Mediator of DNA damage checkpoint 1 (MDC1) contributes to high NaCl-induced activation of the osmoprotective transcription factor TonEBP/OREBP.
BACKGROUND
Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins.


METHODS
We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation.


RESULTS
We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity.


CONCLUSIONS
MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of that factor's transcriptional activity.
Kunin
Margarita
M
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States of America.
Dmitrieva
Natalia I
NI
Gallazzini
Morgan
M
Shen
Rong-Fong
RF
Wang
Guanghui
G
Burg
Maurice B
MB
Ferraris
Joan D
JD
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D055785:Gene Knockdown Techniques; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology*; D012995:Solubility; D015534:Trans-Activators / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C413163:MDC1 protein, human; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D012965:Sodium Chloride; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases