title: Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase.
abstract: The NEIL1 DNA glycosylase is one of eleven mammalian DNA glycosylases that partake in the first step of the base excision repair (BER) pathway. NEIL1 recognizes and cleaves mainly oxidized pyrimidines from DNA. The past decade has witnessed the identification of an increasing number of post-translational modifications (PTMs) in BER enzymes including phosphorylation, acetylation, and sumoylation, which modulate enzyme function. In this work, we performed the first comprehensive analysis of phosphorylation sites in human NEIL1 expressed in human cells. Mass spectrometry (MS) analysis revealed phosphorylation at three serine residues: S207, S306, and a third novel site, S61. We expressed, purified, and characterized phosphomimetic (glutamate) and phosphoablating (alanine) mutants of the three phosphorylation sites in NEIL1 revealed by the MS analysis. All mutant enzymes were active and bound tightly to DNA, indicating that phosphorylation does not affect DNA binding and enzyme activity at these three serine sites. We also characterized phosphomimetic mutants of two other sites of phosphorylation, Y263 and S269, reported previously, and observed that mutation of Y263 to E yielded a completely inactive enzyme. Furthermore, based on sequence motifs and kinase prediction algorithms, we identified the c-Jun N-terminal kinase 1 (JNK1) as the kinase involved in the phosphorylation of NEIL1. JNK1, a member of the mitogen activated protein kinase (MAPK) family, was detected in NEIL1 immunoprecipitates, interacted with NEIL1 in vitro, and was able to phosphorylate the enzyme at residues S207, S306, and S61.
authors: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL, 36604-1405, United States of America.
authors: Aishwarya
authors: A
authors: Prakash
authors: Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, Vermont, 05405-0068, United States of America.
authors: Vy Bao
authors: VB
authors: Cao
authors: Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, Vermont, 05405-0068, United States of America.
authors: Sylvie
authors: S
authors: Doublié
mesh_terms: D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D048055:Mitogen-Activated Protein Kinase 8 / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational
chemical_list: D004247:DNA; D048055:Mitogen-Activated Protein Kinase 8; D045647:DNA Glycosylases; C473634:NEIL1 protein, human