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. 2012 Jun 29;31(13):2952-64.
doi: 10.1038/emboj.2012.122. Epub 2012 May 8.

The p57 CDKi Integrates Stress Signals Into Cell-Cycle Progression to Promote Cell Survival Upon Stress

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Free PMC article

The p57 CDKi Integrates Stress Signals Into Cell-Cycle Progression to Promote Cell Survival Upon Stress

Manel Joaquin et al. EMBO J. .
Free PMC article

Abstract

The p57(Kip2) cyclin-dependent kinase inhibitor (CDKi) has been implicated in embryogenesis, stem-cell senescence and pathologies, but little is known of its role in cell cycle control. Here, we show that p57(Kip2) is targeted by the p38 stress-activated protein kinase (SAPK). Phosphorylation of p57(Kip2) at T143 by p38 enhances its association with and inhibition of Cdk2, which results in cell-cycle delay upon stress. Genetic inactivation of the SAPK or the CDKi abolishes cell-cycle delay upon osmostress and results in decreased cell viability. Oxidative stress and ionomycin also induce p38-mediated phosphorylation of p57 and cells lacking p38 or p57 display reduced viability to these stresses. Therefore, cell survival to various stresses depends on p57 phosphorylation by p38 that inhibits CDK activity. Together, these findings provide a novel molecular mechanism by which cells can delay cell cycle progression to maximize cell survival upon stress.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
p38 SAPK phosphorylates the CDKi p57 at T143 in vitro. (A) All proteins were purified from E. coli. GST–p57 was expressed as a doublet protein being the shorter a cleavage fragment of full-length GST–p57. GST–p38 SAPK phosphorylates human GST–p21 and mouse GST–p57 but not human GST–p27. (B) GST–ATF2 and GST–p57 phosphorylation is prevented by the p38 SAPK inhibitor SB203580. (C) The mouse p57 protein harbours five putative phosphorylation sites following the minimum SAPK S/TP motif clustered in two regions. A GST–p57 N-term, containing three putative sites, and a GST–p57 C-term, containing two putative sites, mutants were made and assayed in vitro in the presence of GST–p38 SAPK. The GST–p57 N-term variant is phosphorylated as well as wild-type GST–p57 whereas the GST–p57 C-term variant is not phosphorylated by the p38 SAPK. (D) The three putative p38 SAPK sites T167, T143 and T139 were mutated to either glycine or alanine and assayed in vitro. The GST–p57 T143A mutant is not phosphorylated by GST–p38 SAPK in vitro. (E) HeLa cells were transfected with Flag-tagged wild-type p57 and p57T143A. Forty-eight hours post transfections, cells were lysed and immunoprecipitated with anti-Flag agarose beads. Immunoprecipitates were assayed in vitro with GST–p38 SAPK in the presence or the absence of the p38 inhibitor SB203580. Only wild-type Flag–p57 was phosphorylated by p38 SAPK. Representative kinase assays and coomassie blue stained gels are shown.
Figure 2
Figure 2
p38 SAPK and p57Kip2 form a stable complex in vivo. (A) HA–p38 and Flag–p57 were transfected into HeLa cells for 48 h. Cell lysates were then immunoprecipitated with either anti-Flag agarose beads or anti-HA coupled sepharose beads and analysed by western blot with anti-HA and anti-Flag antibodies. (B) Flag–p57 was transfected into HeLa cells for 48 h. Cell lysates were then immunoprecipitated with anti-Flag agarose beads and analysed by western blot with anti-p38 and anti-Flag antibodies. (C) HeLa cell extracts were immunoprecipitated with a control IgG (Con IP), anti-p57 or anti-p38 coupled sepharose beads and analysed by western blot with anti-p38 and anti-p57 antibodies. (D) Wild-type, p38−/− and p57−/− MEF cell lysates were immunoprecipiated with mouse anti-p57 coupled sepharose beads and analysed by western blot with anti-p38 and rabbit anti-p57 antibodies. Tubulin was used to monitor the input protein levels. Representative western blots are shown.
Figure 3
Figure 3
The CDKi p57 is phosphorylated at T143 in vivo by stress-activated p38 SAPK. (A) HeLa cells were transfected with wild-type Flag-p57 and Flag–p57T143A in the presence or absence of HA–p38 SAPK and myc-MKK6DD for 48 h. Cell lysates were immunoprecipitated with anti-Flag agarose beads and analysed by western blot with anti-pp38, anti-p38, anti-myc, anti-phospoS/T and anti-Flag antibodies. (B) HeLa cells were transfected with Flag–p57 in the presence or absence of HA–p38 SAPK and myc-MKK6DD for 48 h. The p38 SAPK inhibitor SB203580 was added to a final concentration of 10 μM 24 h prior harvesting the cells. Cell lysates were analysed as in (A). (C) HeLa cells were transfected with Flag–p57. Forty-eight hours post transfection, cells were treated with 100 mM NaCl for the indicated times. Cell lysates were analysed as in (A). Representative western blots are shown. (D) Wild-type and p38−/− MEF cells were grown on glass covers and treated with 100 mM NaCl, 600 μM H2O2 or 7.5 mM ionomycin for 60 min prior to fixation. p57 phosphorylation at T143 was detected by indirect immunofluorescence. Nuclear DNA was stained with Hoeschst 33342. Pictures were taken using an inverted Olympus CKX 41 microscope and the Olympus Cell̂D imaging software. Representative pictures are shown.
Figure 4
Figure 4
