Tau acetylates and stabilizes β‐catenin thereby promoting cell survival
2020; Springer Nature; Volume: 21; Issue: 3 Linguagem: Inglês
10.15252/embr.201948328
ISSN1469-3178
AutoresEnjie Liu, Qiuzhi Zhou, Ao‐Ji Xie, Xiaoguang Li, Mengzhu Li, Jinwang Ye, Shihong Li, Dan Ke, Qun Wang, Zhipeng Xu, Li Li, Yongqiang Yang, Gong‐Ping Liu, Xiaochuan Wang, Honglian Li, Jian‐Zhi Wang,
Tópico(s)Nerve injury and regeneration
ResumoArticle13 January 2020free access Source DataTransparent process Tau acetylates and stabilizes β-catenin thereby promoting cell survival Enjie Liu Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China Search for more papers by this author Qiuzhi Zhou Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Ao-Ji Xie Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xiaoguang Li Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Mengzhu Li Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Jinwang Ye Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Shihong Li Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Dan Ke Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Qun Wang Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Zhi-Peng Xu Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Li Li Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Ying Yang Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Gong-Ping Liu Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xiao-Chuan Wang orcid.org/0000-0001-8207-0042 Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Hong-Lian Li Corresponding Author [email protected] Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Histology and Embryology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Jian-Zhi Wang Corresponding Author [email protected] orcid.org/0000-0002-6216-8525 Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China Search for more papers by this author Enjie Liu Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China Search for more papers by this author Qiuzhi Zhou Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Ao-Ji Xie Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xiaoguang Li Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Mengzhu Li Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Jinwang Ye Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Shihong Li Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Dan Ke Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Qun Wang Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Zhi-Peng Xu Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Li Li Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Ying Yang Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Gong-Ping Liu Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xiao-Chuan Wang orcid.org/0000-0001-8207-0042 Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Hong-Lian Li Corresponding Author [email protected] Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Histology and Embryology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Jian-Zhi Wang Corresponding Author [email protected] orcid.org/0000-0002-6216-8525 Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China Search for more papers by this author Author Information Enjie Liu1,2,‡, Qiuzhi Zhou1,‡, Ao-Ji Xie1, Xiaoguang Li1, Mengzhu Li1, Jinwang Ye1, Shihong Li1, Dan Ke1, Qun Wang1, Zhi-Peng Xu1, Li Li1, Ying Yang1, Gong-Ping Liu1, Xiao-Chuan Wang1, Hong-Lian Li *,3 and Jian-Zhi Wang *,1,4 1Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 3Key Laboratory of Ministry of Education of China for Neurological Disorders, Department of Histology and Embryology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 4Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 027 83692612; E-mail: [email protected] *Corresponding author. Tel: +86 027 83693881; E-mail: [email protected] EMBO Rep (2020)21:e48328https://doi.org/10.15252/embr.201948328 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 Abstract Overexpressing Tau counteracts apoptosis and increases dephosphorylated β-catenin levels, but the underlying mechanisms are elusive. Here, we show that Tau can directly and robustly acetylate β-catenin at K49 in a concentration-, time-, and pH-dependent manner. β-catenin K49 acetylation inhibits its phosphorylation and its ubiquitination-associated proteolysis, thus increasing β-catenin protein levels. K49 acetylation further promotes nuclear translocation and the transcriptional activity of β-catenin, and increases the expression of survival-promoting genes (bcl2 and survivin), counteracting apoptosis. Mutation of