The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stress
2012; Springer Nature; Volume: 31; Issue: 13 Linguagem: Inglês
10.1038/emboj.2012.122
ISSN1460-2075
AutoresManel Joaquin, Albert Gubern, Daniel González-Nuñez, E. Josué Ruiz, Isabel Ferreiro, Eulàlia de Nadal, Ángel R. Nebreda, Francesc Posas,
Tópico(s)DNA Repair Mechanisms
ResumoArticle8 May 2012Open Access The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stress Manel Joaquin Corresponding Author Manel Joaquin Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Albert Gubern Albert Gubern Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Daniel González-Nuñez Daniel González-Nuñez Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author E Josué Ruiz E Josué Ruiz Signalling and Cell Cycle Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain Search for more papers by this author Isabel Ferreiro Isabel Ferreiro Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Eulalia de Nadal Eulalia de Nadal Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Angel R Nebreda Angel R Nebreda Signalling and Cell Cycle Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA) and Signalling and Cell Cycle Laboratory, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Francesc Posas Corresponding Author Francesc Posas Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Manel Joaquin Corresponding Author Manel Joaquin Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Albert Gubern Albert Gubern Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Daniel González-Nuñez Daniel González-Nuñez Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author E Josué Ruiz E Josué Ruiz Signalling and Cell Cycle Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain Search for more papers by this author Isabel Ferreiro Isabel Ferreiro Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Eulalia de Nadal Eulalia de Nadal Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Angel R Nebreda Angel R Nebreda Signalling and Cell Cycle Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA) and Signalling and Cell Cycle Laboratory, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Francesc Posas Corresponding Author Francesc Posas Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Author Information Manel Joaquin 1,‡, Albert Gubern1,‡, Daniel González-Nuñez1, E Josué Ruiz2, Isabel Ferreiro1, Eulalia de Nadal1, Angel R Nebreda2,3 and Francesc Posas 1 1Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Barcelona, Spain 2Signalling and Cell Cycle Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain 3Institució Catalana de Recerca i Estudis Avançats (ICREA) and Signalling and Cell Cycle Laboratory, Institute for Research in Biomedicine, Barcelona, Spain ‡These authors contributed equally to this work. *Corresponding authors. Departament de Ciències Experimentals i de la Salut, Cell Signaling Research Group, Univeristat Pompeu Fabra, Dr Aiguader 88, Barcelona 08003, Spain. Tel.:+34 93 316 1820; Fax:+34 93 316 0901; E-mail: [email protected] or Tel.:+34 93 316 0849; Fax:+34 93 316 0901; E-mail: [email protected] The EMBO Journal (2012)31:2952-2964https://doi.org/10.1038/emboj.2012.122 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The p57Kip2 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 p57Kip2 is targeted by the p38 stress-activated protein kinase (SAPK). Phosphorylation of p57Kip2 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. Introduction Mammalian cell cycle progression throughout the G1 phase is controlled by signalling pathways that regulate the activities of G1 cyclin-dependent kinases (CDKs) Cdk4/6-CyclinD and Cdk2-CyclinE/A, which are responsible for modulating the expression, activity and stability of many cell-cycle regulatory proteins (Malumbres and Barbacid, 2005). CDK activity is regulated by two unrelated families, INK and Cip/Kip, of CDK inhibitors (CDKis) (Vidal and Koff, 2000; Besson et al, 2008). The Cip/Kip family includes p21Cip1, p27Kip1 and the p57Kip2 proteins (Vidal and Koff, 2000). Although all Cip/Kip family members share a high homology in the N-terminal CDKi domain and the C-term region, p57 harbours a large central domain enriched in proline residues, which may confer unique functions not shared by