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High NaCl-induced activation of CDK5 increases phosphorylation of the osmoprotective transcription factor TonEBP/OREBP at threonine 135, which contributes to its rapid nuclear localization.

Gallazzini M, Heussler GE, Kunin M, Izumi Y, Burg MB, Ferraris JD - Mol. Biol. Cell (2011)

Bottom Line: Inhibition of CDK5 activity reduces the rapid high NaCl-induced nuclear localization of TonEBP/OREBP but does not affect its transactivating activity.Inhibition of CDK5 reduces the increase in TonEBP/OREBP transcriptional activity that has occurred by 4 h after NaCl is raised, associated with less nuclear TonEBP/OREBP at that time, but does not reduce either activity or nuclear TonEBP/OREBP after 16 h.Thus high NaCl-induced increase of the overall abundance of TonEBP/OREBP, by itself, eventually raises its effective level in the nucleus, but its rapid CDK5-dependent nuclear localization accelerates the process, speeding transcription of osmoprotective target genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA.

ABSTRACT
When activated by high NaCl, tonicity-responsive enhancer-binding protein/osmotic response element-binding protein (TonEBP/OREBP) increases transcription of osmoprotective genes. High NaCl activates TonEBP/OREBP by increasing its phosphorylation, nuclear localization, and transactivating activity. In HEK293 cells, mass spectrometry shows phosphorylation of TonEBP/OREBP-S120, -S134, -T135, and -S155. When those residues are individually mutated to alanine, nuclear localization is greater for S155A, less for S134A and T135A, and unchanged for S120A. High osmolality increases phosphorylation at T135 in HEK293 cells and in rat renal inner medullas in vivo. In HEK293 cells, high NaCl activates cyclin-dependent kinase 5 (CDK5), which directly phosphorylates TonEBP/OREBP-T135. Inhibition of CDK5 activity reduces the rapid high NaCl-induced nuclear localization of TonEBP/OREBP but does not affect its transactivating activity. High NaCl induces nuclear localization of TonEBP/OREBP faster (≤2 h) than it increases its overall protein abundance (≥6 h). Inhibition of CDK5 reduces the increase in TonEBP/OREBP transcriptional activity that has occurred by 4 h after NaCl is raised, associated with less nuclear TonEBP/OREBP at that time, but does not reduce either activity or nuclear TonEBP/OREBP after 16 h. Thus high NaCl-induced increase of the overall abundance of TonEBP/OREBP, by itself, eventually raises its effective level in the nucleus, but its rapid CDK5-dependent nuclear localization accelerates the process, speeding transcription of osmoprotective target genes.

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MS2 spectra showing phosphorylation of TonEBP/OREBP at (A) S120, (B) S134 and T135, and (C) S155. Ions that are determining for the specific phosphorylation sites are indicated by italics.
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Figure 1: MS2 spectra showing phosphorylation of TonEBP/OREBP at (A) S120, (B) S134 and T135, and (C) S155. Ions that are determining for the specific phosphorylation sites are indicated by italics.

Mentions: Native TonEBP/OREBP is not sufficiently abundant in human embryonic kidney (HEK) 293 cells for analysis by protein mass spectrometry. Therefore, in order to discover amino acids that are phosphorylated, we stably expressed TonEBP/OREBP-1–547-V5 in the cells (Chen et al., 2007) and immunoprecipitated it from cytoplasmic and nuclear extracts of cells bathed in medium kept at 300 mOsm/kg or changed for 2 h to 200 or 500 mOsm/kg by adjusting NaCl. This region of TonEBP/OREBP contains nuclear localization, DNA binding, and dimerization domains (Burg et al., 2007). To maximize coverage of phosphorylation sites by mass spectrometry, we used two different proteolytic enzymes, trypsin and endoproteinase Arg C. We used the SEQUEST (Eng et al., 1994) algorithm for initial identification of phosphorylated amino acids, and Ascore (Beausoleil et al., 2006) for confirmation. Ascore (http://Ascore.med.harvard.edu) estimates the probability of correct identification of phosphorylation sites from the presence and intensity of site-determining ions in tandem mass spectometry (MS/MS) spectra. Ascore > 19 predicts >99% probability of correct identification of phosphorylation sites. We found numerous peptides from TonEBP/OREBP in both nuclear and cytoplasmic fractions—up to 12 unique peptides in a single sample. We identified four likely phosphorylation sites in three different phosphopeptides (Table 1). We found phospho-S120 in nuclear extracts at 200 and 500 mOsm/kg and cytoplasmic extracts at 500 mOsm/kg. We found phospho-S134 and -T135, always together, in nuclear extracts at 500 mOsm/kg. We found phospho-S155 in cytoplasmic extracts at 200 mOsm/kg. Each peptide was detected in several independently prepared samples. Representative MS2 spectra of the phosphopeptides are shown in Figure 1.