Tau's acetyltransferase domain or co-expressing non-acetylatable β-catenin-K49R prevents increased β-catenin signaling and abolishes the anti-apoptotic function of Tau. Our data reveal that Tau preserves β-catenin by acetylating K49, and upregulated β-catenin/survival signaling in turn mediates the anti-apoptotic effect of Tau. Synopsis Tau acetylates β-catenin at K49, which stabilizes β-catenin by inhibiting its phosphorylation and ubiquitination-associated proteolysis. β-catenin mediates the anti-apoptotic effects of Tau by increasing the expression of survival-promoting genes. Tau can acetylate β-catenin at K49 in a concentration-, time- pH- and acetyltransferase domain-dependent manner. K49-acetylation by Tau preserves β-catenin by inhibiting its phosphorylation and ubiquitination-associated proteolysis, resulting in increased nuclear translocation and enhanced transcriptional activity of β-catenin. β-catenin K49-acetylation mediates the anti-apoptotic effects of Tau by augmenting the expression of survival-promoting genes. Introduction Intracellular Tau accumulation forming neurofibrillary tangles is positively correlated with neurodegeneration and memory deterioration in Alzheimer's disease (AD) and other tauopathies 1-4. The neurodegeneration observed in the AD brain takes over 20 years after the formation of tangles 5, although these neurons have been extensively exposed to an aging-dependent increase in pro-apoptotic environment. Then, why do these neurons (i.e., with accumulation of Tau) not go to acute apoptosis (occur in hours) but instead take a chronic degeneration? As Tau is the major protein component of the tangles in the degenerated neurons, the role of Tau has been extensively studied. Previous reports show that overexpressing human wild-type full-length Tau (Tau40) renders the cells more resistant to apoptosis induced by various pro-apoptotic factors both in vitro and in vivo 6-12. Meanwhile, the intracellular Tau accumulation also induces calcium overload, endoplasmic reticulum stress, mitochondrial dysfunction, and synaptic impairments 13-18. Based on these observations, it has been proposed that Tau hyperphosphorylation and accumulation may play a dual role in leading the neurons to escape the acute apoptosis and simultaneously triggering a chronic degeneration 19, 20; importantly, the Tau-induced apoptosis escape may serve as a precondition of AD neurodegeneration 21. Therefore, understanding the molecular mechanisms underlying apoptosis escape is important for designing strategies to prevent the apoptosis-escaped neurons from degeneration. Accompanying the Tau-induced apoptosis escape, the total β-catenin level was remarkably increased with a reduced phosphorylation level of β-catenin 6. It is currently not understood how Tau accumulation affects β-catenin phosphorylation and preserves β-catenin, and whether and how the increased β-catenin mediates the anti-apoptotic effects of Tau. β-catenin was originally discovered as a cell adhesion factor, mainly located in the cytoplasm 22. Subsequent studies reveal that β-catenin participates in several cellular signaling pathways, such as Wnt signaling to regulate cell differentiation and proliferation 23-25. Gene mutation or upregulation of β-catenin has been identified in cancers 26. Studies also show that elevation and the nuclear translocation of β-catenin can prime cellular survival pathways 27-29. The N-terminal (1–49 amino acids) of β-catenin is the key region for its stability and the degradation by ubiquitin–proteasome system 30, and phosphorylation of β-catenin at the N-terminal Ser33, Ser37, Thr41, and Ser45 promotes its ubiquitination and degradation 30-32. Adjacent to the phosphorylation sites in the N-terminal of β-catenin, there are two lysine residuals (K49 and K19) which can be ubiquitinated and/or acetylated 30, 33-36. Furthermore, overexpressing Tau decreases phosphorylation level with upregulation of β-catenin 6, and Tau protein per se has the activity of acetyltransferase 37. It is well accepted that ubiquitination is the prerequisite for the proteasome-associated degradation of proteins, and the ubiquitination and acetylation occur at the same amino acid residual in the proteins. Based on these observations, we speculate that Tau may directly acetylate β-catenin, by which it inhibits the ubiquitination and/or phosphorylation of β-catenin. Consequently, β-catenin is preserved to prime the survival signaling and to mediate the anti-apoptotic effect of Tau. To test this idea, we demonstrate in the present