p21 or p27 (Lee et al, 1995; Pateras et al, 2009). Notably, p57 is the only CDKi which play an essential role in mouse embryogenesis and p57−/− mice display several developmental defects and a phenotype that resembles the Beckwith–Wiedeman syndrome (Yan et al, 1997; Zhang et al, 1997). Loss of p57 contributes to the occurrence of soft tissue carcinomas, Wilm's tumours and, in certain cells, a decrease in its expression has been related to increased invasiveness and metastasis, which suggests a role of p57 as a putative tumour suppressor (Matsuoka et al, 1995; Orlow et al, 1996; Pateras et al, 2009; Borriello et al, 2011). In addition, it has been shown that p57 mediates cell-cycle progression through diverse mechanisms such as the inhibition of G1 CDKs, particularly Cdk2 (Hashimoto et al, 1998). Remarkably, p57 has recently been shown to maintain haematopoietic stem cells (HSCs) quiescence by retaining CyclinD into the cytoplasm (Matsumoto et al, 2011; Zou et al, 2011). However, the regulation of p57 as well as its biological role in cell-cycle control is not well defined yet, possibly due to its essentiality and the lack of proper tools for its detection and study. Stress-activated protein kinases (SAPKs) play a key role in controlling different cell-cycle checkpoints (Ambrosino and Nebreda, 2001; Bulavin and Fornace, 2004). Mammalian p38 SAPK has been implicated in cell cycle arrest induced by several stimuli at both G2/M and G1/S phases, at least in part, through the stabilization of p21Cip1 mRNA or p27Kip1 protein (Bulavin et al, 2001; Dmitrieva et al, 2002; Bulavin and Fornace, 2004; Pedraza-Alva et al, 2006; Reinhardt et al, 2007; Cuadrado et al, 2009; Lafarga et al, 2009). In budding yeast, the p38-related SAPK Hog1 controls cell cycle at different phases such as S, G2/M (Clotet et al, 2006; Yaakov et al, 2009) and G1 (Escoté et al, 2004; Adrover et al, 2011). In G1, Hog1 directly phosphorylates and controls the activity of the CDKi Sic1, which is related to the members of the mammalian Cip/Kip family, and prevents entry into S phase until proper cellular adaptation to osmostress is achieved (Escoté et al, 2004). The functional and structural conservation of Hog1 and p38 (Galcheva-Gargova et al, 1994; Han et al, 1994; de Nadal et al, 2002) prompted us to test whether p38 was able to phosphorylate and regulate the activity of the mammalian Cip/Kip family of CDKis. Here, we report that stress-activated p38 phosphorylates and regulates the activity of the p57 CDKi. Phosphorylated p57 delays cell cycle and this delay is critical for cell survival in response to stress. This defines a novel role for the p57 CDKi as an integrator of stress signals to regulate cell-cycle progression. Results p38 SAPK phosphorylates the p57Kip2 CDKi in vitro To analyse whether p38 SAPK was able to regulate some members of the Cip/Kip family of CDKis, we initially expressed in bacteria GST-fused p21Cip1, p27Kip1 and the p57Kip2 proteins. Purified proteins were subjected to an in vitro phosphorylation assay with activated p38. In-vitro activated p38 SAPK was able to phosphorylate the CDKis p21Cip1 and p57Kip2 but not p27Kip1 (Figure 1A). Since p21 was already known to be a p38 target (Kim et al, 2002; Todd et al, 2004) we focussed our efforts to further characterize p57 as a novel putative substrate for the p38 SAPK. 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. Download figure Download PowerPoint The phosphorylation of p57 in vitro by p38 was fully prevented by the p38 inhibitor SB203580. ATF2, a known p38 substrate, was used as positive control (Figure 1B). The p57 protein contains five putative S/TP MAPK consensus sites. Thus, we generated two p57 truncated variants; the N-term containing three S/TP sites and the C-term containing two S/TP sites. In vitro kinase assays showed that the N-terminal p57 fragment was phosphorylated to the same extent as the full-length protein whereas the C-term fragment was not phosphorylated at all (Figure 1C). The three S/TP sites found at the p57 N-term fragment were then mutated in full-length p57 to either glycine or alanine and assayed in vitro. Mutation at T143 completely abolished in-vitro phosphorylation of p57 by p38 whereas mutation of p57 at T139 or