Dataset Information

H3K79 methyltransferase Dot1p/DOT1L is phosphorylated by proline-directed MAP kinases Hog1p and p38/MAPK11

ABSTRACT: Disruptor of telomeric silencing (Dot1p) is an exquisitely conserved histone methyltransferase, and is the sole enzyme responsible for H3K79 methylation in the budding yeast, Saccharomyces cerevisiae. It has been shown to be highly phosphorylated in vivo; however, the upstream kinases that act on Dot1p are unknown in yeast and all other eukaryotes. Here, we used in vitro and in vivo kinase discovery approaches to show that the mitogen-activated protein kinase Hog1p is a bona fide kinase of Dot1p methyltransferase. In in vitro kinase assays, Hog1p phosphorylates Dot1p at multiple sites, including at several proline-adjacent sites that are consistent with its known substrate preference. Hog1p kinase activity was specifically enhanced at these proline-adjacent sites on Dot1p upon its activation by the osmostress-responsive kinase, Pbs2p. We then show that genomic deletion of HOG1 markedly reduces phosphorylation at specific sites on Dot1p in vivo, thus providing evidence for Hog1p kinase activity on Dot1p in budding yeast cells. Phenotypic analysis of knockout and phosphosite mutant yeast strains revealed the importance of Hog1p-catalysed phosphorylation of Dot1p for cellular responses to ultraviolet-induced DNA damage. This kinase-substrate relationship is conserved in mammalian systems, where human DOT1L (ortholog of Dot1p) can be phosphorylated by the proline-directed kinase p38β/MAPK11 (ortholog of Hog1p) at multiple sites in vitro. Taken together, our findings establish Hog1p and p38β/MAPK11 as the first kinases known to act on H3K79 methyltransferase enzymes in any eukaryotic species.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Ryan Separovich

LAB HEAD: Marc R. Wilkins

PROVIDER: PXD036747 | Pride | 2024-01-29

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	DOT1L_ID_trypsin.dat	Other
	DOT1L___p38_no_ATP_trypsin.dat	Other
	DOT1L___p38_rep1_trypsin.dat	Other
	DOT1L___p38_rep2_trypsin.dat	Other
	DOT1L_noEnzyme_trypsin_1ul.raw	Raw

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Publications

Proline-directed yeast and human MAP kinases phosphorylate the Dot1p/DOT1L histone H3K79 methyltransferase.

Separovich Ryan J RJ Karakatsanis Nicola M NM Gao Kelley K Fuh David D Hamey Joshua J JJ Wilkins Marc R MR

The FEBS journal 20240125 12

Disruptor of telomeric silencing 1 (Dot1p) is an exquisitely conserved histone methyltransferase and is the sole enzyme responsible for H3K79 methylation in the budding yeast Saccharomyces cerevisiae. It has been shown to be highly phosphorylated in vivo; however, the upstream kinases that act on Dot1p are almost entirely unknown in yeast and all other eukaryotes. Here, we used in vitro and in vivo kinase discovery approaches to show that mitogen-activated protein kinase HOG1 (Hog1p) is a bona f ...[more]

PMID: 38270553

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