Preparation of Recombinant Histone H3 as a Substrate for Protein Kinase Assays

Himpel, Sunke; Joost, Hans‐Georg; Becker, Walter

doi:10.1006/abio.1999.4205

Cited by 3 publications

(3 citation statements)

References 16 publications

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“…3 To avoid potential problems due to post-translational modifications or sequence microheterogeneity of histone H3 isoforms, we decided to use recombinant histone H3 (rH3) for the following experiments. Construction of the expression plasmid and the method to prepare bacterially expressed rH3 are presented elsewhere (20).…”

Section: Resultsmentioning

confidence: 99%

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Specificity Determinants of Substrate Recognition by the Protein Kinase DYRK1A

Himpel¹,

Tegge²,

Frank³

et al. 2000

Journal of Biological Chemistry

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223

221

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DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K m ‫؍‬ 35 M) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogenactivated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P ؊3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.DYRK1A is a nuclear protein kinase that is ubiquitously expressed in rat tissues (1, 2). We have recently characterized a closely related isoform, DYRK1B, that is predominantly expressed in testis and muscle (3). The homolog of DYRK1A and DYRK1B in Drosophila, the protein kinase MNB, is encoded by the minibrain gene whose mutation results in specific defects in the development of the central nervous system (4). The human gene for DYRK1A is located in the "Down syndrome critical region" of chromosome 21, and the similarity of DYRK1A and MNB suggests that the triplication of the DYRK1A gene may play a role in mental retardation of patients with Down syndrome (5-9).DYRK1A, DYRK1B, and MNB belong to a subfamily of protein kinases with structurally related catalytic domains and similar enzymatic properties (2, 10). At least 7 different DYRKrelated kinases exist in mammals, of which DYRK1A and DYRK1B are targeted to the nucleus, whereas DYRK2 and DYRK3 are located in the cytoplasm (2, 3).1 Members of the DYRK family have also been found in lower eukaryotes, such as Yak1p in Saccharomyces cerevisiae (11), Pom1p in Schizosaccharomyces pombe (12), and YAKA in Dictyostelium discoideum (13). Although mutations in YAK1, pom1, and yakA have diverse phenotypic consequences, it appears reasonable to generalize that DYRK-related kinases are involved in the regulation of growth and development.The enzymatic activity of DYRK1A has been shown to depend on the presence of tyrosine residues in the activation loop, a result that suggests an activation mechanism similar to that of the MAP 2 kinases (1). However, the participation of DYRK1A or other DYRK-related kinases in a particular signal transduction pathway has not been eluci...

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Section: Resultsmentioning

confidence: 99%

“…Recombinant histone H3 (rH3) was prepared from bacterial inclusion bodies as described elsewhere (20). Point mutants of rH3 were generated by overlap extension polymerase chain reaction (21).…”

Section: Methodsmentioning

confidence: 99%

Specificity Determinants of Substrate Recognition by the Protein Kinase DYRK1A

Himpel¹,

Tegge²,

Frank³

et al. 2000

Journal of Biological Chemistry

Self Cite

223

221

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“…Proteins were eluted from the beads under native conditions (reduced glutathione or imidazole). His 6 -tagged cyclin L2 expressed from the pQE-30NST vector (RZPD clone N16596) was purified from inclusion bodies as described previously (37). The purified protein was refolded by gel filtration through a Sephadex G25 column (NAP TM -5 column, Amersham Biosciences) with 10 mM Tris-HCl, pH 7.4, 100 mM NaCl as equilibration and elution buffer.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Cyclin L2, a Novel Cyclin with an Arginine/Serine-rich Domain

Graaf

Hekerman

Spelten

et al. 2004

Journal of Biological Chemistry

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110

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A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2 S ) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2 S , was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domaincontaining proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.

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Characterization of Posttranslational Modifications on Histone Variants

Saavedra

Marty-Lombardi

Loyola

2018

Methods in Molecular Biology

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The study of histone variants and histone posttranslational modifications (PTMs) is a trending topic in different fields including developmental biology, neurobiology, and immunology; as well as in the understanding of molecular mechanisms leading to diverse diseases, such as cancer. Since the establishment of histone PTMs starts immediately after their synthesis and it continues once they are assembled into chromatin, here we describe a classic protocol of subcellular fractionation aiming to study histones at different stages of maturation. This includes newly synthesized histones enriched in cytosolic fractions; a pool of newly synthesized, evicted, and stored histones enriched in nuclear soluble fractions; and chromatin-associated histones enriched in chromatin pellet. To study specific histone variants and the establishment of their PTMs, we describe a protocol for obtaining histone variants expressed in bacteria. In addition, we describe a Triton-Acetic acid-Urea (TAU) gel electrophoresis protocol adapted to work on mini-gels, which can be coupled to Western blot to analyze PTMs on histone variants. Finally, we describe a Chromatin immunoprecipitation (ChIP) assay for studying histone PTMs, or tagged histone variants, on specific DNA sequences.

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Preparation of Recombinant Histone H3 as a Substrate for Protein Kinase Assays

Cited by 3 publications

References 16 publications

Specificity Determinants of Substrate Recognition by the Protein Kinase DYRK1A

Specificity Determinants of Substrate Recognition by the Protein Kinase DYRK1A

Characterization of Cyclin L2, a Novel Cyclin with an Arginine/Serine-rich Domain

Characterization of Posttranslational Modifications on Histone Variants

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