Ser46 phosphorylation and prolyl-isomerase Pin1- mediated isomerization of p53 are key events in p53- dependent apoptosis induced by mutant huntingtin Alice Grisona,1,2, Fiamma Mantovania,b,1, Anna Comela,b, Elena Agostonic, Stefano Gustincichc,d,e, Francesca Persichettic,f, and Giannino Del Sala,b,3 aLaboratorio Nazionale Consorzio Interuniversitario per le Biotecnologie, 34149 Trieste, Italy; bDepartment of Life Sciences, University of Trieste, 34100 Trieste, Italy; cSector of Neurobiology, International School for Advanced Studies, 34136 Trieste, Italy; dInternational School for Advanced Studies Unit, Italian Institute of Technology, 34136 Trieste, Italy; eGiovanni Armenise–Harvard Foundation Laboratory, International School for Advanced Studies, 34136 Trieste, Italy; and fDepartment of Environmental and Life Sciences, University of Eastern Piedmont, 15121 Alessandria, Italy Edited* by Carol Prives, Columbia University, New York, NY, and approved September 13, 2011 (received for review April 18, 2011) Huntington disease (HD) is a neurodegenerative disorder caused by and PKCδ. The subsequent transduction of stress-dependent a CAG repeat expansion in the gene coding for huntingtin protein. phosphorylation into specific conformational changes of p53 that Several mechanisms have been proposed by which mutant hunting- fully unleash its apoptotic activity is performed by the prolyl tin (mHtt) may trigger striatal neurodegeneration, including mito- isomerase Pin1. This enzyme catalyzes cis/trans isomerization of chondrial dysfunction, oxidative stress, and apoptosis. Furthermore, proline bonds preceded by phosphorylated serine or threonine mHtt induces DNA damage and activates a stress response. In this residues (pSer/Thr-Pro), thereby altering structure and functions context, p53 plays a crucial role in mediating mHtt toxic effects. Here of its substrates (10, 11). Upon genotoxic insults, Pin1 binds mul- we have dissected the pathway of p53 activation by mHtt in human tiple sites on p53, promoting its accumulation in stressed cells, the activation of its transcriptional functions, and the induction of its neuronal cells and in HD mice, with the aim of highlighting critical – nodes that may be pharmacologically manipulated for therapeutic apoptotic activity (12 14). intervention. We demonstrate that expression of mHtt causes in- Phosphorylation-dependent prolyl isomerization triggered by creased phosphorylation of p53 on Ser46, leading to its interaction Pin1 represents an essential mechanism in modulating several signaling pathways involved in DNA damage and apopotosis. In with phosphorylation-dependent prolyl isomerase Pin1 and conse- the CNS, Pin1 is highly expressed and regulates several substrates, quent dissociation from the apoptosis inhibitor iASPP, thereby including the hyperphosphorylated form of tau in Alzheimer’s inducing the expression of apoptotic target genes. Inhibition of disease (15), whereas in Parkinson disease (PD) Pin1 facilitates Ser46 phosphorylation by targeting homeodomain-interacting pro- α δ formation of -synuclein inclusions (16). tein kinase 2 (HIPK2), PKC , or ataxia telangiectasia mutated kinase, Based on these considerations, we reasoned that Pin1 could be as well as inhibition of the prolyl isomerase Pin1, prevents mHtt- critical for mediating p53-dependent mHtt toxicity. Therefore, dependent apoptosis of neuronal cells. These results provide a ratio- by investigating its role in this context, we may highlight crucial CELL BIOLOGY nale for the use of small-molecule inhibitors of stress-responsive upstream events involved in HD pathogenesis and unveil at- protein kinases and Pin1 as a potential therapeutic strategy for HD tractive targets for development of novel therapeutic options to treatment. counteract HD. untington disease is a dominantly inherited neurodegenera- Results Htive disorder due to an expanded CAG repeat sequence in the Expression of Mutant Huntingtin Promotes the Interaction of p53 HD gene that elongates a segment of glutamine residues in the with Pin1. Analysis of postmortem brains of HD patients revealed protein huntingtin (Htt) (1). The most striking pathological man- high levels of p53 relative to healthy controls (Fig. 1A), in agree- ifestation of HD is a gradual loss of neurons, predominantly in the ment with previous reports (5). To study the stress pathways re- striatum, causing motor abnormalities and cognitive decline (2). sponsible for p53 activation in HD neurons, we then analyzed p53 Toxic properties of mutant huntingtin (mHtt) are believed to cause phosphorylation. Interestingly, in HD brains, p53 was phosphory- HD. Among them, mitochondrial dysfunction and generation of lated on Ser46 (Fig.1A), a modification that has been associated