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Quantitative proteomic analysis based on the isobaric tags for relative and absolute quantitation (iTRAQ) labeling in WT and NudCL2 KO-1 HEK-293 cells

ABSTRACT: Cytokinesis is a key step in the later stage of cell division to partition cellular contents into daughter cells, which undergoes precise regulation in temporal and spatial. However, the underlying mechanisms are still unclear. Here, we show that Hsp90 cochaperone, NudCL2 (NudC-like protein 2), is required for cytokinesis in mammalian cells. NudCL2 localizes to midbody during cytokinesis. Knockout (KO) of NudCL2 using CRISPR/Cas9-based genome editing causes cytokinesis failure and leads to the accumulation of multinucleated cells. To investigate the potential regulator involved in NudCL2-mediated cytokinesis regulation, we performed isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis in WT and NudCL2 KO cells and found hundreds of differentially expressed proteins (KO/WT fold change > 1.2 or < 0.83, p < 0.05) in NudCL2 KO cells. We found that the protein level of RCC2 (a midbody-associated protein) is obviously decrease in NudCL2 KO cells. Further studies display that knockout of NudCL2 induces an increase in RCC2 protein instability and degradation. Our data show that depletion of RCC2 leads to the similar defects as NudCL2 KO. Ectopic expression of RCC2 effectively rescues the cytokinesis failure caused by the loss of NudCL2. Interestingly, our data reveals that Hsp90 localizes to midbody in cytokinesis, and interacts with NudCL2 and RCC2. Inhibition of Hsp90 ATPase activity also causes the RCC2 instability and cytokinesis failure as NudCL2 KO. Moreover, the abnormal phenotypes induced by NudCL2 KO are able to rescue by the ectopic expression of Hsp90, but not vice versa. Taken together, our data suggest that NudCL2 is required to cytokinesis by stabilizing RCC2 at midbody through the Hsp90 pathway, providing a hitherto unrecognized mechanism crucial for cytokinesis regulation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Xiaoyang Xu

LAB HEAD: Tianhua Zhou

PROVIDER: PXD043570 | Pride | 2024-03-21

REPOSITORIES: Pride

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Project description:This 89-node Boolean model of mammalian growth factor signaling can reproduce oscillations in PI3K signaling in cycling cells, and links these oscillations to the regulatory networks that drive each phase of cell cycle progression, as well as apoptosis. It builds on our previous work on modeling cell cycle progression as the interaction of two linked multi-stable switches (Deritei et al, Sci Rep 6:21957, 2016) and extends it to capture the role of Plk1 in cell cycle progression. The resulting model reproduces the following experimentally documented cell behaviors: — cyclic PI3K/AKT1 activity in dividing cells, which remain in sync with the cell cycle — apoptosis in response to prolonged mitosis or mitotic catastrophe — four distinct, experimentally documented cell fates caused by Plk1 inhibition, depending on the timing of Plk1 loss; namely, G2 arrest, mitotic catastrophe, premature anaphase and chromosome mis-segregation leading to aneuploidy, and failure to complete cytokinesis following telophase, which can lead to genome duplication — failure of cytokinesis and accumulation of binucleate telophase cells driven by hyperactive PI3K, hyperactive Ak1, or FoxO inhibition. — the effect of a large number of knockdown / forced activation mutations Testable predictions: — PI3K degradation in response to high PI3K activation is driven by the Neddl4 ubiquitin ligase activated by PLCγ, while its re-synthesis requires nuclear re-accumulation of FoxO3. — The degradation/re-synthesis cycle of PI3K occurs twice per division cycle, synchronized by Plk1-mediated inhibition of FoxO3 during metaphase/anaphase. — Cells in which PI3K is inhibited after the start of DNA synthesis can nevertheless pre-commit to another cell cycle in the presence of saturating growth stimulation (passing the restriction point in late metaphase), albeit at lower rates than wild-type cells. — Cell cycle defects in response to PI3K/Ak1 over-activation or FoxO knockdown are driven by a loss of Plk1 in telophase.

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Quantitative proteomic analysis based on the isobaric tags for relative and absolute quantitation (iTRAQ) labeling in WT and NudCL2 KO-1 HEK-293 cells

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