T167 did not alter phosphorylation of p57 by p38 (Figure 1D). To further confirm that p57 was a direct substrate for p38, we expressed Flag-tagged wild-type p57 and mutant p57T143A in HeLa cells. Flag immunoprecipitates were assayed in vitro with active p38 SAPK in the absence or the presence of SB203580. Wild-type p57 but not p57T143A was specifically phosphorylated by active p38 (Figure 1E). Therefore, p38 directly phosphorylates p57 at T143 in vitro. p38 SAPK interacts with the p57Kip2 CDKi Most SAPKs interact with their corresponding substrates in cells. Thus, we tested whether p38 was able to interact with p57 by performing immunoprecipitation experiments in extracts from HeLa cells expressing Flag-tagged p57 and HA-tagged p38. Binding of HA–p38 was observed when Flag–p57 was precipitated from cell extracts (Figure 2A). Correspondingly, Flag–p57 was also able to co-immunoprecipitate when HA–p38 was precipitated using anti-HA antibodies (Figure 2A). Notably, we were also able to co-immunoprecipitate Flag-tagged p57 with endogenous p38 SAPK (Figure 2B). By using specific antibodies against endogenous p57 and p38 proteins, we were able to confirm the interaction of the two proteins in HeLa cells (Figure 2C). This interaction was also confirmed in wild-type MEF cells and it was abolished in p38−/− or p57−/− cells (Figure 2D). These results show that the CDKi p57 and the p38 SAPK do interact in vivo and form a stable complex. 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. Download figure Download PowerPoint The p38 SAPK phosphorylates the p57Kip2 CDKi in vivo Due to the fact that T143 is a novel p38 target site not described to date, to detect p38 SAPK-mediated p57 phosphorylation in vivo, we took advantage of a generic anti-phospho S/T antibody that was able to specifically recognize p57 phosphorylation at T143. Thus, E. coli purified GST–p57 and GST–p57T143A proteins were incubated in vitro with cold ATP in the absence or presence of activated p38 and analysed by western blot. Only wild type p57, but not p57T143A was recognized by the anti-phospho S/T antibody (Supplementary Figure S1A). We next transfected HeLa cells with wild-type Flag-tagged p57 or Flag-tagged p57T143A in the presence of HA-tagged p38 and myc-tagged MKK6DD (a constitutively active form of the MKK6 MAPKK). The analysis of Flag immunoprecipitates revealed that wild-type p57 was strongly phosphorylated when p38 SAPK was activated by MKK6DD. In contrast, the p57T143A mutant was not phosphorylated by p38 (Figure 3A). Importantly, incubation of the cells with the p38 SAPK inhibitor SB203580 precluded p57 phosphorylation indicating that in vivo p57 phosphorylation required p38 activation (Figure 3B). To rule out that p57 phosphorylation was due to p38 and MKK6DD overexpression, we then assessed p57 phosphorylation upon osmostress. HeLa cells expressing Flag–p57 or Flag–p57T143A were subjected to osmostress and we found that only p57 but not p57T143A was phosphorylated (Figure 3C). The importance of finding a novel in-vivo p38 substrate prompted us to generate specific antibodies targeting phosphorylated p57 at T143. Thus, a phosphopeptide surrounding the p57 T143 site was used to immunize rabbits and the collected anti-sera was affinity purified. The antibody specifically recognized the phosphopeptide but not the non-phosphorylated peptide. Next, we phosphorylated in vitro purified wild-type GST–p57 and GST–p57T143A in the presence of p38 and MKK6DD with cold ATP. The purified anti-pp57 antibody was able to specifically recognize p57 phosphorylation at T143A (Supplementary Figure S1B). Then, we expressed wild-type Flag-tagged p57 in HeLa cells in the absence or the presence of the p38 SAPK inhibitor Birb 0796. Cells were osmostressed and analysed by western blot. The anti-pp57 antibody was able to specifically recognize p57 phosphorylation in vivo upon p38 SAPK activation (Supplementary Figure S1C). Correspondingly, phosphorylation of Flag–p57 upon osmostress was also abolished in p38−/− cells (Supplementary Figure S1D). We next assessed in vivo phosphorylation p57 by immunofluorescence using the specific phospho-p57 antibody. Wild-type and p38−/− MEFs were subjected to osmostress and found that whereas no phosphorylation of p57 was detected in the absence of stress, strong nuclear fluorescence was detected upon osmostress. The increase on p57 phosphorylation upon osmostress was not observed in p38−/− cells (Figure 3D). Altogether, these results show that p57 is phosphorylated at T143 in vivo by the p38 SAPK. 