23 research outputs found
Mediator of DNA Damage Checkpoint 1 (MDC1) Contributes to High NaCl-Induced Activation of the Osmoprotective Transcription Factor TonEBP/OREBP
Background: Hypertonicity, such as induced by high NaCl, increases the activity of the transcription factor TonEBP/OREBP whose target genes increase osmoprotective organic osmolytes and heat shock proteins. Methodology: We used mass spectrometry to analyze proteins that coimmunoprecipitate with TonEBP/OREBP in order to identify ones that might contribute to its high NaCl-induced activation. Principal Findings: We identified 20 unique peptides from Mediator of DNA Damage Checkpoint 1 (MDC1) with high probability. The identification was confirmed by Western analysis. We used small interfering RNA knockdown of MDC1 to characterize its osmotic function. Knocking down MDC1 reduces high NaCl-induced increases in TonEBP/OREBP transcriptional and transactivating activity, but has no significant effect on its nuclear localization. We confirm six previously known phosphorylation sites in MDC1, but do not find evidence that high NaCl increases phosphorylation of MDC1. It is suggestive that MDC1 acts as a DNA damage response protein since hypertonicity reversibly increases DNA breaks, and other DNA damage response proteins, like ATM, also associate with TonEBP/OREBP and contribute to its activation by hypertonicity. Conclusions/Significance: MDC1 associates with TonEBP/OREBP and contributes to high NaCl-induced increase of tha
Régulation de l'expression du canal potassique ROMK dans les cellules de la branche large ascendante médullaire de l'anse de Henle
PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF
c-Abl mediates high NaCl-induced phosphorylation and activation of the transcription factor TonEBP/OREBP
The transcription factor TonEBP/OREBP promotes cell survival during osmotic stress. High NaCl-induced phosphorylation of TonEBP/OREBP at tyrosine-143 was known to be an important factor in increasing its activity in cell culture. We now find that TonEBP/OREBP also is phosphorylated at tyrosine-143 in rat renal inner medulla, dependent on the interstitial osmolality. c-Abl seemed likely to be the kinase that phosphorylates TonEBP/OREBP because Y143 is in a consensus c-Abl phosphorylation site. We now confirm that, as follows. High NaCl increases c-Abl activity. Specific inhibition of c-Abl by imatinib, siRNA, or c-Abl kinase dead drastically reduces high NaCl-induced TonEBP/OREBP activity by reducing its nuclear location and transactivating activity. c-Abl associates with TonEBP/OREBP (coimmunoprecipitation) and phosphorylates TonEBP/OREBP-Y143 both in cell and in vitro. High NaCl-induced activation of ataxia telangiectasia mutated, previously known to contribute to activation of TonEBP/OREBP, depends on c-Abl activity. Thus, c-Abl is the kinase responsible for high NaCl-induced phosphorylation of TonEBP/OREBP-Y143, which contributes to its increased activity.—Gallazzini, M., Yu, M.-J., Gunaratne, R., Burg, M. B., Ferraris, J. D. c-Abl mediates high NaCl-induced phosphorylation and activation of the transcription factor TonEBP/OREBP
Figure 5
<p><b>A.</b> Lack of effect of siRNA-mediated knockdown of MDC1 on nuclear localization of TonEBP/OREBP. HEK 293 cells were transiently transfected with 25 nM MDC1 siRNA or control siRNA. 48 h later the osmolality of medium was changed to 200 or 500 mosmol/kg or kept at 300 mosmol/kg for 1 h. Nuclear and cytoplasmic extracts were prepared. TonEBP/OREBP nuclear/cytoplasmic ratio was calculated from its abundance in nuclear and cytoplasmic extracts. Antibodies to BRG1 and aldose reductase serve as controls for nuclear and cytoplasmic fractionation, respectively. <b>B</b>. High NaCl decreases the nuclear localization of MDC1. Osmolality was changed to 200 or 500 mosmol/kg or kept at 300 mosmol/kg for 1 h. Nuclear and cytoplasmic extracts were prepared. MDC1 nuclear/cytoplasmic ratio was calculated from its abundance in nuclear and cytoplasmic extracts. <b>C.</b> Lack of effect of siRNA-mediated knockdown of MDC1 on phosphorylation of ATM on Ser1981. As in (A), except that abundance of ATM was measured in whole cell extracts by Western analysis using a non-phosphospecific antibody and its phosphorylation on Ser1981 was measured using phosphospecific anti-ATM 1981S-P antibody. Results are presented as the ratio of phosphorylated to non phosphorylated ATM. (* P<.05, compared to 300 mosmol).</p
MDC1 peptides identified by mass spectrometry.
<p>Xcorr is the SEQUEST peptide cross-correlation score. XCorr values above 2.0 indicate a good identification. The charges on the ions are indicated.</p
Effect of high NaCl on MDC1 abundance and distribution.
<p>HEK293 cells were exposed to media of the indicated total osmolalities (NaCl varied) for 2 h. <b>A.</b> High NaCl increases the abundance of MDC1 in the soluble fraction and decreases it in the chromatin-bound fraction. (n = 3, *P<0.05, t test). <b>B.</b> High NaCl decreases total MDC1 abundance (n = 3, *P<0.05, t test). <b>C.</b> After 2 h of exposure to high NaCl medium osmolality was decreased to 300 mosmol/kg for 30 min. MDC1 returns to the chromatin-bound fraction within that time (representative of 2 experiments).</p
MDC1 phosphopeptides identified by mass spectrometry.
<p>Xcorr is peptide cross-correlation score. Ascore measures the probability of correct identity of the phosphorylation sites. Asterisks indicate phosphorylation of the preceding residues.</p
Confirmation by Western analysis of identification of MDC1 in immunoprecipitates of TonEBP/OREBP-1-547-V5.
<p>“Input” is the protein extract before immunoprecipitation. <b>A.</b> Osmolality bathing HEK 293 cells stably transfected with TonEBP/OREBP-1-547-V5 or empty vector-V5 (EV) was raised from 300 to 500 mosmol/kg by adding NaCl for 1, 3 and 6 h. Proteins were immunoprecipitated with rabbit IgG or anti-V5 antibodies and immunoblotted with anti-V5 or anti-MDC1 antibody. <b>B</b>. Osmolality bathing HEK 293 cells stably transfected with TonEBP/OREBP-1-547-V5 or empty vector-V5 was raised from 300 to 500 mosmol/kg by adding NaCl for 2 h. Proteins were immunoprecipitated from nuclear lysates with rabbit IgG or anti-MDC1 and immunoblotted with anti-MDC1 or anti-V5 antibody. <b>C</b>. 100 µg/ml ethidium bromide was added during immunoprecipitation with anti-MDC1 from nuclear extracts of the stably transfected HEK293 cells expressing TonEBP/OREBP-1-547-V5.</p
Identification of MDC1 in immunoprecipitates of TonEBP/OREBP-1-547-V5 by mass spectrometry.
<p>MS<sup>2</sup> spectra of four MDC1 peptides. The arrows indicate ions that are site determining for phosphorylation.</p