Identification of Phosphorylation Sites on the Yeast Ribonucleotide Reductase Inhibitor Sml1

Tomoaki Uchiki, Lezlee T. Dice, Robert L. Hettich, Chris Dealwis

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

Sml1 is a small protein in Saccharomyces cerevisiae which inhibits the activity of ribonucleotide reductase (RNR). RNR catalyzes the rate-limiting step of de novo dNTP synthesis. Sml1 is a downstream effector of the Mec1/Rad53 cell cycle checkpoint pathway. The phosphorylation by Dun1 kinase during S phase or in response to DNA damage leads to diminished levels of Sml1. Removal of Sml1 increases the population of active RNR, which raises cellular dNTP levels. In this study using mass spectrometry and site-directed mutagenesis, we have identified the region of Sml1 phosphorylation to be between residues 52 and 64 containing the sequence GSSAS-ASASSLEM. This is the first identification of a phosphorylation sequence of a Dun1 biological substrate. This sequence is quite different from the consensus Dun1 phosphorylation sequence reported previously from peptide library studies. The specific phosphoserines were identified to be Ser56, Ser58, and Ser60 by chemical modification of these residues to S-ethylcysteines followed by collision activated dissociation. To investigate further Sml1 phosphorylation, we constructed the single mutants S56A, S58A, S60A, and the triple mutant S56A/S58A/S60A and compared their degrees of phosphorylation with that of wild type Sml1. We observed a 90% decrease in the relative phosphorylation of S60A compared with that of wild type, a 25% decrease in S58A, and little or no decrease in the S56A mutant. There was no observed phosphate incorporation in the triple mutant, suggesting that Ser56, Ser58, and Ser60 in Sml1 are the sites of phosphorylation. Further mutagenesis studies reveal that Dun1 kinase requires an acidic residue at the +3 position, and there is cooperativity between the phosphorylation sites. These results show that Dun1 has a unique phosphorylation motif.

Original languageEnglish
Pages (from-to)11293-11303
Number of pages11
JournalJournal of Biological Chemistry
Volume279
Issue number12
DOIs
StatePublished - Mar 19 2004

Funding

FundersFunder number
National Cancer InstituteR01CA100827

    Fingerprint

    Dive into the research topics of 'Identification of Phosphorylation Sites on the Yeast Ribonucleotide Reductase Inhibitor Sml1'. Together they form a unique fingerprint.

    Cite this