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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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CDK5 contributes to high NaCl–induced regulation of TonEBP/OREBP transcriptional activity but not to regulation of its transactivating activity. Osmolality bathing HEK293 cells stably expressing a TonEBP/OREBP transcription reporter (ORE-X) was increased from 300 to 500 mOsm/kg (NaCl added). (A) Transcriptional activity increases at 4 h under control conditions (DMSO), but not in the presence of 10 μM CDK1/5 inhibitor (CDK1/5). TonEBP protein expression does not change during the 4 h that osmolality is increased (top). However, after 16 h (B) the inhibitor no longer reduces TonEBP/OREBP transcriptional activity and TonEBP protein expression has increased, also not affected by the inhibitor (top). These effects do not occur with a control reporter that lacks ORE elements (IL2) (mean ± SEM, *P < 0.05, n = 3). (C) Inhibition of CDK5 does not affect the increase of TonEBP/OREBP in the nucleus 16 h after NaCl is raised. HEK293 cells were incubated with CDK1/5 inhibitor (CDK1/5 inh) or vehicle and then maintained at 300 mOsm/kg or increased to 500 mOsm/kg (NaCl added) for 16 h. Western analysis of nuclear and cytoplasmic extracts was performed using anti-TonEBP/OREBP antibody. Aldose reductase (AR, cytoplasmic marker) and BRG1 (nuclear marker) are controls for the purity of the cytoplasmic and nuclear extracts. Top, representative Western blot. Bottom, TonEBP/OREBP relative abundance (mean ± range, n = 2). (D) High NaCl does not affect TonEBP/OREBP transactivating activity. Osmolality bathing HEK293 cells stably expressing a dual luciferase reporter of TonEBP/OREBP transactivating activity (TAD) was increased from 300 to 500 mOsm/kg (NaCl added) for 16 h. An otherwise identical reporter lacking the TonEBP/OREBP transactivation domain (GAL4) serves as control (mean ± SEM, n = 3).
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Figure 6: CDK5 contributes to high NaCl–induced regulation of TonEBP/OREBP transcriptional activity but not to regulation of its transactivating activity. Osmolality bathing HEK293 cells stably expressing a TonEBP/OREBP transcription reporter (ORE-X) was increased from 300 to 500 mOsm/kg (NaCl added). (A) Transcriptional activity increases at 4 h under control conditions (DMSO), but not in the presence of 10 μM CDK1/5 inhibitor (CDK1/5). TonEBP protein expression does not change during the 4 h that osmolality is increased (top). However, after 16 h (B) the inhibitor no longer reduces TonEBP/OREBP transcriptional activity and TonEBP protein expression has increased, also not affected by the inhibitor (top). These effects do not occur with a control reporter that lacks ORE elements (IL2) (mean ± SEM, *P < 0.05, n = 3). (C) Inhibition of CDK5 does not affect the increase of TonEBP/OREBP in the nucleus 16 h after NaCl is raised. HEK293 cells were incubated with CDK1/5 inhibitor (CDK1/5 inh) or vehicle and then maintained at 300 mOsm/kg or increased to 500 mOsm/kg (NaCl added) for 16 h. Western analysis of nuclear and cytoplasmic extracts was performed using anti-TonEBP/OREBP antibody. Aldose reductase (AR, cytoplasmic marker) and BRG1 (nuclear marker) are controls for the purity of the cytoplasmic and nuclear extracts. Top, representative Western blot. Bottom, TonEBP/OREBP relative abundance (mean ± range, n = 2). (D) High NaCl does not affect TonEBP/OREBP transactivating activity. Osmolality bathing HEK293 cells stably expressing a dual luciferase reporter of TonEBP/OREBP transactivating activity (TAD) was increased from 300 to 500 mOsm/kg (NaCl added) for 16 h. An otherwise identical reporter lacking the TonEBP/OREBP transactivation domain (GAL4) serves as control (mean ± SEM, n = 3).

Mentions: We measured TonEBP/OREBP transcriptional activity in HEK293 cells stably expressing an ORE-X reporter. Four hours after osmolality is increased from 300 to 500 mOsm/kg by adding NaCl, the transcriptional activity increases 2.7-fold (Figure 6A, bottom). CDK1/5 inhibitor prevents the increase (Figure 6A, bottom).