p57 phosphorylation at T143 regulates Cdk2 activity. (A) All proteins were purified from E. coli. Wild-type GST–p57 and GST–p57T143A was phosphorylated in vitro with activated GST–p38 SAPK using cold ATP. Phosphorylated proteins were then incubated with purified GST–CDK2/His–CyclinA complexes. GST–CDK2 was immunoprecipitated with anti-CDK2 antibodies and the presence of GST–p57 was assessed by western blot with anti-p57 antibodies. Inputs were analysed by western blot with the shown antibodies. The graph represents the average and s.e.m. of three independent experiments. (B) Increasing amounts of the CDK inhibitors GST–p57, GST–p57T143A and GST–p27 were phosphorylated or not with activated GST–p38 SAPK using cold ATP. The phosphorylated proteins were then incubated with purified GST–CDK2/His–CyclinA complexes. CDK2 activity was assayed in the presence of radiolabelled 32P-γ-ATP and Histone H1 as substrates and analysed by phosphoimaging. (C) Wild-type, p38−/− and p57−/− MEFs were incubated with 100 mM NaCl for 4 h. Cell lysates were then immunoprecipitated with anti-CDK2 antibodies. Endogenous CDK2 activity was assayed as in (B). (D) p57−/− MEFs were infected with lentiviruses carrying an empty vector, a wild-type p57 or p57T143A. Infected MEFs were then treated and assayed as in (C). Representative kinase assays and western blots are shown. The average CDK2 activity of two independent experiments is shown in (BD).
Figure 5
Figure 5
Osmostress mediates a G1 cell-cycle delay in a p38 SAPK- and p57-dependent manner. (A) Wild-type, p38−/− and p57−/− MEFs were stressed with 100 mM NaCl. One hour later, nocodazole was added to trap the cells at the G2/M transition. Cell-cycle progression was monitored by FACS by collecting samples every 2 h. Representative DNA profiles are shown. (B) The percentage of wild-type, p38−/− and p57−/− MEFs in G1 from three independent experiments is shown. Solid circles represent osmostressed MEF cells. Open circles are non-stressed control MEF cells.
Figure 6
Figure 6
The osmostress-induced G1 delay is rescued in p57−/− MEFs infected with wild-type p57 but not with p57T143A. (A) p57−/− MEFs infected with lentiviruses carrying an empty vector, a wild-type p57 or p57T143A were stressed with 100 mM NaCl. One hour later, nocodazole was added to trap the cells at the G2/M transition. Cell-cycle progression was monitored by FACS by collecting samples every 2 h. Representative DNA profiles are shown (B). The percentage of p57−/− infected with lentiviruses carrying an empty vector, a wild-type p57 or p57T143A in G1 from three independent experiments is shown. Solid circles represent osmostressed MEF cells. Open circles are non-stressed control MEF cells.
Figure 7
Figure 7
p38 SAPK and p57 promote cell survival upon stress. (A) Cell viability was assessed by FACS in wild-type, p38−/− and p57−/− MEFs 24 h after the addition of the indicated amounts of NaCl. Statistical significance was determined by one-way ANOVA followed by a Dunnett's multiple comparison test. A value of P<0.05 was considered statistically significant and represented by (*) in the bar graph. (B) Cell viability was assessed on wild-type, p38−/− and p57−/− MEF cells 24 and 48 h after the addition of 200 mM NaCl. (C) Cell viability upon 200 mM NaCl treatment is compromised when p38 SAPK is inhibited by SB203580 in wild-type MEFs. Reintroduction of wild-type p57 but not p57T143A into p57−/− rescued cell viability 24 h after the addition of NaCl. (D) HeLa cells were transfected with Flag–p57. Forty-eight hours post transfection, cells were incubated in the presence or the absence of the specific p38 SAPK inhibitor Birb 0796. Birb 0796 was added to a final concentration of 0.5 μM 2 h prior to the treatment with 600 μM H2O2 and 7.5 mM ionomycin for 60 min. Cell lysates were analysed by western blot with anti-pp38, anti-p38, anti-pHSP27, anti-pp57 and anti-Flag antibodies. (E, F) Cell viability is compromised in p38−/− and p57−/− MEFs after oxidative and ionomycin stress. Reintroduction of wild-type p57 but not p57T143A into p57−/− MEF cell rescued cell viability 24 h after oxidative and ionomycin stress. (G) HeLa cells were transfected with Flag–p57. Forty-eight hours post transfection, cells were incubated in the presence or the absence of the specific p38 SAPK inhibitor Birb 0796. Birb 0796 was added to a final concentration of 0.5 μM 2 h prior to the treatment with 5 mJ of UV for 60 min. Cell lysates were analysed as in (D). (H) Cell viability is compromised in p38−/− MEFs but not in wild type and p57−/− 24 h after irradiating the cells with 5 mJ of UV. Data are represented as mean±s.e.m.

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References

    1. Adrover M, Zi Z, Duch A, Schaber J, González-Novo A, Jimenez J, Nadal-Ribelles M, Clotet J, Klipp E, Posas F (2011) Time-dependent quantitative multicomponent control of the G1-S network by the stress-activated protein kinase Hog1 upon osmostress. Sci Signal 4: ra63. - PubMed
    1. Ambrosino C, Mace G, Galban S, Fritsch C, Vintersten K, Black E, Gorospe M, Nebreda AR (2003) Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase. Mol Cell Biol 23: 370–381 - PMC - PubMed
    1. Ambrosino C, Nebreda AR (2001) Cell cycle regulation by p38 MAP kinases. Biol Cell 93: 47–51 - PubMed
    1. Besson A, Dowdy S, Roberts J (2008) CDK inhibitors: cell cycle regulators and beyond. Dev Cell 14: 159–169 - PubMed
    1. Borriello A, Caldarelli I, Bencivenga D, Criscuolo M, Cucciolla V, Tramontano A, Oliva A, Perrotta S, Della Ragione F (2011) p57(Kip2) and cancer: time for a critical appraisal. Mol Cancer Res 9: 1269–1284 - PubMed

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