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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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Phosphorylation of TonEBP/OREB-T135 is regulated by osmolality in the rat renal inner medullary cells in vivo. Osmolality of urine and renal inner medullary interstitial fluid is low in Brattleboro rats because of their congenital lack of vasopressin but is rapidly restored following administration of dDAVP. Injection of 2 nmol dDAVP (dDAVP) increases phosphorylation of TonEBP/OREBP at T135 in the renal inner medulla compared with control injection (vehicle). Top, representative Western blots from inner medullas of two rats from each group, using anti-TonEBP/OREBP and anti–TonEBP/OREBP-phospho-T135 antibodies. Bottom, TonEBP/OREBP–phospho-T135/TonEBP (mean ± SEM, n = 3, *P < 0.05).
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Figure 7: Phosphorylation of TonEBP/OREB-T135 is regulated by osmolality in the rat renal inner medullary cells in vivo. Osmolality of urine and renal inner medullary interstitial fluid is low in Brattleboro rats because of their congenital lack of vasopressin but is rapidly restored following administration of dDAVP. Injection of 2 nmol dDAVP (dDAVP) increases phosphorylation of TonEBP/OREBP at T135 in the renal inner medulla compared with control injection (vehicle). Top, representative Western blots from inner medullas of two rats from each group, using anti-TonEBP/OREBP and anti–TonEBP/OREBP-phospho-T135 antibodies. Bottom, TonEBP/OREBP–phospho-T135/TonEBP (mean ± SEM, n = 3, *P < 0.05).

Mentions: Brattleboro rats, being deficient in vasopressin, do not maintain a high renal medullary interstitial osmolality, nor do they concentrate their urine. One hour after injection of 2 nmol 1-desamino-8-d-arginine vasopressin (dDAVP), their urine osmolality increases from 203 to 575 mOsm/kg (Gallazzini et al., 2010). We find that phosphorylation of TonEBP/OREBP at T135 is significantly higher in renal inner medullas of Brattleboro rats 1 h after injection with dDAVP, compared with control rats injected with saline (Figure 7). This is consistent with high NaCl–induced increase of phosphorylation of TonEBP/OREBP on T135 in the renal inner medulla in vivo as well as in cell culture. We cannot exclude direct effects of vasopressin in vivo, but elevated vasopressin evidently does not contribute to the effect of high NaCl on HEK293 cells because it is not added in those experiments.


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)

Phosphorylation of TonEBP/OREB-T135 is regulated by osmolality in the rat renal inner medullary cells in vivo. Osmolality of urine and renal inner medullary interstitial fluid is low in Brattleboro rats because of their congenital lack of vasopressin but is rapidly restored following administration of dDAVP. Injection of 2 nmol dDAVP (dDAVP) increases phosphorylation of TonEBP/OREBP at T135 in the renal inner medulla compared with control injection (vehicle). Top, representative Western blots from inner medullas of two rats from each group, using anti-TonEBP/OREBP and anti–TonEBP/OREBP-phospho-T135 antibodies. Bottom, TonEBP/OREBP–phospho-T135/TonEBP (mean ± SEM, n = 3, *P < 0.05).
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Figure 7: Phosphorylation of TonEBP/OREB-T135 is regulated by osmolality in the rat renal inner medullary cells in vivo. Osmolality of urine and renal inner medullary interstitial fluid is low in Brattleboro rats because of their congenital lack of vasopressin but is rapidly restored following administration of dDAVP. Injection of 2 nmol dDAVP (dDAVP) increases phosphorylation of TonEBP/OREBP at T135 in the renal inner medulla compared with control injection (vehicle). Top, representative Western blots from inner medullas of two rats from each group, using anti-TonEBP/OREBP and anti–TonEBP/OREBP-phospho-T135 antibodies. Bottom, TonEBP/OREBP–phospho-T135/TonEBP (mean ± SEM, n = 3, *P < 0.05).
Mentions: Brattleboro rats, being deficient in vasopressin, do not maintain a high renal medullary interstitial osmolality, nor do they concentrate their urine. One hour after injection of 2 nmol 1-desamino-8-d-arginine vasopressin (dDAVP), their urine osmolality increases from 203 to 575 mOsm/kg (Gallazzini et al., 2010). We find that phosphorylation of TonEBP/OREBP at T135 is significantly higher in renal inner medullas of Brattleboro rats 1 h after injection with dDAVP, compared with control rats injected with saline (Figure 7). This is consistent with high NaCl–induced increase of phosphorylation of TonEBP/OREBP on T135 in the renal inner medulla in vivo as well as in cell culture. We cannot exclude direct effects of vasopressin in vivo, but elevated vasopressin evidently does not contribute to the effect of high NaCl on HEK293 cells because it is not added in those experiments.

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