SUMOylation at K340 inhibits tau degradation through deregulating its phosphorylation and ubiquitination Hong-Bin Luoa,1, Yi-Yuan Xiaa,1, Xi-Ji Shub,1, Zan-Chao Liua, Ye Fenga, Xing-Hua Liua, Guang Yua, Gang Yina, Yan-Si Xionga, Kuan Zenga, Jun Jianga, Keqiang Yec, Xiao-Chuan Wanga,d,2, and Jian-Zhi Wanga,d,2 aDepartment of Pathophysiology, School of Basic Medicine, Key Laboratory of Ministry of Education of China for Neurological Disorders, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China; bDepartment of Pathology and Pathophysiology, School of Medicine, Jianghan University, Wuhan 430056, China; cDepartment of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322; and dDivision of Neurodegenerative Disorders, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, JS 226001, China Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 15, 2014 (received for review September 11, 2014) Intracellular accumulation of the abnormally modified tau is hall- tau that mimics tau cleaved at Asp421 (tauΔC) is removed by mark pathology of Alzheimer’s disease (AD), but the mechanism macroautophagic and lysosomal mechanisms (13). Lysosomal leading to tau aggregation is not fully characterized. Here, we stud- perturbation inhibits the clearance of tau with accumulation and ied the effects of tau SUMOylation on its phosphorylation, ubiquiti- aggregation of tau in M1C cells (14). Cathepsin D released from nation, and degradation. We show that tau SUMOylation induces lysosome can degrade tau in cultured hippocampal slices (15). In- tau hyperphosphorylation at multiple AD-associated sites, whereas hibition of the autophagic vacuole formation leads to a noticeable site-specific mutagenesis of tau at K340R (the SUMOylation site) or accumulation of tau (14). Studies also suggest that tau protein is simultaneous inhibition of tau SUMOylation by ginkgolic acid abol- degraded in an ubiquitin-, ATP-, and 26S proteasome-, but not ishes the effect of small ubiquitin-like modifier protein 1 (SUMO-1). a 20S proteasome-dependent manner under normal conditions Conversely, tau hyperphosphorylation promotes its SUMOylation; (16). When the cells are exposed to the stresses, CHIP, a ubiquitin the latter in turn inhibits tau degradation with reduction of solubil- ligase that interacts directly with Hsp70/90, can induce tau ity and ubiquitination of tau proteins. Furthermore, the enhanced ubiquitination and thus selectively reduce the level of detergent SUMO-immunoreactivity, costained with the hyperphosphorylated insoluble tau (17). The compensatory activation of autophagy- NEUROSCIENCE tau, is detected in cerebral cortex of the AD brains, and β-amyloid lysosomal or ubiqutin-proteasomal system can antagonize tau exposure of rat primary hippocampal neurons induces a dose- aggregation; therefore, tau accumulation does not show in the dependent SUMOylation of the hyperphosphorylated tau. Our early stage of AD. During the evolution of AD, a gradual impair- findings suggest that tau SUMOylation reciprocally stimulates ment of autophagy-lysosomal system and ubiqutin-proteasomal its phosphorylation and inhibits the ubiquitination-mediated tau system has been detected at later stage of the disease (18–20). degradation, which provides a new insight into the AD-like tau Studies suggest that the ubiquitin-mediated degradation pathway accumulation. seems ineffective in removing the tau-positive fibrillar structures in the AD brains (21–23); however, the mechanisms underlying SUMOylation | tau | phosphorylation | ubiquitination | degradation the impairment of the ubiqutin-proteasomal system are elusive. Ubiquitin is an important component of the cellular defense lzheimer’s disease (AD) is the most common neurodegen- system that tags abnormal proteins for their degradation by ATP- Aerative disorder in the elderly. Intracellular accumulation of dependent nonlysosomal proteases (24). Monoclonal antibodies neurofibrillary tangles (NFTs) and extracellular precipitation of 3-39 and 5-25 raised against paired helical filaments of NFTs have senile plaques are the most prominent pathological hallmarks been shown to recognize ubiquitin (25). Meanwhile, tau can be of AD (1–3). The clinical-to-pathological correlation studies sumoylated at K340 in vitro by SUMO-1 (small ubiquitin-like have demonstrated that the number of NFTs consisting of hyper- modifier protein-1) and to a lesser extent by SUMO2 and SUMO3 phosphorylated tau correlates with the degree of dementia in (9–11). Moreover, SUMO-1 immunoreactivity was colocalized AD (4–6). Tau is the major microtubule-associated protein that normally contains 2–3 mol of phosphate per mole of tau protein. Significance In