Phosphoproteome analysis of the human mitotic spindle
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q-Tof Ultima, instrument model
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Hela Cell
SUBMITTER: 
DAVE THORNEYCROFT
LAB HEAD: DAVE THORNEYCROFT
PROVIDER: PRD000007 | Pride | 2006-02-24
REPOSITORIES: Pride
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Project description:Abstract: Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, methodologies to enrich the heterogeneous types of phosphopeptides are critical towards comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinctive binding affinity of Ga3+ and Fe3+ towards phosphopeptides, we designed a tip-based metal-directed immobilized metal ion affinity (MD-IMAC) chromatography for sequential enrichment of phosphopeptides. On the analysis of Raji B cell, this sequential Ga3+-Fe3+-IMAC strategy demonstrated 1.5-3.5 fold superior phosphoproteomic coverage compared to the single IMAC (Fe3+, Ti4+, Ga3+ and Al3+). In addition, as high as 92% among the 6283 phosphopeptides was uniquely enriched by either 1st Ga3+-IMAC fraction (41%) or 2nd Fe3+-IMAC fraction (51%). The complementary property of Ga3+ and Fe3+ was further shown on the exclusively superior efficiency to enriched almost all the 1214 multiply phosphorylated peptides (99.4%) by 1st Ga3+-IMAC, while as low as 10% of 5069 monophosphorylated phosphopeptides was commonly enriched by both fractions. Application of our sequential Ga3+-Fe3+-IMAC approach to a human lung cancer tissue allowed the identification of 2560 unique phosphopeptides with only 8% overlapping. The fractionation ability was shown not only on the mono phosphopeptides and multiply phosphopeptides but also on the basic and acidic phosphopeptides; acidiphilic phosphorylation sites were predominately present in 1st Ga3+-IMAC (72%) and 85% Pro-directed and 79% basophilic phosphorylation sites were enriched by 2nd Fe3+-IMAC. Most interestingly, this strategy complementarily mapped different kinase substrate on the protein as well as site levels in multiple cellular pathways related to cancer invasion and metastasis of lung cancer ., Given the demonstrated fractionation ability, reproducibility, sensitivity and ease of tip preparation, we hope that this Ga3+-Fe3+-IMAC allow more comprehensive characterization of phosphoproteome in vitro and in vivo. Database search: The raw MS/MS data obtained by TripleTOF 5600 were processed using AB_SCIEX MS Data Converter with default parameters. All MS/MS files were analyzed using Mascot (Matrix Science, London, UK; version 2.3) against the SwissPort database (version 57.8) with the following constraints: an allowance for tryptic peptides of up to two missed cleavage sites, a fragment ion mass tolerance of 0.05 Da, and a parent ion tolerance of 10 ppm. Phosphorylation (S, T, Y) and oxidation (M) were selected as variable modifications. Searching on a randomized decoy database created by Mascot was required to evaluate the false discovery rate associated with protein identification. The false discovery rates with a Mascot score (p< 0.05) ranged between 0% and 1% in this study.
2014-01-29 | PXD000314 | Pride
Project description:During cell division, the mitotic spindle segregates the sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Errors in spindle formation can result in both chromosome missegregation and cytokinesis defects and hence lead to genomic instability. To ensure the correct function of the spindle, the activity and localization of spindle associated proteins has to be tightly regulated in time and space. Reversible phosphorylation has been shown to be one of the key regulatory mechanisms for the organization of the mitotic spindle. The relatively low number of identified in vivo phosphorylation sites of spindle components, however, has hampered functional analysis of regulatory spindle networks. A more complete inventory of the phosphorylation sites of spindle-associated proteins would therefore constitute an important advance. Here, we describe the mass spectrometry-based identification of in vivo phosphorylation sites from purified human mitotic spindles. In total, 736 phosphorylation sites were identified, of which 312 could be attributed to known spindle proteins. Among these are phosphorylation sites that were previously shown to be important for the regulation of spindle-associated proteins. Importantly, this data set also comprises 279 novel phosphorylation sites of known spindle proteins for future functional studies. This inventory of spindle phosphorylation sites should thus make an important contribution to a better understanding of the molecular mechanisms that regulate the formation, function, and integrity of the mitotic spindle.
| S-EPMC1459365 | biostudies-literature
Project description:Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle.