reactive oxygen species (ROS) lead to DNA lesions (3, 4). In- with activation of its apoptotic function upon stress (14, 17). Nu- terestingly, expression of full-length mHtt protein and N-terminal clear accumulation of mHtt N-terminal fragments is observed in fragments containing the polyQ expansion elicit a DNA damage HD brains (18) and animal models (18). Expression of these response, with activation of the ATM/ATR pathways (3, 4) and truncated forms recapitulates many molecular and neurological their downstream effectors, including the tumor suppressor p53 (3, HD phenotypes (19). The N-terminal fragment (residues 1–171) of 5, 6). p53 mediates mitochondrial dysfunction and cytotoxicity in either wild-type or mutant Htt (bearing 21 and 150 polyQ, re- HD cells and in transgenic animal models, whereas its inhibition spectively) were thus expressed in SH-SY5Y human neuroblas- prevents these phenotypes (5). p53 governs a wide array of pathways involved in genomic sta- bility, antioxidant activities, and energy metabolism in addition to Author contributions: F.M. and G.D.S. designed research; A.G., F.M., A.C., and E.A. per- promoting either cytostatic or cytotoxic responses to intrinsic and formed research; E.A., S.G., and F.P. contributed new reagents/analytic tools; F.M. and exogenous sources of cellular stress (7). Given the complexity of G.D.S. analyzed data; and F.M., F.P., and G.D.S. wrote the paper. p53 functions within the cell, a better understanding of how sig- The authors declare no conflict of interest. naling networks converge on this hub to modulate mHtt-de- pendent toxicity is required to shed light on the reduced ability of *This Direct Submission article had a prearranged editor. the brain neurons to survive. Regulation of p53 activities relies on Freely available online through the PNAS open access option. a complex network of posttranslational modifications and protein 1A.G and F.M. contributed equally to this work. interactions (8, 9), which ultimately determine the outcome of the 2Present address: Sector of Neurobiology, International School for Advanced Studies, p53 response. This process entails site-specific phosphorylation by 34136 Trieste, Italy. several DNA damage-activated protein kinases, including, among 3To whom correspondence should be addressed. E-mail: [email protected]. others, ataxia telangiectasia mutated (ATM), ATM and Rad3-re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lated (ATR), homeodomain-interacting protein kinase 2 (HIPK2), 1073/pnas.1106198108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1106198108 PNAS | November 1, 2011 | vol. 108 | no. 44 | 17979–17984 Downloaded by guest on September 24, 2021 ABC D Fig. 1. Expression of mutant huntingtin induces the interaction of p53 with Pin1. (A) Phosphorylation of p53 on Ser46 in postmortem brains was compared between HD patients (HD1 and HD2) and healthy controls (C) by Western blot. (Middle and Bottom) Levels of total p53 and actin as loading control. (B) SH- SY5Y cells were transfected with constructs expressing the N-terminal 1–171 Htt fragment with either 21Q or 150Q. p53 was immunoprecipitated from equal amounts of total cell lysates and analyzed by Western blot with antibodies specific for phosphorylated Ser46 and total p53. The levels of actin and Htt proteins in input lysates are shown. (C) SH-SY5Y were transfected with indicated constructs. Cell lysates normalized for p53 protein levels were subjected to coimmunoprecipitation to analyze interaction of endogenous p53 and Pin1. (D) H1299 cells were transfected with indicated constructs, and the interaction of p53 with recombinant Pin1 protein was analyzed by GST pull-down of cell lysates normalized for p53 levels. toma cells to verify whether p53 Ser46 phosphorylation was a We then analyzed activation of the p53 response in the brains of consequence of mHtt expression. Interestingly, mutant but not Hdh CAG knock-in mice in which the glutamine tract of mouse Htt wild-type Htt induced the phosphorylation of endogenous p53 on is extended to 111 residues (HdhQ111) (20). These mice show striatal Ser46, in addition to the previously reported phosphorylation of neurodegeneration, reactive gliosis, and gait abnormalities at older Ser15 (6) (Fig. 1B). Because Ser46 phosphorylation generates age (after 24 mo) (21). However, we observed stabilization of p53 in a target site for the prolyl isomerase Pin1 (12, 14), we asked whether brain extracts and the consequent transcriptional induction of the Pin1 might play a role in mediating activation of p53 upon mHtt p53 target gene p21WAF1 in the striatum of 12-mo-old HdhQ111 mice expression in neuronal cells. Strikingly, expression of the mHtt- compared with their WT littermates, HdhQ7 (Fig. 2D). This finding