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GSK3 TIPping Off p53 to Unleash PUMA

2011; Elsevier BV; Volume: 42; Issue: 5 Linguagem: Inglês

10.1016/j.molcel.2011.05.022

1097-4164

Florian J. Bock, Andreas Villunger,

Ubiquitin and proteasome pathways

Knowledge about “life vs. death” decisions made by p53 after DNA damage is limited but critical to preventing side effects during therapeutic application and to improve anticancer activity. Here, Charvet et al. define a signaling network that explains the protective effects of cytokines on cells exposed to γ-radiation. Knowledge about “life vs. death” decisions made by p53 after DNA damage is limited but critical to preventing side effects during therapeutic application and to improve anticancer activity. Here, Charvet et al. define a signaling network that explains the protective effects of cytokines on cells exposed to γ-radiation. Why certain tissues, such as bone marrow or the gastrointestinal tract, are exquisitely sensitive to ionizing radiation while others are not remains poorly understood. The cyclin-dependent kinase inhibitor p21 and PUMA, a proapoptotic “BH3-only” member of the Bcl-2 family, are prime targets of the transcription factor and tumor suppressor p53. Both genes are induced in most cell types after DNA damage, but what signals tilt the cellular response in favor of arrest and repair or cell death remains to be fully clarified (Olsson et al., 2007Olsson A. Manzl C. Strasser A. Villunger A. Cell Death Differ. 2007; 14: 1561-1575Crossref PubMed Scopus (148) Google Scholar). Growth factors and cytokines present in the microenvironment clearly influence the outcome of radiation damage, for example in the intestinal mucosa (Booth and Potten, 2001Booth D. Potten C.S. J. Natl. Cancer Inst. Monogr. 2001; 29: 16-20Crossref PubMed Scopus (78) Google Scholar). They may do so by triggering signaling pathways that culminate in p53-independent gene expression or posttranslational modifications on p53 that impact on protein stability and/or promoter binding selectivity. Alternatively, such signals may act indirectly by modification of one or more of p53's many interaction partners involved in the coordination of target gene transcription, such as the acetyltransferases MYST1/MOF or TIP60 (Sykes et al., 2006Sykes S.M. Mellert H.S. Holbert M.A. Li K. Marmorstein R. Lane W.S. McMahon S.B. Mol. Cell. 2006; 24: 841-851Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, Tang et al., 2006Tang Y. Luo J. Zhang W. Gu W. Mol. Cell. 2006; 24: 827-839Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). In the present study, the authors link cytokine-triggered, AKT/PKB-dependent inhibition of glycogen synthase kinase 3 (GSK3) with impaired apoptosis induction by p53 after DNA damage. Reduced cytokine support enables efficient GSK3-mediated phosphorylation and activation of the lysine acetyltransferase activity of TIP60/KAT5 after DNA damage. This culminates in K120 acetylation on p53, acetylation of histone H4, and puma transcription, triggering DNA-damage-mediated apoptosis. Early studies have suggested that the PTEN/PI3K/AKT signaling network can influence the outcome of p53 stabilization and fibroblasts from PTEN-deficient mice were surprisingly resistant to p53-induced apoptosis (Stambolic et al., 2001Stambolic V. MacPherson D. Sas D. Lin Y. Snow B. Jang Y. Benchimol S. Mak T.W. Mol. Cell. 2001; 8: 317-325Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar). Hence, the authors tested the consequences of pharmacological inhibition of this signaling axis and realized that maximal induction of PUMA upon radiation damage was only observed when PI3K was inhibited, while blocking GSK3-impaired PUMA expression. Of note, neither accumulation of p53 nor induction of p21, proapoptotic Bax or Noxa appeared affected by this treatment in U2OS osteosarcoma cells. These results were confirmed in IL-3-dependent BAF3 and FL5.12 pro-B cells. Importantly, transient GSK3 inhibition prior to γ-radiation increased also their clonal survival. Similar responses were documented in IL-2 responsive splenocytes and GSK3 inhibition also prevented PUMA-induction after γ-radiation in vivo. Interestingly, a recent study documenting IGF- or bFGF-triggered, PI3K/AKT-dependent inhibition of mucosal radiation damage by suppression of PUMA transcription also noted strong changes in p53 accumulation and p21 induction (Qiu et al., 2010Qiu W. Leibowitz B. Zhang L. Yu J. Oncogene. 2010; 29: 1622-1632Crossref PubMed Scopus (92) Google Scholar). This indicates that the molecular basis of the observed effect may be fine-tuned differently downstream of IGF/bFGF and cytokine receptors and/or may be cell-type dependent. The acetyltransferase TIP60 has been implicated previously in regulating preferential expression of proapoptotic p53 genes by targeting the K120 residue within the DNA-binding domain, a residue occasionally also found mutated in human cancers (http://p53.free.fr/Database/p53_database_distr.html). By reconstituting p53-deficient cells with an inducible p53 variant, either WT or K120 acetylation defective, the authors demonstrated a critical role for TIP60-mediated K120 acetylation on p53, downstream of GSK3. Additionally, they identified a conserved phosphorylation consensus site (S86XXXS90), indicating that TIP60 might be a direct GSK3 substrate, which was elegantly confirmed. Usually, GSK3 substrates require