| S-EPMC3013462 | biostudies-literature
Project description:To ensure an even segregation of chromosomes during somatic cell division, eukaryotes rely on specific microtubule structures called mitotic spindles. There are however striking differences in overall spindle organization among eukaryotic super groups, and in particular little is known about how spindle architecture is determined in plants. As a foundation for our work, we have measured prime characteristics of Arabidopsis mitotic spindles and built a three-dimensional dynamic model of the Arabidopsis mitotic spindle using Cytosim. Next, we identified the cell-cycle regulator CYCLIN-DEPENDENT KINASE B1 (CDKB1) together with its cyclin partner CYCB3;1 as key regulators of spindle shape and organization in Arabidopsis. Loss of CDKB1 function resulted in a high number of astral microtubules that are normally absent from plant spindles, as opposed to animal ones. We identified an augmin complex member, ENDOSPERM DEFECTIVE1 (EDE1), as a substrate of the CDKB1;1-CYCB3;1 complex. A non-phosphorylatable mutant of EDE1 displayed spindles with extended pole-to-pole distance, resembling the phenotypes of cycb3;1 and cdkb1 mutants, and the mutated EDE1 associated less efficiently with spindle microtubules. Consistently, reducing the level of augmin in Cytosim simulations largely recapitulated the spindle phenotypes observed in cycb3;1 and cdkb1 mutants. Our results emphasize the importance of cell cycle-dependent phospho-control of the mitotic spindle in plant cells. They also support the validity of our computational model as a framework for the exploration of mechanisms controlling the organization of plant spindles
2024-08-27 | PXD046697 | Pride
Project description:Staufen1 (STAU1) is an RNA-binding protein involved in maturation, localization, translation and decay of mRNAs. STAU1 expression is modulated during the cell cycle and decreases during mitosis. In prometaphase, STAU155 binds specific classes of mRNAs that code for proteins implicated in transcription and cell cycle regulation. In this paper, we report that STAU155 co-localizes with microtubules on the mitotic spindle in human colorectal cancer cell line HCT116, and map the molecular determinant required for this association within the N-terminal 88 amino acids (aa 25-37). Interestingly, STAU1 co-purifies with ribosomal proteins and co-localizes with active sites of translation on the mitotic spindle. To characterize STAU1-dependent mRNA transport and localization on the spindle, we used RNAseq analysis to identify spindle-associated mRNAs on purified spindles of wild-type and STAU1-KO CRISPR cell lines. Our datasets identify 161 protein-coding transcripts that are less abundant on the mitotic spindle of STAU1-KO cells compared to WT, and 660 that are more abundant. Altogether, these data demonstrate that STAU1 controls the transport and the localization of specific sub-populations of mRNAs to the mitotic spindle of cancer cells and suggest that at least some spindle-localized mRNAs undergo local translation during mitosis.
2019-10-05 | GSE138441 | GEO
Project description:Purpose: Matrin3 (MATR3) is a DNA and RNA-binding protein and one of the major components of the nuclear matrix. Matrin3 is upregulated in colorectal cancer (CRC) compared to normal tissues, indicating potential oncogenic function in CRC. We found that depletion of Matrin3 results in decreased proliferation and colony formation in two CRC cell lines. To understand the molecular mechanism(s) by which Matrin3 mediates these effects, we aimed to find Matrin3 direct RNA targets and the effects of Matrin3 on them. Methods: We performed PAR-CLIP for endogenous Matrin3 and RNA-seq after Matrin3 silencing in HCT116 cells, a colorectal cancer cell line. Results: We uncovered Matrin3-mediated regulation of spindle dynamics in colorectal cancer (CRC) cells. We identified bound and regulated Matrin3 target RNAs transcriptome-wide in CRC cells, and found that Matrin3 broadly regulates alternative splicing. Among the top Matrin3 targets, we focused on CDC14B and found that Matrin3 loss resulted in misprocessing of the CDC14B transcript that has a premature termination codon and simultaneous down-regulation of the standard CDC14B transcript in our model. Selective knockdown of the CDC14B standard variant phenocopied the loss of Matrin3 and resulted in reduced CRC cell proliferation, stabilized microtubules, and defects in mitotic spindle formation with tumbled mitotic spindles, suggesting that CDC14B is a key downstream effector of Matrin3. Conclusions: Our data show that by regulating the abundance of CDC14B standard variant, Matrin3 contributes to maintenance of microtubules dynamics, spindle morphology and proper mitotic spindle orientation and suggest that defects in this pathway may contribute to the reduced proliferation of cells after depletion of Matrin3.