study that Tau can directly and robustly acetylate β-catenin both in vitro and in vivo. Acetylation of β-catenin at K49 by Tau inhibits its phosphorylation, ubiquitination, and proteolysis and thus promotes its nuclease translocation leading to upregulation of β-catenin/Wnt/survival signaling. The upregulation of β-catenin/Wnt/survival signaling in turn mediates the anti-apoptotic function of Tau. Results Tau can acetylate β-catenin at K49 in vitro and in vivo We reported previously that overexpressing Tau could upregulate β-catenin 6, but the mechanism has been elusive. To clarify this, we first measured the effect of Tau on the transcription of β-catenin. Overexpressing Tau did not significantly change the mRNA level of β-catenin (Fig EV1A and B), although a significantly increased protein level of β-catenin was shown in neuroblastoma (N2a) cells, the cultured primary hippocampal neurons, and human Tau transgenic mice (Fig EV1C–I). These data suggest that Tau-induced β-catenin elevation involves post-translational modifications but not the transcription of β-catenin. Click here to expand this figure. Figure EV1. Overexpressing Tau increases β-catenin protein level without affecting the mRNA level of β-catenin A, B. Overexpressing Tau did not affect mRNA level of β-catenin. HEK293 (A) and N2a (B) cells were transfected respectively with Tau40 (Tau) or the empty vector (Vec) for 48 h. β-catenin mRNA level (ctnnb1) was detected by RT–PCR (n = 6–9 biological replicates each group). C–H. Overexpressing Tau increased β-cat protein level in N2a cells (transfected with Tau40 or Vec for 48 h; C, D), cultured hippocampal neurons (cultured with 7 div and then transfected with AAV-eGFP-vector or AAV-eGFP-Tau for 5 div; E, F), and in the hippocampi of 10-month-old Tau transgenic mice (Tau+) (G, H) measured by Western blotting (n = at least three biological replicates each group). I. The protein level of β-cat protein (upper panels) was significantly increased in hippocampal cornu ammonis 1(CA1), cornu ammonis 3 (CA3), and dentate gyrus (DG) subsets of 10-month-old Tau transgenic mice (Tau+) compared with Tau knockout mice (Tau−), with significantly increased total Tau (HT7, middle panels) and the phosphorylated Tau (AT8, lower panels). Scale bar, 50 μm. Data information: Data are presented as mean ± SEM; **P < 0.01; ***P < 0.001 vs. Vec (A, B, D, and F) or vs. Tau− (H). Data were analyzed by Student's t-test. Source data are available online for this figure. Download figure Download PowerPoint The N-terminal (1–49 amino acids) of β-catenin is the key region for its stability and the degradation by ubiquitin–proteasome system 30, and there are two lysine (K49 and K19) and four serine/therein (Ser33, Ser37, Thr41, and Ser45) residuals in this region of β-catenin. A recent study shows that Tau has acetyltransferase activity and can promote its self-acetylation 37. We speculate that Tau may acetylate β-catenin and thus affects its phosphorylation and the ubiquitination-related proteolysis. To test this, we first measured the acetylation of β-catenin in Tau-overexpressing systems. By immunoprecipitation, we observed that overexpressing Tau significantly increased acetylation level of β-catenin compared with the empty vector control (Fig 1A and B). To identify the site acetylated by Tau, we used site-specific mutagenesis to replace K49 and K19 with acetylation-resistant arginine (K19R and K49R) in β-catenin. We found that co-expressing K49R or K49R/K19R double mutant almost abolished Tau-induced acetylation whereas mutation at K19R only negligibly changed the acetylation level (Fig 1C and D), suggesting that Tau may predominantly acetylate β-catenin at K49. Indeed, the following studies using K49-specific antibody showed a significantly increased acetylation of β-catenin at K49 in human Tau-expressing N2a cells (Fig EV2A and B), in cultured hippocampal neurons (7 div) infected with AAV-eGFP-Tau for 5 div (Fig EV2C and D), in AAV-eGFP-Tau-infected mouse hippocampus (Fig EV2E), and in human Tau transgenic mice (Fig 1E and F), while expressing the acetyltransferase activity domain-deleted Tau-K18(−) 37 abolished β-catenin acetylation (Fig 1G and H). Furthermore, co-expression of Tau and β-catenin further increased the acetylation level of β-catenin at K49 compared with expressing β-catenin alone (Fig EV2F and G). The remarkably increased levels of total and/or K49-acetylated β-catenin were also detected in the hippocampal extracts of AD patients (Fig 1I and J), in which a positive correlation was detected between