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)

CDK5 contributes to high NaCl–induced regulation of TonEBP/OREBP transcriptional activity but not to regulation of its transactivating activity. Osmolality bathing HEK293 cells stably expressing a TonEBP/OREBP transcription reporter (ORE-X) was increased from 300 to 500 mOsm/kg (NaCl added). (A) Transcriptional activity increases at 4 h under control conditions (DMSO), but not in the presence of 10 μM CDK1/5 inhibitor (CDK1/5). TonEBP protein expression does not change during the 4 h that osmolality is increased (top). However, after 16 h (B) the inhibitor no longer reduces TonEBP/OREBP transcriptional activity and TonEBP protein expression has increased, also not affected by the inhibitor (top). These effects do not occur with a control reporter that lacks ORE elements (IL2) (mean ± SEM, *P < 0.05, n = 3). (C) Inhibition of CDK5 does not affect the increase of TonEBP/OREBP in the nucleus 16 h after NaCl is raised. HEK293 cells were incubated with CDK1/5 inhibitor (CDK1/5 inh) or vehicle and then maintained at 300 mOsm/kg or increased to 500 mOsm/kg (NaCl added) for 16 h. Western analysis of nuclear and cytoplasmic extracts was performed using anti-TonEBP/OREBP antibody. Aldose reductase (AR, cytoplasmic marker) and BRG1 (nuclear marker) are controls for the purity of the cytoplasmic and nuclear extracts. Top, representative Western blot. Bottom, TonEBP/OREBP relative abundance (mean ± range, n = 2). (D) High NaCl does not affect TonEBP/OREBP transactivating activity. Osmolality bathing HEK293 cells stably expressing a dual luciferase reporter of TonEBP/OREBP transactivating activity (TAD) was increased from 300 to 500 mOsm/kg (NaCl added) for 16 h. An otherwise identical reporter lacking the TonEBP/OREBP transactivation domain (GAL4) serves as control (mean ± SEM, n = 3).
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Figure 6: CDK5 contributes to high NaCl–induced regulation of TonEBP/OREBP transcriptional activity but not to regulation of its transactivating activity. Osmolality bathing HEK293 cells stably expressing a TonEBP/OREBP transcription reporter (ORE-X) was increased from 300 to 500 mOsm/kg (NaCl added). (A) Transcriptional activity increases at 4 h under control conditions (DMSO), but not in the presence of 10 μM CDK1/5 inhibitor (CDK1/5). TonEBP protein expression does not change during the 4 h that osmolality is increased (top). However, after 16 h (B) the inhibitor no longer reduces TonEBP/OREBP transcriptional activity and TonEBP protein expression has increased, also not affected by the inhibitor (top). These effects do not occur with a control reporter that lacks ORE elements (IL2) (mean ± SEM, *P < 0.05, n = 3). (C) Inhibition of CDK5 does not affect the increase of TonEBP/OREBP in the nucleus 16 h after NaCl is raised. HEK293 cells were incubated with CDK1/5 inhibitor (CDK1/5 inh) or vehicle and then maintained at 300 mOsm/kg or increased to 500 mOsm/kg (NaCl added) for 16 h. Western analysis of nuclear and cytoplasmic extracts was performed using anti-TonEBP/OREBP antibody. Aldose reductase (AR, cytoplasmic marker) and BRG1 (nuclear marker) are controls for the purity of the cytoplasmic and nuclear extracts. Top, representative Western blot. Bottom, TonEBP/OREBP relative abundance (mean ± range, n = 2). (D) High NaCl does not affect TonEBP/OREBP transactivating activity. Osmolality bathing HEK293 cells stably expressing a dual luciferase reporter of TonEBP/OREBP transactivating activity (TAD) was increased from 300 to 500 mOsm/kg (NaCl added) for 16 h. An otherwise identical reporter lacking the TonEBP/OREBP transactivation domain (GAL4) serves as control (mean ± SEM, n = 3).
Mentions: We measured TonEBP/OREBP transcriptional activity in HEK293 cells stably expressing an ORE-X reporter. Four hours after osmolality is increased from 300 to 500 mOsm/kg by adding NaCl, the transcriptional activity increases 2.7-fold (Figure 6A, bottom). CDK1/5 inhibitor prevents the increase (Figure 6A, bottom).

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