High NaCl-induced activation of CDK5 increases phosphorylation of the osmoprotective transcription factor TonEBP/OREBP at threonine 135, which contributes to its rapid nuclear localization.

Gallazzini M, Heussler GE, Kunin M, Izumi Y, Burg MB, Ferraris JD - Mol. Biol. Cell (2011)

MS2 spectra showing phosphorylation of TonEBP/OREBP at (A) S120, (B) S134 and T135, and (C) S155. Ions that are determining for the specific phosphorylation sites are indicated by italics.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: MS2 spectra showing phosphorylation of TonEBP/OREBP at (A) S120, (B) S134 and T135, and (C) S155. Ions that are determining for the specific phosphorylation sites are indicated by italics.
Mentions: Native TonEBP/OREBP is not sufficiently abundant in human embryonic kidney (HEK) 293 cells for analysis by protein mass spectrometry. Therefore, in order to discover amino acids that are phosphorylated, we stably expressed TonEBP/OREBP-1–547-V5 in the cells (Chen et al., 2007) and immunoprecipitated it from cytoplasmic and nuclear extracts of cells bathed in medium kept at 300 mOsm/kg or changed for 2 h to 200 or 500 mOsm/kg by adjusting NaCl. This region of TonEBP/OREBP contains nuclear localization, DNA binding, and dimerization domains (Burg et al., 2007). To maximize coverage of phosphorylation sites by mass spectrometry, we used two different proteolytic enzymes, trypsin and endoproteinase Arg C. We used the SEQUEST (Eng et al., 1994) algorithm for initial identification of phosphorylated amino acids, and Ascore (Beausoleil et al., 2006) for confirmation. Ascore (http://Ascore.med.harvard.edu) estimates the probability of correct identification of phosphorylation sites from the presence and intensity of site-determining ions in tandem mass spectometry (MS/MS) spectra. Ascore > 19 predicts >99% probability of correct identification of phosphorylation sites. We found numerous peptides from TonEBP/OREBP in both nuclear and cytoplasmic fractions—up to 12 unique peptides in a single sample. We identified four likely phosphorylation sites in three different phosphopeptides (Table 1). We found phospho-S120 in nuclear extracts at 200 and 500 mOsm/kg and cytoplasmic extracts at 500 mOsm/kg. We found phospho-S134 and -T135, always together, in nuclear extracts at 500 mOsm/kg. We found phospho-S155 in cytoplasmic extracts at 200 mOsm/kg. Each peptide was detected in several independently prepared samples. Representative MS2 spectra of the phosphopeptides are shown in Figure 1.

Bottom Line: Inhibition of CDK5 activity reduces the rapid high NaCl-induced nuclear localization of TonEBP/OREBP but does not affect its transactivating activity.Inhibition of CDK5 reduces the increase in TonEBP/OREBP transcriptional activity that has occurred by 4 h after NaCl is raised, associated with less nuclear TonEBP/OREBP at that time, but does not reduce either activity or nuclear TonEBP/OREBP after 16 h.Thus high NaCl-induced increase of the overall abundance of TonEBP/OREBP, by itself, eventually raises its effective level in the nucleus, but its rapid CDK5-dependent nuclear localization accelerates the process, speeding transcription of osmoprotective target genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, USA.

ABSTRACT
When activated by high NaCl, tonicity-responsive enhancer-binding protein/osmotic response element-binding protein (TonEBP/OREBP) increases transcription of osmoprotective genes. High NaCl activates TonEBP/OREBP by increasing its phosphorylation, nuclear localization, and transactivating activity. In HEK293 cells, mass spectrometry shows phosphorylation of TonEBP/OREBP-S120, -S134, -T135, and -S155. When those residues are individually mutated to alanine, nuclear localization is greater for S155A, less for S134A and T135A, and unchanged for S120A. High osmolality increases phosphorylation at T135 in HEK293 cells and in rat renal inner medullas in vivo. In HEK293 cells, high NaCl activates cyclin-dependent kinase 5 (CDK5), which directly phosphorylates TonEBP/OREBP-T135. Inhibition of CDK5 activity reduces the rapid high NaCl-induced nuclear localization of TonEBP/OREBP but does not affect its transactivating activity. High NaCl induces nuclear localization of TonEBP/OREBP faster (≤2 h) than it increases its overall protein abundance (≥6 h). Inhibition of CDK5 reduces the increase in TonEBP/OREBP transcriptional activity that has occurred by 4 h after NaCl is raised, associated with less nuclear TonEBP/OREBP at that time, but does not reduce either activity or nuclear TonEBP/OREBP after 16 h. Thus high NaCl-induced increase of the overall abundance of TonEBP/OREBP, by itself, eventually raises its effective level in the nucleus, but its rapid CDK5-dependent nuclear localization accelerates the process, speeding transcription of osmoprotective target genes.

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