Contribution of SHP-1 protein tyrosine phosphatase to osmotic regulation of the transcription factor TonEBP/OREBP.
Hypertonicity activates the transcription factor TonEBP/OREBP, resulting in increased expression of osmoprotective genes, including those responsible for accumulation of organic osmolytes and heat-shock proteins. Phosphorylation of TonEBP/OREBP contributes to its activation. Several of the kinases that are involved were previously identified, but the phosphatases were not. In the present studies we screened a genomewide human phosphatase siRNA library in human embryonic kidney (HEK)293 cells for effects on TonEBP/OREBP transcriptional activity. We found that siRNAs against 57 phosphatases significantly alter TonEBP/OREBP transcriptional activity during normotonicity (290 mosmol/kg) or hypertonicity (500 mosmol/kg, NaCl added) or both. Most siRNAs increase TonEBP/OREBP activity, implying that the targeted phosphatases normally reduce that activity. We further studied in detail SHP-1, whose knockdown by its specific siRNA increases TonEBP/OREBP transcriptional activity at 500 mosmol/kg. We confirmed that SHP-1 is inhibitory by overexpressing it, which reduces TonEBP/OREBP transcriptional activity at 500 mosmol/kg. SHP-1 dephosphorylates TonEBP/OREBP at a known regulatory site, Y143, both in vivo and in vitro. It inhibits TonEBP/OREBP by both reducing TonEBP/OREBP nuclear localization, which is Y143 dependent, and by lowering high NaCl-induced TonEBP/OREBP transactivating activity. SHP-1 coimmunoprecipitates with TonEBP/OREBP and vice versa, suggesting that they are physically associated in the cell. High NaCl inhibits the effect of SHP-1 on TonEBP/OREBP by increasing phosphorylation of SHP-1 on Ser591, which reduces its phosphatase activity and localization to the nucleus. Thus, TonEBP/OREBP is extensively regulated by phosphatases, including SHP-1, whose inhibition by high NaCl increases phosphorylation of TonEBP/OREBP at Y143, contributing to the nuclear localization and activation of TonEBP/OREBP.
Zhou
Xiaoming
X
Division of Nephrology, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA. xiazhou@usuhs.mil
Gallazzini
Morgan
M
Burg
Maurice B
MB
Ferraris
Joan D
JD
D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation; D015723:Gene Library; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009995:Osmosis; D010766:Phosphorylation; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D014157:Transcription Factors / Q000378:metabolism*
D006360:Heat-Shock Proteins; C118125:NFAT5 protein, human; D034741:RNA, Small Interfering; D014157:Transcription Factors; D012965:Sodium Chloride; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6