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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 regulation of TonEBP/OREBP nuclear localization. (A) and (B) Inhibition of CDK5 kinase activity abolishes high NaCl–induced nuclear localization of TonEBP/OREBP. Western analysis was performed on proteins extracted from cytoplasm and nucleus, using antibodies against TonEBP/OREBP. (A) HEK293 cells transiently transfected with CDK5 wild-type (CDK5-WT) or dominant-negative kinase dead (CDK5-DN) were incubated for 2 h at 200, 300, or 500 mOsm/kg (NaCl varied). Top, representative Western blot. Bottom, summary data. Cytoplasmic (aldose reductase) and nuclear (Brg1) markers rule out important cross-contamination between the respective extracts. (B) HEK293 cells were incubated for 2 h at 200, 300, or 500 mOsm/kg (NaCl varied), with or without 10 μM CDK1/5 inhibitor (CDK1/5 inh). Top, representative Western blot. Bottom, summary data (mean ± SEM, n = 3, *P < 0.05 vs. 300 mOsm/kg control, †P < 0.05 control vs. experimental. (C) HeLa cells were preincubated for 1 h at 300 mOsm/kg with DMSO or with 10 μM CDK1/5 inhibitor, and then osmolality was increased to 500 mOsm/kg (NaCl added) for 1 h or kept at 300 mOsm/kg in the continued presence of CDK1/5 inhibitor or vehicle. Cells were fixed and stained with anti-TonEBP/OREBP and anti-CDK5 antibodies. Under control (DMSO) conditions at 300 mOsm/kg, TonEBP/OREBP is present in the cytoplasm, where it colocalizes with CDK5. Under control conditions at 500 mOsm/kg, TonEBP/OREBP moves into the nucleus, where it colocalizes in speckles with CDK5. The CDK1/5 inhibitor decreases both nuclear localization of TonEBP/OREBP at 500 mOsm/kg and its colocalization with CDK5 in the nucleus.
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Figure 5: CDK5 contributes to regulation of TonEBP/OREBP nuclear localization. (A) and (B) Inhibition of CDK5 kinase activity abolishes high NaCl–induced nuclear localization of TonEBP/OREBP. Western analysis was performed on proteins extracted from cytoplasm and nucleus, using antibodies against TonEBP/OREBP. (A) HEK293 cells transiently transfected with CDK5 wild-type (CDK5-WT) or dominant-negative kinase dead (CDK5-DN) were incubated for 2 h at 200, 300, or 500 mOsm/kg (NaCl varied). Top, representative Western blot. Bottom, summary data. Cytoplasmic (aldose reductase) and nuclear (Brg1) markers rule out important cross-contamination between the respective extracts. (B) HEK293 cells were incubated for 2 h at 200, 300, or 500 mOsm/kg (NaCl varied), with or without 10 μM CDK1/5 inhibitor (CDK1/5 inh). Top, representative Western blot. Bottom, summary data (mean ± SEM, n = 3, *P < 0.05 vs. 300 mOsm/kg control, †P < 0.05 control vs. experimental. (C) HeLa cells were preincubated for 1 h at 300 mOsm/kg with DMSO or with 10 μM CDK1/5 inhibitor, and then osmolality was increased to 500 mOsm/kg (NaCl added) for 1 h or kept at 300 mOsm/kg in the continued presence of CDK1/5 inhibitor or vehicle. Cells were fixed and stained with anti-TonEBP/OREBP and anti-CDK5 antibodies. Under control (DMSO) conditions at 300 mOsm/kg, TonEBP/OREBP is present in the cytoplasm, where it colocalizes with CDK5. Under control conditions at 500 mOsm/kg, TonEBP/OREBP moves into the nucleus, where it colocalizes in speckles with CDK5. The CDK1/5 inhibitor decreases both nuclear localization of TonEBP/OREBP at 500 mOsm/kg and its colocalization with CDK5 in the nucleus.

Mentions: Within 2 h after NaCl is increased, the nuclear to cytoplasmic ratio of TonEBP/OREBP increases two- to threefold in HEK293 cells (Figure 5, A and B). Possible cross contamination between the nuclear and cytoplasmic extracts is excluded by clear separation of cytoplasmic (aldose reductase) and nuclear (Brg1) markers (Figure 5A). We inhibited CDK5 activity by overexpression of dominant-negative CDK5 kinase dead (CDK5-DN) (Figure 5A) or by addition of CDK1/5 inhibitor (Figure 5, B and C). Both ways of inhibiting CDK5 activity reduce the rapid increase of nuclear to cytoplasmic ratio of TonEBP/OREBP (Figure 5, A and B). Roscovitine, which is a less specific inhibitor of CDK5, also reduces the high NaCl–induced increase of TonEBP/OREBP nuclear localization (Supplemental Figure 3). Interestingly, under control conditions (DMSO) when osmolality is increased from 300 to 500 mOsm/kg by adding NaCl, bright foci of CDK5 appear in nuclei, colocalized with native TonEBP/OREBP. CDK1/5 inhibitor prevents formation of the foci (Figure 5C).


