Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

Grosstessner-Hain, Karin; Hegemann, Björn; Novatchkova, Maria; Rameseder, Jonathan; Joughin, Brian A.; Hudecz, Otto; Roitinger, Elisabeth; Pichler, Peter; Kraut, Norbert; Yaffe, Michael B.; Peters, Jan‐Michael; Mechtler, Karl

doi:10.1074/mcp.m111.008540

Cited by 67 publications

(76 citation statements)

References 48 publications

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“…CDK1/cyclin B has a large number of mitotic substrates, including lamins, various lamina-associated proteins, and subunits of the NPC (for review see Guttinger et al 2009). Other relevant kinases are polo-like kinase 1 (PLK-1), which has multiple targets in mitosis, including subunits of the NPC (Grosstessner-Hain et al 2011;Laurell et al 2011); VRK1 (VRK-1), which phosphorylated BAF (Nichols et al 2006;Gorjanacz et al 2007); and the AIR-1 aurora kinase (Portier et al 2007). Counteracting the activity of these kinases are a number of phosphatases, including protein phosphatase 1 (PP1) and PP2A (Wurzenberger and Gerlich 2011).…”

Section: The Dynamic Nature Of the Nucleusmentioning

confidence: 99%

Cell Biology of theCaenorhabditis elegansNucleus

Cohen-Fix

Askjaer

2017

Genetics

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Studies on the Caenorhabditis elegans nucleus have provided fascinating insight to the organization and activities of eukaryotic cells. Being the organelle that holds the genetic blueprint of the cell, the nucleus is critical for basically every aspect of cell biology. The stereotypical development of C. elegans from a one cell-stage embryo to a fertile hermaphrodite with 959 somatic nuclei has allowed the identification of mutants with specific alterations in gene expression programs, nuclear morphology, or nuclear positioning. Moreover, the early C. elegans embryo is an excellent model to dissect the mitotic processes of nuclear disassembly and reformation with high spatiotemporal resolution. We review here several features of the C. elegans nucleus, including its composition, structure, and dynamics. We also discuss the spatial organization of chromatin and regulation of gene expression and how this depends on tight control of nucleocytoplasmic transport. Finally, the extensive connections of the nucleus with the cytoskeleton and their implications during development are described. Most processes of the C. elegans nucleus are evolutionarily conserved, highlighting the relevance of this powerful and versatile model organism to human biology.KEYWORDS Caenorhabditis elegans; chromatin; gene expression; lamin; LEM-domain proteins; LINC; P granule; nuclear pore complex; nuclear envelope; nucleocytoplasmic transport; nucleolus; WormBook TABLE OF CONTENTSAbstract 25 C. elegans as a model system to study cell biology of the nucleus 26 Structural components of the nucleus 27

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Section: The Dynamic Nature Of the Nucleusmentioning

confidence: 99%

Cell Biology of theCaenorhabditis elegansNucleus

Cohen-Fix

Askjaer

2017

Genetics

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“…Given the inherent complexity of cellular proteome, prefractionation is prerequisite to improve the coverage of cellular phosphoproteome. For example, the combination of SCX and IMAC or MOAC has been proven to be an excellent strategy for phosphoproteome analysis 12,[44][45][46][47][48][49][50][51] .…”

Section: Introductionmentioning

confidence: 99%

Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

et al. 2013

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Mass spectrometry (Ms)-based proteomics has become the preferred tool for the analysis of protein phosphorylation. to be successful at such an endeavor, there is a requirement for an efficient enrichment of phosphopeptides. this is necessary because of the substoichiometric nature of phosphorylation at a given site and the complexity of the cell. recently, new alternative materials have emerged that allow excellent and robust enrichment of phosphopeptides. these monodisperse microsphere-based immobilized metal ion affinity chromatography (IMac) resins incorporate a flexible linker terminated with phosphonate groups that chelate either zirconium or titanium ions. the chelated zirconium or titanium ions bind specifically to phosphopeptides, with an affinity that is similar to that of other widely used metal oxide affinity chromatography materials (typically tio 2 ). Here we present a detailed protocol for the preparation of monodisperse microsphere-based ti 4 + -IMac adsorbents and the subsequent enrichment process. Furthermore, we discuss general pitfalls and crucial steps in the preparation of phosphoproteomics samples before enrichment and, just as importantly, in the subsequent mass spectrometric analysis. Key points such as lysis, preparation of the chromatographic system for analysis and the most appropriate methods for sequencing phosphopeptides are discussed. Bioinformatics analysis specifically relating to site localization is also addressed. Finally, we demonstrate how the protocols provided are appropriate for both single-protein analysis and the screening of entire phosphoproteomes. It takes ~2 weeks to complete the protocol: 1 week to prepare the ti 4 + -IMac material, 2 d for sample preparation, 3 d for Ms analysis of the enriched sample and 2 d for data analysis.

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“…A number of studies have taken a proteomics approach to identify mitotic PLK1 PBD-dependent binding partners and substrates [20,21]. However, these studies have identified few novel regulators of G2/M phase progression.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

“…We have identified a number of proteins previously identified as PLK1 binding proteins, including JIP4, as well as a number of novel interactors. The Polobox binding site identified in JIP4 was phospho-Thr217, but a phosphoproteomics screen for PLK1 phosphorylation sites found this site was not responsive to the PLK1 inhibitor BI-4834 [21]. Two other sites were identified as PLK1 phosphorylation sites on JIP4, Ser183/185 and Ser 311 [23], although neither of these match consensus Polobox binding sites [20].…”

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JIP4 is a PLK1 binding protein that regulates p38MAPK activity in G2 phase

Pinder

Loo

Harrington

et al. 2015

Cellular Signalling

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Cell cycle progression from G2 phase into mitosis is regulated by a complex network of mechanisms, all of which finally control the timing of Cyclin B/CDK1 activation. PLK1 regulates a network of events that contribute to regulating G2/M phase progression. Here we have used a proteomics approach to identify proteins that specifically bind to the Polobox domain of PLK1. This identified a panel of proteins that were either associated with PLK1 in G2 phase and/or mitosis, the strongest interaction being with the MAPK scaffold protein JIP4. PLK1 binding to JIP4 was found in G2 phase and mitosis, and PLK1 binding was selfprimed by PLK1 phosphorylation of JIP4. PLK1 binding is required for JIP4-dependent p38MAPK activation in G2 phase during normal cell cycle progression, but not in either G2 phase or mitotic stress response. Finally, JIP4 is a target for caspase-dependent cleavage in mitotically arrested cells. The role for the PLK1-JIP4 regulated p38MAPK activation in G2 phase is unclear, but it does not affect either progression into or through mitosis.

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Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome

Cited by 67 publications

References 48 publications

Cell Biology of theCaenorhabditis elegansNucleus

Cell Biology of theCaenorhabditis elegansNucleus

Robust phosphoproteome enrichment using monodisperse microsphere–based immobilized titanium (IV) ion affinity chromatography

JIP4 is a PLK1 binding protein that regulates p38MAPK activity in G2 phase

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