis

Cited by 15 publications

References 45 publications

Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis

Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis

Systems-level Analysis Reveals Multiple Modulators of Epithelial-mesenchymal Transition and Identifies DNAJB4 and CD81 as Novel Metastasis Inducers in Breast Cancer

Proteomics in Cell Division

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