the increased phosphorylated Tau at pS199 or AT8 epitopes and the increased K49-acetylated β-catenin (Fig 1K and L). Furthermore, expressing pseudo-phosphorylated Tau (TauS199E) further increased the K49 acetylation of β-catenin (Fig 1M and N). These in vitro and in vivo data together strongly indicate that Tau may preserve β-catenin by enhancing its acetylation at K49 and phosphorylation of Tau enhances its acetyltransferase activity toward K49-β-catenin. Figure 1. Tau acetylates β-catenin at K49 in cells, mice, and human brains, and phosphorylation further increases its acetyltransferase activity A, B. Tau increased β-catenin acetylation (Ace-β-cat) measured by immunoprecipitation using anti-β-cat, or Western blotting using anti-β-cat, anti-acetylated lysine (Ace-lys), and HT7 (probes specifically human total Tau). HEK293 cells transiently transfected with wild-type Tau40 (Tau) or the empty vector (Vec) (n = 3 from three independent experiments). C, D. β-cat mutation at K49 (K49R) but not K19 (K19R) abolished Tau-induced acetylation. HEK293 cells were co-transfected with eGFP-β-cat (WT, or K19R, or K49R, or K19R/K49R) and Tau or the empty vector for 48 h, and then immunoprecipitated using anti-GFP and Western blotting using anti-β-cat, anti-Ace-lys, and HT7 (n = 3 from three independent experiments). E, F. The increased β-cat (K49) acetylation was detected in the hippocampal extracts of human Tau transgenic mice (Tau+) compared with the endogenous Tau knockout mice (Tau−), measured by Western blotting using acetylation site-specific antibody (Ace-β-cat-K49); arrowhead in panel (E) denoted non-specific band identified by molecular weight. The remarkably increased levels of phosphorylated Tau at Ser199 (pS199) and AT8 epitopes were detected in Tau+ mice (at least three mice per group were used). G, H. K18 deletion of Tau (Tau-k18(−)) attenuated Tau-induced β-cat acetylation. HEK293 cells were transfected with empty Vec, or Tau40 or Tau-k18(−) for 48 h, and the ace-β-cat was measured by immunoprecipitation using anti-β-cat and Western blotting using anti-ace-lys, anti-β-cat, and Tau5 (detecting total Tau; n = 3 from three independent experiments). I–L. The increased total and Ace-β-cat (K49) levels positively correlated with increased phosphorylated Tau pS199 and AT8 as detected by Western blotting in the hippocampal extracts of AD patients compared with age-matched controls (n = 10 each group, detailed information for human brain samples was shown in Appendix Table S1). Pearson's analysis was used for correlation analysis (K and L). M, N. Expressing pseudo-phosphorylated Tau (TauS199E) further increased the K49 acetylation of β-catenin. N2a cells were transiently transfected with Vec, Tau40, or TauS199E for 48 h, levels of K49-acetylated β-cat, total β-cat, and total Tau were detected by Western blotting (n = 3 biological replicates each group). O, P. Inhibiting acetyltransferases CBP/P300 by TPOP146 (134 nM) or PCAF by L-45 (126 nM) for 24 h did not significantly decrease the K49-acetylated β-cat level in Tau-overexpressing N2a cells (n = 3 biological replicates each group). Data information: Data are presented as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001 vs. Vec (B, N and P), or vs. β-cat WT (D), or vs. Tau− (F), or vs. Tau (H), or vs. Ctrl (J); #P < 0.05; ##P < 0.01; ###P < 0.001 vs. β-cat WT plus Tau (D), or vs. Tau (N); &&P < 0.01; &&&P < 0.001 vs. β-cat K19R plus Tau (D); the absence of asterisk indicates that the difference is not significant. Data were analyzed by Student's t-test (B, F and J) or one-way ANOVA (D, H, N and P). Source data are available online for this figure. Source Data for Figure 1 [embr201948328-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Tau has a predominant role in acetylating β-catenin at K49 A–D. N2a cells were transfected with Tau40 for 48 h (A, B), or the culture hippocampal neurons (7 div) were transfected with AAV-eGFP-Tau or AAV-eGFP-vector for 5 div (C, D). An increased Ace-β-cat (K49) was detected in the cell extracts by using anti-K49-Ace-β-cat antibody. The arrowhead in panel (A) denoted non-specific bands identified by molecular weight (n = at least three biological replicates each group). E. The increased Ace-β-cat (K49) in C57 mice after overexpressing AAV-eGFP-Tau40 for 1 month tested by immunohistochemical staining (n = 3, scale bars, 100 μm). F, G. Co-expressing Tau40 and β-cat further increased β-cat K49 acetylation measured by Western blotting using Ace-β-cat (K49) antibody in HEK293 cells (n = at least three biological