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 CellD imaging software. Representative pictures are shown. Download figure Download PowerPoint p57 phosphorylation at T143 by p38 regulates p57 activity towards Cdk2 in vitro We then analysed whether p57 phosphorylation by p38 was modulating p57 activity. It has been shown that protein phosphorylation can alter the stability or localization of Cip/Kip CDKis (Tsvetkov et al, 1999; Ishida et al, 2002; Kim et al, 2002; Liang et al, 2002; Shin et al, 2002; Kotake et al, 2005; Kossatz et al, 2006). Thus, we initially monitored endogenous p57 half live in HeLa cells treated with NaCl or anisomycin (a known activator of p38). Protein synthesis was stopped by the addition of cycloheximide 30 min prior to stressing the cells. p57 protein levels were followed over time by western blot. Neither osmostress nor anisomycin altered p57 half live (Supplementary Figure S2A), albeit this was under the control of the proteosome as previously reported (Supplementary Figure S2B; Kamura et al, 2003). To further confirm that p57 protein half life was not affected by cell stress, we expressed wild type Flag-tagged p57 in HeLa cells. As observed with endogenous p57, Flag-tagged p57 protein half life was neither affected by osmostress nor anisomycin (Supplementary Figure S2C). We then monitored whether p57 localization was altered upon osmostress by following the localization of a p57-DsRed construct. p57 was found to be localized mainly in the nucleus and it did not change its localization upon stress (Supplementary Figure S3B). Similar results were obtained when endogenous p57 was followed in cell fractionation (Supplementary Figure S3A). Of note, CyclinD was also mainly nuclear (Supplementary Figure S3A). The localization of p38 did not change significantly under the conditions tested but there was a significant amount of active p38 present in the nuclei of the cells (Supplementary Figure S3A and B). Altogether, phosphorylation of p57 by p38 neither affects its stability nor its localization. p57 preferentially binds to Cdk2 (Hashimoto et al, 1998) and thus, we asked whether T143 phosphorylation altered the ability of p57 to interact with and inhibit Cdk2. Wild type GST–p57 and mutant GST–p57T143A purified from bacteria were phosphorylated in vitro by activated p38 SAPK and binding to Cdk2 was assessed. Binding of p57 to Cdk2 increased almost four-fold when phosphorylated by p38. Remarkably, binding of the p57T143A mutant to Cdk2 was not affected after incubation with p38 (Figure 4A). Therefore, phosphorylation of p57 by p38 increased the association of p57 with Cdk2. 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 (B–D). Download figure Download PowerPoint An increase in the affinity of p57 towards CDK2 could result in a decrease on Cdk2 activity. Thus, we tested whether phosphorylation of p57 could inhibit more efficiently Cdk2 activity. We incubated increasing amounts of purified GST–p57 (wild-type and the p57T143A mutant) previously incubated or not with active p38 and then, analysed Cdk2/CyclinA activity in vitro. Increasing amounts of GST–p57 inhibited gradually the activity of Cdk2 as it was observed by incubation of Cdk2 with p27. Remarkably, the inhibition of Cdk2 activity was more pronounced when p57 was phosphorylated by p38 (Figure 4B). Correspondingly, the ability of p57T143A to inhibit Cdk2 did not increase by preincubation with p38 (Figure 4B). Thus, phosphorylated p57 at T143 inhibits more efficiently Cdk2–CyclinA activity than non-phosphorylated p57. Osmostress regulates Cdk2 activity in vivo through p57 phosphorylation at T143 If phosphorylation of p57 by p38 increases its ability to inhibit Cdk2, then osmostress should be expected to cause a decrease in Cdk2 activity in vivo. To assess Cdk2 activity, wild-type, p38−/− and p57−/− MEFs were challenged with 100 mM NaCl for 4 h and cell lysates were then immunoprecipitated with anti-Cdk2 