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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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Effect on its nuclear to cytoplasmic ratio of mutating to alanine TonEBP/OREBP-V5- (A) S120, (B) S134, (C) T135, or (D) S155. HEK293 cells were transiently transfected with wild-type TonEBP/OREBP-V5 or a mutant at 300 mOsm/kg, and then the osmolality was changed to 200, 300, or 500 mOsm/kg by varying NaCl for 1 h. The relative amounts of TonEBP/OREBP-V5 in the cytoplasmic and nuclear fractions and the nuclear/cytoplasmic ratio were calculated from Western blots with anti-V5 antibody (mean ± SEM, *P ≤ 0.05, n = 3).
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Figure 2: Effect on its nuclear to cytoplasmic ratio of mutating to alanine TonEBP/OREBP-V5- (A) S120, (B) S134, (C) T135, or (D) S155. HEK293 cells were transiently transfected with wild-type TonEBP/OREBP-V5 or a mutant at 300 mOsm/kg, and then the osmolality was changed to 200, 300, or 500 mOsm/kg by varying NaCl for 1 h. The relative amounts of TonEBP/OREBP-V5 in the cytoplasmic and nuclear fractions and the nuclear/cytoplasmic ratio were calculated from Western blots with anti-V5 antibody (mean ± SEM, *P ≤ 0.05, n = 3).

Mentions: We measured the nuclear to cytoplasmic ratio of TonEBP/OREBP after changing osmolality from 300 to 200, 300, or 500 mOsm/kg for 1 h by altering NaCl. Raising NaCl increases nuclear to cytoplasmic ratio of wild-type (WT) TonEBP/OREBP-V5 approximately fourfold (Figure 2). Mutation of the amino acids in TonEBP/OREBP that are sites of phosphorylation has varying effects. Mutating S120 to alanine has no significant effect at any osmolality (Figure 2A). Mutating S134 (Figure 2B) or T135 (Figure 2C) to alanine decreases nuclear to cytoplasmic ratio at 500 mOsm/kg but has no effect at 200 or 300 mOsm/kg. Mutating S155 to alanine increases nuclear to cytoplasmic ratio at all osmolalities (Figure 2D).


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)

Effect on its nuclear to cytoplasmic ratio of mutating to alanine TonEBP/OREBP-V5- (A) S120, (B) S134, (C) T135, or (D) S155. HEK293 cells were transiently transfected with wild-type TonEBP/OREBP-V5 or a mutant at 300 mOsm/kg, and then the osmolality was changed to 200, 300, or 500 mOsm/kg by varying NaCl for 1 h. The relative amounts of TonEBP/OREBP-V5 in the cytoplasmic and nuclear fractions and the nuclear/cytoplasmic ratio were calculated from Western blots with anti-V5 antibody (mean ± SEM, *P ≤ 0.05, n = 3).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Effect on its nuclear to cytoplasmic ratio of mutating to alanine TonEBP/OREBP-V5- (A) S120, (B) S134, (C) T135, or (D) S155. HEK293 cells were transiently transfected with wild-type TonEBP/OREBP-V5 or a mutant at 300 mOsm/kg, and then the osmolality was changed to 200, 300, or 500 mOsm/kg by varying NaCl for 1 h. The relative amounts of TonEBP/OREBP-V5 in the cytoplasmic and nuclear fractions and the nuclear/cytoplasmic ratio were calculated from Western blots with anti-V5 antibody (mean ± SEM, *P ≤ 0.05, n = 3).
Mentions: We measured the nuclear to cytoplasmic ratio of TonEBP/OREBP after changing osmolality from 300 to 200, 300, or 500 mOsm/kg for 1 h by altering NaCl. Raising NaCl increases nuclear to cytoplasmic ratio of wild-type (WT) TonEBP/OREBP-V5 approximately fourfold (Figure 2). Mutation of the amino acids in TonEBP/OREBP that are sites of phosphorylation has varying effects. Mutating S120 to alanine has no significant effect at any osmolality (Figure 2A). Mutating S134 (Figure 2B) or T135 (Figure 2C) to alanine decreases nuclear to cytoplasmic ratio at 500 mOsm/kg but has no effect at 200 or 300 mOsm/kg. Mutating S155 to alanine increases nuclear to cytoplasmic ratio at all osmolalities (Figure 2D).

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.

Show MeSH
Related in: MedlinePlus