2023-03-17 | GSE205565 | GEO
Project description:Purpose: Matrin3 (MATR3) is a DNA and RNA-binding protein and one of the major components of the nuclear matrix. Matrin3 is upregulated in colorectal cancer (CRC) compared to normal tissues, indicating potential oncogenic function in CRC. We found that depletion of Matrin3 results in decreased proliferation and colony formation in two CRC cell lines. To understand the molecular mechanism(s) by which Matrin3 mediates these effects, we aimed to find Matrin3 direct RNA targets and the effects of Matrin3 on them. Methods: We performed PAR-CLIP for endogenous Matrin3 and RNA-seq after Matrin3 silencing in HCT116 cells, a colorectal cancer cell line. Results: We uncovered Matrin3-mediated regulation of spindle dynamics in colorectal cancer (CRC) cells. We identified bound and regulated Matrin3 target RNAs transcriptome-wide in CRC cells, and found that Matrin3 broadly regulates alternative splicing. Among the top Matrin3 targets, we focused on CDC14B and found that Matrin3 loss resulted in misprocessing of the CDC14B transcript that has a premature termination codon and simultaneous down-regulation of the standard CDC14B transcript in our model. Selective knockdown of the CDC14B standard variant phenocopied the loss of Matrin3 and resulted in reduced CRC cell proliferation, stabilized microtubules, and defects in mitotic spindle formation with tumbled mitotic spindles, suggesting that CDC14B is a key downstream effector of Matrin3. Conclusions: Our data show that by regulating the abundance of CDC14B standard variant, Matrin3 contributes to maintenance of microtubules dynamics, spindle morphology and proper mitotic spindle orientation and suggest that defects in this pathway may contribute to the reduced proliferation of cells after depletion of Matrin3.
2023-03-17 | GSE203520 | GEO
Project description:Purpose: Matrin3 (MATR3) is a DNA and RNA-binding protein and one of the major components of the nuclear matrix. Matrin3 is upregulated in colorectal cancer (CRC) compared to normal tissues, indicating potential oncogenic function in CRC. We found that depletion of Matrin3 results in decreased proliferation and colony formation in two CRC cell lines. To understand the molecular mechanism(s) by which Matrin3 mediates these effects, we aimed to find Matrin3 direct RNA targets and the effects of Matrin3 on them. Methods: We performed PAR-CLIP for endogenous Matrin3 and RNA-seq after Matrin3 silencing in HCT116 cells, a colorectal cancer cell line. Results: We uncovered Matrin3-mediated regulation of spindle dynamics in colorectal cancer (CRC) cells. We identified bound and regulated Matrin3 target RNAs transcriptome-wide in CRC cells, and found that Matrin3 broadly regulates alternative splicing. Among the top Matrin3 targets, we focused on CDC14B and found that Matrin3 loss resulted in misprocessing of the CDC14B transcript that has a premature termination codon and simultaneous down-regulation of the standard CDC14B transcript in our model. Selective knockdown of the CDC14B standard variant phenocopied the loss of Matrin3 and resulted in reduced CRC cell proliferation, stabilized microtubules, and defects in mitotic spindle formation with tumbled mitotic spindles, suggesting that CDC14B is a key downstream effector of Matrin3. Conclusions: Our data show that by regulating the abundance of CDC14B standard variant, Matrin3 contributes to maintenance of microtubules dynamics, spindle morphology and proper mitotic spindle orientation and suggest that defects in this pathway may contribute to the reduced proliferation of cells after depletion of Matrin3.
2023-03-17 | GSE203519 | GEO
Project description:Purpose: Matrin3 (MATR3) is a DNA and RNA-binding protein and one of the major components of the nuclear matrix. Matrin3 is upregulated in colorectal cancer (CRC) compared to normal tissues, indicating potential oncogenic function in CRC. We found that depletion of Matrin3 results in decreased proliferation and colony formation in two CRC cell lines. To understand the molecular mechanism(s) by which Matrin3 mediates these effects, we aimed to find Matrin3 direct RNA targets and the effects of Matrin3 on them. Methods: We performed PAR-CLIP for endogenous Matrin3 and RNA-seq after Matrin3 silencing in HCT116 cells, a colorectal cancer cell line. Results: We uncovered Matrin3-mediated regulation of spindle dynamics in colorectal cancer (CRC) cells. We identified bound and regulated Matrin3 target RNAs transcriptome-wide in CRC cells, and found that Matrin3 broadly regulates alternative splicing. Among the top Matrin3 targets, we focused on CDC14B and found that Matrin3 loss resulted in misprocessing of the CDC14B transcript that has a premature termination codon and simultaneous down-regulation of the standard CDC14B transcript in our model. Selective knockdown of the CDC14B standard variant phenocopied the loss of Matrin3 and resulted in reduced CRC cell proliferation, stabilized microtubules, and defects in mitotic spindle formation with tumbled mitotic spindles, suggesting that CDC14B is a key downstream effector of Matrin3. Conclusions: Our data show that by regulating the abundance of CDC14B standard variant, Matrin3 contributes to maintenance of microtubules dynamics, spindle morphology and proper mitotic spindle orientation and suggest that defects in this pathway may contribute to the reduced proliferation of cells after depletion of Matrin3.
2023-03-17 | GSE203518 | GEO
Project description:Yeast SILAC Bottom-Up Phosphoproteomics. Phorphopeptides purified following tryptic digest using IMAC (Fe3+) and analyzed on nano-LC-MS/MS (RSLC-Nano)
2024-04-16 | MSV000094550 | MassIVE