a priming phosphorylation (in this case S90), but the kinase mediating this event on TIP60 after γ radiation remained unidentified. It is tempting to speculate that this must be a cyclin-dependent kinase that could effectively link cell-cycle arrest with cell death, yet pharmacological inhibition of CDK1, previously implicated in phosphorylating S90 on TIP60 (Lemercier et al., 2003Lemercier C. Legube G. Caron C. Louwagie M. Garin J. Trouche D. Khochbin S. J. Biol. Chem. 2003; 278: 4713-4718Crossref PubMed Scopus (47) Google Scholar), did have no effect on S86 phosphorylation. Alternatively, checkpoint kinases Chk1 and/or Chk2 could do the job, as the priming site of TIP60 harbors a consensus site for those kinases (RXXS90), but S86 phosphorylation was appeared to be detectable already prior DNA damage. Similarly to pharmacological inhibition of GSK3, overexpression of a constitutive active version of its antagonist, AKT/PKB, prevented TIP60 phosphorylation on S86 and induction of PUMA mRNA. Remarkably, under all these conditions, induction of p21 was never significantly affected, in line with the idea that GSK3 acts as a switch to preferentially eliminate damaged cells when cytokine support is limited. Notably, when GSK3 was inhibited or a constitutive version of AKT overexpressed, leading to repressed induction of PUMA, p21 protein often appeared even more strongly induced, contrasting findings made in epithelial cells exposed to IGF or bFGF prior to radiaton (Qiu et al., 2010Qiu W. Leibowitz B. Zhang L. Yu J. Oncogene. 2010; 29: 1622-1632Crossref PubMed Scopus (92) Google Scholar). As mentioned, although S86 phosphorylation on TIP60 required PI3K/AKT signaling, it was independent of DNA damage to occur, yet critical for p53-mediated PUMA induction. As TIP60 can also acetylate histone H4 and is recruited to the puma promoter in a p53K120 acetylation-dependent manner (Tang et al., 2006Tang Y. Luo J. Zhang W. Gu W. Mol. Cell. 2006; 24: 827-839Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar) it remained possible that S86 phosphorylation by GSK3 might also confer some substrate specificity to TIP60. However, this was not the case, as acetylation of p53, as well as H4, depended on S86 phosphorylation while the interaction of TIP60 with p53 did not. Noteworthy, while inhibition of PI3K increased binding of p53 to the puma promoter, inhibition of GSK3 did not, but influenced H4 acetylation at the promoter, allowing more efficient transcription. This suggests that additional PI3K signals can influence p53 recruitment to the puma promoter, independent of proposed effects on p53 stabilization, while GSK3-modified TIP60 needs to hitch a ride with p53. What remains unclear so far is what discriminates the puma promoter from others driving expression of proapoptotic genes such as Noxa or Bax, the latter also preferentially activated by p53K120 (Sykes et al., 2006Sykes S.M. Mellert H.S. Holbert M.A. Li K. Marmorstein R. Lane W.S. McMahon S.B. Mol. Cell. 2006; 24: 841-851Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). Hence one has to implicate “protein X,” maybe Axin, ASPP family members or MYST1/MOF, the latter equally proficient in acetylating K120 on p53 (Sykes et al., 2006Sykes S.M. Mellert H.S. Holbert M.A. Li K. Marmorstein R. Lane W.S. McMahon S.B. Mol. Cell. 2006; 24: 841-851Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar), in the fine tuning of this response. Of note, it was previously reported by Maurer and colleagues that GSK3-mediated phosphorylation of antiapoptotic MCL-1 on S159, a prime target for the proapoptotic action of PUMA, promotes its proteasomal degradation under conditions of trophic factor withdrawal (Maurer et al., 2006Maurer U. Charvet C. Wagman A.S. Dejardin E. Green D.R. Mol. Cell. 2006; 21: 749-760Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar), assigning at least a dual prodeath role to GSK3 (Figure 1). As such, inhibition of GSK3 appears to be an interesting target that may help to prevent unwanted side effects during radiation or chemotherapy. Of note, MCL-1 appears most critical for hematopoietic stem cell survival and can protect from DNA-damage-mediated apoptosis (Opferman et al., 2005Opferman J.T. Iwasaki H. Ong C.C. Suh H. Mizuno S. Akashi K. Korsmeyer S.J. Science. 2005; 307: 1101-1104Crossref PubMed Scopus (442) Google Scholar). Along similar lines, DNA-damaging regimens combined with AKT inhibitors may prove more potent in tumor-cell killing. However, one also needs to keep in mind that inhibition of apoptosis during the acute DNA-damage response can actively drive malignant disease (Labi and Villunger, 2010Labi V. Villunger A. Cell Cycle. 2010; 9: 4269-4275Crossref PubMed Scopus (14) Google Scholar), posing a certain caveat for the use of GSK3 or PUMA inhibitors. Nonetheless, the detailed knowledge about these molecular events will help to improve the efficacy of radiation therapy and other DNA-damaging anticancer agents and opens up new possibilities to limit their negative side effects. Phosphorylation of Tip60 by GSK-3 Determines the Induction of PUMA and Apoptosis by p53Charvet et al.Molecular CellJune 10, 2011In BriefActivation of p53 by DNA damage results in either cell-cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here, we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the proapoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. Full-Text PDF Open Archive