antibodies. Endogenous Cdk2 activity was assayed in the presence of radiolabelled 32P-γ-ATP and Histone H1 as substrate. Cdk2 activity from wild-type MEFs was reduced to <20% upon osmostress (Figure 4C). This effect was dependent on p38 SAPK and p57 since it was not observed in p38−/− or p57−/− MEF cells (Figure 4C). Therefore, Cdk2 activity was inhibited in response to osmostress depending on p38 and p57. To analyse whether phosphorylation of p57 by p38 was promoting the decrease in Cdk2 activity, we infected p57−/− MEF cells with lentiviruses expressing wild-type or the p57T143A mutant. Both proteins were expressed to a similar extend in p57−/− MEF cells (Supplementary Figure S4). Osmostress provoked a reduction on endogenous Cdk2 activity in cells carrying wild type p57 but not in cells carrying the p57T143A mutant (Figure 4D). Therefore, p57 phosphorylation at T143 is essential to regulate Cdk2 activity in vivo. Osmostress regulates G1 progression in a p38- and p57-dependent manner Since phosphorylation of p57 by p38 induced a reduction on Cdk2 activity, we analysed whether p57 was important to mediate a cell-cycle delay in G1 upon osmostress. Wild-type, p38−/− and p57−/− knockout MEFs cultures were subjected to osmostress (100 mM NaCl and 200 mM) and cell-cycle progression was followed by FACS. Nocodazole was added 1 h after osmostress to trap the cells at G2/M. In response to osmostress, wild-type cells clearly delayed the transition to G2/M (Figure 5A and B). In contrast, p38−/− and p57−/− cells were not able to delay cell-cycle progression to the same extent upon osmostress (Figure 5A and B; Supplementary Figure S9). Both cells express p18, p21 and p27 CDKis and it is worth noting that p21 is upregulated in the absence of p57 (Supplementary Figure S5A). Of note, p21 protein levels in p57−/− cells were not affected upon osmostress and were downregulated upon the reintroduction of p57 (Supplementary Figure S5B and C). Taken together, our data indicate that both p38 and p57 are required for cell-cycle delay in response to osmostress. 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. Download figure Download PowerPoint To address the involvement of p57 phosphorylation at T143 on cell-cycle progression, we then infected p57−/− MEFs with viruses expressing wild-type p57 or p57T143A and subjected them to stress (100 mM NaCl). p57−/− cells expressing wild-type p57 arrested at G1 upon osmostress as well as wild-type cells (Figure 6A and B versus 5A and B, 5). In contrast, albeit cells arrested a little longer than p57−/− cells, expression of p57T143A was not able to proper delay cell-cycle progression upon stress (Figure 6A and B). Therefore, phosphorylation of p57 at T143 by p38 is critical to impose a G1 delay upon osmostress. 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. Download figure Download PowerPoint The p38 SAPK and p57 CDKi promote cell survival upon osmostress We reasoned that delaying cell-cycle progression in G1 could be necessary to guarantee cell adaptation and survival to osmostress. To assess the biological relevance of this cell-cycle delay induced by p57 phosphorylation, we monitored cell viability by propidium iodide (PI) staining by FACS in response to osmostress. We subjected wild-type, p38−/− and p57−/− cells to increasing amounts of NaCl and found that cell viability was compromised at increasing NaCl concentrations. Notably, both p38−/− and p57−/− cells displayed a strong reduction on their ability to survive to osmostress when compared with wild type (Figure 7A and B). Similar results were obtained when cell viability was assessed using an MTT assay (Supplementary Figure S6). MK2 and Cdt1 have been defined as targets for p38 SAPK involved in cell cycle (Manke et al, 2005; Reinhardt et al, 2007; Chandrasekaran et al, 2011). To assess the relevance of those proteins in response to osmostress, MK2 and Cdt1 were downregulated by the use of a chemical inhibitor (MK2 inhibitor III) or an siRNA against Cdt1. The downregulation of either MK2 or Cdt1 did not alter significantly cell survival in response to osmostress in wild-type or p57−/− and p38−/− cells (Supplementary Figures S10 and S11). There
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