replicates each group); the "upper" denotes exogenous β-catenin, and the "lower" denotes endogenous β-cat. H, I. Inhibiting acetyltransferases CBP/P300 significantly decreased acetylated H4 (Ace-H4) level in N2a cells transfected with Tau40 or Vec for 48 h, and then treated with TPOP146 (134 nM) or L-45 (126 nM) for 24 h (n = 3 biological replicates each group). The samples used for this figure were the same as those used for Fig 1O, and the blots of HT7 and β-actin were shared. J. Application of the inhibitor TPOP146 (134 nM) or L-45 (126 nM) for 1 h did not change β-cat acetylation by Tau measured by Western blotting using anti-Ace-lys in test tube (n = 3 from three independent experiments). K, L. Overexpressing Tau decreased the protein levels of acetyltransferases CBP, P300, and PCAF in N2a cells transfected with Tau40 or Vec for 48 h (n = 3 biological replicates each group). Data information: Data are presented as mean ± SEM; *P < 0.05; **P < 0.01 vs. Vec (B, D, I, and L); or vs. β-cat+vec (G); #P < 0.05 vs. Tau (I); the absence of asterisk indicates that the difference is not significance. Data were analyzed by Student's t-test (B, D, G, and L) or one-way ANOVA (I). Source data are available online for this figure. Download figure Download PowerPoint Several acetyltransferases, including CREB-binding protein (CBP), P300, and p300/CBP-associated factor (PCAF), may also acetylate β-catenin 33-36. To verify whether Tau plays a dominant role in acetylating K49, we used inhibitors (TPOP146 and L-45) of CBP/P300 38 and PCAF 39. Reduction of the acetylated H4 (Ace-H4) confirmed the suppression of CBP, P300, and PCAF activities by TPOP146 or L-45 (Fig EV2H and I). However, inhibiting CBP/P300 or PCAF only slightly decreased the acetylation level of K49-β-catenin by Tau (Fig 1O and P), which suggested a predominant role of Tau in acetylating β-catenin at K49. We also demonstrated that incubating purified Tau with TPOP146 (134 nM) or L-45 (126 nM) for 1 h did not affect its activity in acetylating β-catenin (Fig EV2J). Interestingly, we also observed that overexpressing Tau decreased protein levels of CBP, P300, and PCAF (Fig EV2K and L), which further reduced the contribution of these acetyltransferases in β-catenin acetylation at least in this Tau-overexpressing system. Tau directly acetylates β-catenin in concentration-, time-, pH-, and the acetyltransferase activity domain-dependent manner To confirm the direct effect of Tau on β-catenin acetylation at K49 and characterize its enzymatic kinetics, we purified wild-type Tau and the acetyltransferase activity domain-deleted or domain-mutated Tau, i.e., Tau-k18(−) and Tau-2CA (Fig EV3A) 37, the wild-type β-catenin and β-catenin K49R proteins from eukaryocyte (HEK293) and prokaryocyte (Escherichia coli), respectively (Fig EV3B). Then, we incubated in test tube the purified wild-type Tau with wild-type β-catenin and confirmed that Tau could directly acetylate β-catenin (Fig 2A). However, incubating Tau-k18(−) or Tau2CA with wild-type β-catenin (Fig 2B and C) and incubating wild-type Tau with β-catenin K49R abolished the acetylation of β-catenin by Tau (Fig 2D and E). These data confirm that Tau can directly acetylate β-catenin at K49 with activity domain-dependent manner. Further kinetic studies showed that Tau could acetylate β-catenin K49 in concentration-, time-, and pH-dependent manner (Fig 2F–H), and the Km value for Tau and β-catenin interaction was 5.13 μM (Fig 2I and J). The association of Tau with β-catenin was also detected in HEK293 cells with stable or transient expression of Tau (Fig EV3C and D) and in mouse hippocampus (Fig EV3E and F) measured by immunoprecipitation and co-localization by immunofluorescence staining in cultured primary hippocampal neurons (Fig EV3G). These data confirm that Tau can directly acetylate β-catenin with concentration, pH, and time and its activity domain dependence. Click here to expand this figure. Figure EV3. Affinity-purified Tau and β-catenin proteins and the association of Tau with β-catenin in vitro and in vivo A. The schematic shows full-length Tau (Tau40), and the acetyltransferase activity domain double-mutated Tau (cystine-291/322 to alanine, Tau-2CA) or the activity domain-deleted Tau (Tau-k18(−)). B. The representative Coomassie blue staining pattern of the affinity-purified β-cat WT, β-cat K49R, Tau40, Tau-2CA, and Tau-k18(−) from Escherichia coli using Ni-NTA resin. C, D. Association of Tau with β-cat in vitro. HEK293 cells with stable e
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