We used linker histone-depleted normal human fibroblast nuclei as templates to study how phosphorylation affects histone H5 binding to chromatin in situ. Permeabilized cells were treated with 0.7 M NaCl to extract the native linker histones. Histone H5 was purified from chicken erythrocytes and phosphorylated in vitro by recombinant cdk5/p35 kinase. High performance capillary electrophoresis (HPCE) showed that the phosphorylated protein contained a mixture of multiply phosphorylated forms. Control experiments, using mass spectrometry, revealed that up to five SPXK motifs in the C terminus were phosphorylated, but also that about 10% of the protein contained one phosphoserine in the N-terminus. Reconstitution of H1-depleted fibroblast nuclei with nonphosphorylated or phosphorylated H5 was performed at physiological ionic strength. The bound H5 was then extracted using NaCl concentrations in the range of 0.15 to 0.7 M. The release of the H5 molecules was monitored by DAPI staining and image cytofluorometry. Our results show that H5 phosphorylation substantially reduced its affinity for chromatin in situ, which support previous observations indicating that C-terminal phosphorylation may be essential for the biological functions of linker histones.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1002/cyto.a.22221 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Uncovering novel KCC2 regulatory motifs through a comprehensive transposon-based mutant library.
Front Mol Neurosci
January 2025
Neuroscience Center, HiLIFE, University of Helsinki, Helsinki, Finland.
Introduction: The neuron-specific K-Cl cotransporter KCC2 maintains low intracellular chloride levels, which are crucial for fast GABAergic and glycinergic neurotransmission. KCC2 also plays a pivotal role in the development of excitatory glutamatergic neurotransmission by promoting dendritic spine maturation. The cytoplasmic C-terminal domain (KCC2-CTD) plays a critical regulatory role in the molecular mechanisms controlling the cotransporter activity through dimerization, phosphorylation, and protein interaction.
View Article and Find Full Text PDFSelection of initiator tRNA and start codon by mammalian mitochondrial initiation factor 3 in leaderless mRNA translation.
Nucleic Acids Res
January 2025
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan.
Article Synopsis
- The mammalian mitochondrial protein synthesis system is responsible for synthesizing 13 crucial subunits for energy production, but the exact role of IF-3mt in translation initiation is still unclear.
- Researchers created a mitochondrial translation system to explore IF-3mt's proofreading abilities, revealing it helps distinguish between different start codons for more accurate protein synthesis.
- The findings indicate that IF-3mt not only supports initiation from standard AUG start codons but can also facilitate initiation from non-AUG codons like AUA, highlighting its adaptability in working with leaderless mRNAs.
Characterization of multiple binding sites on microtubule associated protein 2c recognized by dimeric and monomeric 14-3-3ζ.
FEBS J
January 2025
Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain.
View Article and Find Full Text PDFThe Role of Individual Residues in the N-Terminus of Arrestin-1 in Rhodopsin Binding.
Int J Mol Sci
January 2025
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA.
Sequences and three-dimensional structures of the four vertebrate arrestins are very similar, yet in sharp contrast to other subtypes, arrestin-1 demonstrates exquisite selectivity for the active phosphorylated form of its cognate receptor, rhodopsin. The N-terminus participates in receptor binding and serves as the anchor of the C-terminus, the release of which facilitates arrestin transition into a receptor-binding state. We tested the effects of substitutions of fourteen residues in the N-terminus of arrestin-1 on the binding to phosphorylated and unphosphorylated light-activated rhodopsin of wild-type protein and its enhanced mutant with C-terminal deletion that demonstrates higher binding to both functional forms of rhodopsin.
View Article and Find Full Text PDFCasein Kinase I Protein Hrr25 Is Required for Pin4 Phosphorylation and Mediates Cell Wall Integrity Signaling in .
Genes (Basel)
January 2025
Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148, USA.
Background: Casein kinase I protein Hrr25 plays important roles in many cellular processes, including autophagy, vesicular trafficking, ribosome biogenesis, mitochondrial biogenesis, and the DNA damage response in . Pin4 is a multi-phosphorylated protein that has been reported to be involved in the cell wall integrity (CWI) pathway and DNA damage response. Pin4 was reported to interact with Hrr25 in yeast two-hybrid and large-scale pulldown assays.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!