Four Distinct Cyclin-Dependent Kinases Phosphorylate Histone H1 at All of Its Growth-Related Phosphorylation Sites

Swank, Richard A.; Th'ng, John P.H.; Guo, Xiaowen; Valdez, J.G.; Bradbury, E. Morton; Gurley, L.R.

doi:10.1021/bi9714363

Cited by 42 publications

(36 citation statements)

References 36 publications

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“…The phosphorylation activity for these phosphorylation sites appeared to be independent of variant and site identity. The kinase for these phosphorylation sites has not been identified in human cells, but the equivalent peptide (SETAPAAPAAAPPAEK) is phosphorylated at both phosphorylation sites in a cell-cycle-specific manner by p38cdc2 in Chinese hamster ovary cells (50).…”

Section: Figmentioning

confidence: 99%

Stable Isotope Labeling of Phosphoproteins for Large-scale Phosphorylation Rate Determination

Molden

Goya

Khan

et al. 2014

Molecular & Cellular Proteomics

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Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MSbased techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [␥- 18 O 4 ]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min ؊1 to 0.001 min ؊1 . Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680. Molecular & Cellular Proteomics 13: 10.1074/mcp.O113.036145, 1106-1118, 2014.Protein phosphorylation is crucial for modulating protein structure, protein localization, and the protein-protein interactions that form the basis of many cell-signaling networks. Phosphorylation-based signaling often takes the form of a cascade in which sequential protein phosphorylations lead to changes in protein stability, function, and localization. Protein kinases, the enzymes that propagate these signals, catalyze the transfer of ␥ phosphate from ATP onto serine, threonine, or tyrosine residues of substrate proteins. The sites of protein phosphorylation and phosphorylation dynamics are important in determining the biological outcome of a signaling event (1). For instance, protein phosphorylation drives many of the changes during the cell cycle (2, 3). During mitosis, kinases are activated at precise times to direct the course of chromosome segregation and cell division. For example, CDK1 activation at the beginning of mitosis leads to phosphorylation of NUP98 during prophase, w...

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Section: Figmentioning

confidence: 99%

Stable Isotope Labeling of Phosphoproteins for Large-scale Phosphorylation Rate Determination

Molden

Goya

Khan

et al. 2014

Molecular & Cellular Proteomics

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“…We therefore chose this kinase, as well as the closely related Aurora A, as candidates to test in the subsequent assays. Although CDK1 was not predicted to be a putative H1.4S27 kinases, we decided to include it in our analyses as it has been shown that CDKs can phosphorylate serine and threonine residues in non-CDK consensus sites (Swank et al, 1997). We found that both recombinant Aurora A and Aurora B could phosphorylate H1.4 at S27 in vitro, whereas a CDK1-cyclinB1 complex could not (Fig.…”

Section: Aurora B Is a Mitotic H14s27 Kinasementioning

confidence: 99%

Isoform-specific phosphorylation of human linker histone H1.4 in mitosis by the kinase Aurora B

Hergeth

Dundr

Tropberger

et al. 2011

Journal of Cell Science

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The linker histone H1 plays an essential role in maintaining and establishing higher-order chromatin structure. As with core histones, histone H1 is also extensively covalently modified. We showed previously that phosphorylation of S27 in human histone H1.4 (H1.4S27-P), prevents binding of heterochromatin protein 1 (HP1) family members (officially known as chromobox protein homologs) to the neighboring dimethylated K26. Here, we present the first functional characterization of H1.4S27-P in vivo and in vitro. We show that H1.4S27 phosphorylation is cell-cycle-regulated and its levels peak on metaphase chromosomes. We identify further Aurora B as the kinase phosphorylating H1.4S27. We demonstrate that histone H1.4 is the only somatic linker histone variant targeted by Aurora B and that Aurora B exclusively phosphorylates S27. Adjacent K26 dimethylation can regulate Aurora B activity towards S27, uncovering a crosstalk between these modifications. Finally, our fluorescence recovery after photobleaching (FRAP) analysis on histone H1.4 mutants suggests a role of S27 phosphorylation in the regulation of histone H1.4 mobility and chromatin binding in mitosis.

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“…Although they have diverged much more than the core histones during evolution, linker histones in a wide variety of species typically contain numerous (S/T)PXZ motifs (where X is any amino acid and Z is a basic amino acid), and accordingly cyclin-dependent kinases have been investigated for their roles in H1 phosphorylation. Toward this end, the addition of Cdc2 kinase has been shown to cause premature chromatin condensation in some cell types, and decreased H1 phosphorylation following inhibition of cyclin-dependent kinases is associated with chromosome decondensation (22,23). However, because H1 phosphorylation and mitosis do not always occur simultaneously in certain organisms such as in Tetrahymena thermophila, the function of H1 phosphorylation has remained elusive.…”

Section: Molecular and Cellular Proteomics 5: 1593-1609 2006mentioning

confidence: 99%

Comprehensive Phosphoprotein Analysis of Linker Histone H1 from Tetrahymena thermophila

Garcia

Joshi

Thomas

et al. 2006

Molecular & Cellular Proteomics

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Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophila but becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation "signature" for this linker histone. Histone H1 from both growing and starved Tetrahymena was analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymena were obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry. Molecular & Cellular Proteomics 5: 1593-1609, 2006.

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Four Distinct Cyclin-Dependent Kinases Phosphorylate Histone H1 at All of Its Growth-Related Phosphorylation Sites

Cited by 42 publications

References 36 publications

Stable Isotope Labeling of Phosphoproteins for Large-scale Phosphorylation Rate Determination

Stable Isotope Labeling of Phosphoproteins for Large-scale Phosphorylation Rate Determination

Isoform-specific phosphorylation of human linker histone H1.4 in mitosis by the kinase Aurora B

Comprehensive Phosphoprotein Analysis of Linker Histone H1 from Tetrahymena thermophila

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