1 Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet2 Groth Group, BRIC Research Groups, BRIC, Københavns Universitet3 Ploug Group, BRIC Research Groups, BRIC, Københavns Universitet4 Department of Biochemistry and Molecular Biology, SDU5 Groth Group, BRIC Research Groups, BRIC, Københavns Universitet6 Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet7 Ploug Group, BRIC Research Groups, BRIC, Københavns Universitet
During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry and dissect how phosphorylation has an impact on human Asf1 function. The divergent C-terminal tail of Asf1a is phosphorylated at several sites, and this is required for timely progression through S phase. Consistent with this, biochemical analysis of wild-type and phospho-mimetic Asf1a shows that phosphorylation enhances binding to histones and the downstream chaperones CAF-1 and HIRA. Moreover, we find that TLK phosphorylation of Asf1a is induced in cells experiencing deficiency of new histones and that TLK interaction with Asf1a involves its histone-binding pocket. We thus propose that TLK signalling promotes histone supply in S phase by targeting histone-free Asf1 and stimulating its ability to shuttle histones to sites of chromatin assembly.
Nature Communications, 2014, Vol 5
Amino Acid Sequence; Binding Sites; Blotting, Western; Cell Cycle Proteins; Cell Line, Tumor; Chromatin; DNA Replication; HeLa Cells; Histones; Humans; Mass Spectrometry; Microscopy, Confocal; Molecular Chaperones; Mutation; Phosphorylation; Protein Binding; Protein-Serine-Threonine Kinases; RNA Interference; S Phase