AD brains, tau is abnormally hyperphosphorylated (namely – AD-P-tau) and the phosphate level increases to 5 9 mol phos- Intracellular accumulation of the abnormally modified tau is phate per mole tau (4). AD-P-tau does not bind to tubulin and hallmark pathology of AD, but the mechanism leading to tau become incompetent in promoting microtubule assembly and aggregation is not fully characterized. In the present study, we maintaining the stability of the microtubules. The AD-P-tau also studied the effects of tau SUMOylation on its phosphorylation, sequesters normal tau from microtubules (7), and serves as a ubiquitination, degradation, and aggregation. We discovered template for the conversion of normal tau into misfolded protein that sumoylation competes with ubiquitination in modifying in a prion-like manner (8). In addition to hyperphosphorylation, tau, correlating with tau hyperphosphorylation. Identification tau is also contains other posttranslational modifications, such as – of the posttranslational modification on tau provides the new ubiquitination and SUMOylation (5, 9 11). The abnormal mod- insight into the molecular mechanism in tau aggregation. ification of tau also decreases its solubility, and ∼40% of the hyperphosphorylated tau in AD brains has been isolated as sedi- Author contributions: X.-C.W. and J.-Z.W. designed research; H.-B.L., Y.-Y.X., X.-J.S., Z.-C.L., mentable nonfibril cytosolic protein (1, 12). Although the mech- Y.F., X.-H.L., G. Yu, G. Yin, Y.-S.X., K.Z., J.J., and X.-C.W. performed research; H.-B.L., Y.-Y.X., anisms underlying the formation of the NFTs remain unclear, the K.Y., X.-C.W., and J.-Z.W. analyzed data; and X.-C.W. and J.-Z.W. wrote the paper. altered tau modifications and impaired degradation are believed The authors declare no conflict of interest. to play a role. Therefore, clarifying the mechanism that may cause This article is a PNAS Direct Submission. tau accumulation is of great significance for understanding the 1H.-B.L., Y.-Y.X., and X.-J.S. contributed equally to this work. pathogenesis of AD and for developing new therapeutics. 2To whom correspondence may be addressed. Email: [email protected] or Like other proteins, tau can be degraded by autophagy-lyso- [email protected]. somal and ubiqutin-proteasomal systems under physiological con- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ditions. In mouse cortical neurons, a C-terminal–truncated form of 1073/pnas.1417548111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1417548111 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 with tau aggregates in neuritic plaques of APP transgenic mice of SUMO-1 into HEK293/tau cells. Phosphorylation of tau at (11). It is well known that SUMO share similarities with ubiquitin Thr205, Ser262, and Ser396 was gradually reduced with a de- in both the structure and the biochemistry of their conjugation creasing SUMO-1 staining after GA treatment (Fig. S1). In- (26). Therefore, tau SUMOylation may compete against its ubiq- terestingly, the ∼95-kDa (55 kDa tau + 40 kDa GFP-SUMO) uitination and thus suppress tau degradation. In the present study, band was only seen in the cells expressing wild-type tau but not we found that tau SUMOylation reciprocally stimulates its phos- those expressing K340R tau (Fig. 1A), indicating that the ∼95-kDa phorylation and thus inhibits the ubiquitination and degradation of band may represent the SUMOylated tau. Both antibodies against tau proteins. total tau antibody (Tau5) and SUMO-1 antibody reacted with the 95-kDa band, confirming that the 95-kDa band represented the Results SUMOylated tau (Fig. 1C). Immunofluorescence staining dem- Tau SUMOylation Promotes Tau Phosphorylation. To explore the onstrated that the cells transfected with SUMO-1 (green) showed effect of tau SUMOylation on its phosphorylation, we first higher level of tau phosphorylation compared with untransfected D E overexpressed eGFP-labeled SUMO-1 or the vector in HEK293/ cells (Fig. 1 and ). These data together strongly suggest that tau cells that stably express the longest isoform of human tau for SUMOylation promotes tau phosphorylation. 24 h and analyzed the phosphorylation level of tau by Western Tau Phosphorylation Stimulates Tau SUMOylation. To explore blotting. Compared with the controls, expression of SUMO-1 whether tau phosphorylation reciprocally affects its SUMOyla- significantly increased tau phosphorylation at Thr-205, Ser-214, tion, we induced tau hyperphosphorylation by treated the cells Thr-231, Ser-262, Ser-396, and Ser-404 sites, and a 95-kDa band with okadaic acid (OA; 20 nM) to inhibit PP2A (28) [the crucial A B was found (Fig. 1 and ). phosphatase