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 regulation of TonEBP/OREBP nuclear localization. (A) and (B) Inhibition of CDK5 kinase activity abolishes high NaCl–induced nuclear localization of TonEBP/OREBP. Western analysis was performed on proteins extracted from cytoplasm and nucleus, using antibodies against TonEBP/OREBP. (A) HEK293 cells transiently transfected with CDK5 wild-type (CDK5-WT) or dominant-negative kinase dead (CDK5-DN) were incubated for 2 h at 200, 300, or 500 mOsm/kg (NaCl varied). Top, representative Western blot. Bottom, summary data. Cytoplasmic (aldose reductase) and nuclear (Brg1) markers rule out important cross-contamination between the respective extracts. (B) HEK293 cells were incubated for 2 h at 200, 300, or 500 mOsm/kg (NaCl varied), with or without 10 μM CDK1/5 inhibitor (CDK1/5 inh). Top, representative Western blot. Bottom, summary data (mean ± SEM, n = 3, *P < 0.05 vs. 300 mOsm/kg control, †P < 0.05 control vs. experimental. (C) HeLa cells were preincubated for 1 h at 300 mOsm/kg with DMSO or with 10 μM CDK1/5 inhibitor, and then osmolality was increased to 500 mOsm/kg (NaCl added) for 1 h or kept at 300 mOsm/kg in the continued presence of CDK1/5 inhibitor or vehicle. Cells were fixed and stained with anti-TonEBP/OREBP and anti-CDK5 antibodies. Under control (DMSO) conditions at 300 mOsm/kg, TonEBP/OREBP is present in the cytoplasm, where it colocalizes with CDK5. Under control conditions at 500 mOsm/kg, TonEBP/OREBP moves into the nucleus, where it colocalizes in speckles with CDK5. The CDK1/5 inhibitor decreases both nuclear localization of TonEBP/OREBP at 500 mOsm/kg and its colocalization with CDK5 in the nucleus.
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Figure 5: CDK5 contributes to regulation of TonEBP/OREBP nuclear localization. (A) and (B) Inhibition of CDK5 kinase activity abolishes high NaCl–induced nuclear localization of TonEBP/OREBP. Western analysis was performed on proteins extracted from cytoplasm and nucleus, using antibodies against TonEBP/OREBP. (A) HEK293 cells transiently transfected with CDK5 wild-type (CDK5-WT) or dominant-negative kinase dead (CDK5-DN) were incubated for 2 h at 200, 300, or 500 mOsm/kg (NaCl varied). Top, representative Western blot. Bottom, summary data. Cytoplasmic (aldose reductase) and nuclear (Brg1) markers rule out important cross-contamination between the respective extracts. (B) HEK293 cells were incubated for 2 h at 200, 300, or 500 mOsm/kg (NaCl varied), with or without 10 μM CDK1/5 inhibitor (CDK1/5 inh). Top, representative Western blot. Bottom, summary data (mean ± SEM, n = 3, *P < 0.05 vs. 300 mOsm/kg control, †P < 0.05 control vs. experimental. (C) HeLa cells were preincubated for 1 h at 300 mOsm/kg with DMSO or with 10 μM CDK1/5 inhibitor, and then osmolality was increased to 500 mOsm/kg (NaCl added) for 1 h or kept at 300 mOsm/kg in the continued presence of CDK1/5 inhibitor or vehicle. Cells were fixed and stained with anti-TonEBP/OREBP and anti-CDK5 antibodies. Under control (DMSO) conditions at 300 mOsm/kg, TonEBP/OREBP is present in the cytoplasm, where it colocalizes with CDK5. Under control conditions at 500 mOsm/kg, TonEBP/OREBP moves into the nucleus, where it colocalizes in speckles with CDK5. The CDK1/5 inhibitor decreases both nuclear localization of TonEBP/OREBP at 500 mOsm/kg and its colocalization with CDK5 in the nucleus.
Mentions: Within 2 h after NaCl is increased, the nuclear to cytoplasmic ratio of TonEBP/OREBP increases two- to threefold in HEK293 cells (Figure 5, A and B). Possible cross contamination between the nuclear and cytoplasmic extracts is excluded by clear separation of cytoplasmic (aldose reductase) and nuclear (Brg1) markers (Figure 5A). We inhibited CDK5 activity by overexpression of dominant-negative CDK5 kinase dead (CDK5-DN) (Figure 5A) or by addition of CDK1/5 inhibitor (Figure 5, B and C). Both ways of inhibiting CDK5 activity reduce the rapid increase of nuclear to cytoplasmic ratio of TonEBP/OREBP (Figure 5, A and B). Roscovitine, which is a less specific inhibitor of CDK5, also reduces the high NaCl–induced increase of TonEBP/OREBP nuclear localization (Supplemental Figure 3). Interestingly, under control conditions (DMSO) when osmolality is increased from 300 to 500 mOsm/kg by adding NaCl, bright foci of CDK5 appear in nuclei, colocalized with native TonEBP/OREBP. CDK1/5 inhibitor prevents formation of the foci (Figure 5C).

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