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PAK4 Phosphorylates Fumarase and Blocks Tgfb

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PAK4 Phosphorylates Fumarase and Blocks Tgfb Published OnlineFirst January 25, 2019; DOI: 10.1158/0008-5472.CAN-18-2575 Cancer Metabolism and Chemical Biology Research PAK4 Phosphorylates Fumarase and Blocks TGFb- Induced Cell Growth Arrest in Lung Cancer Cells Tao Chen1, Ting Wang2,3, Wenhua Liang1, Qin Zhao2, Qiujing Yu2, Chun-Min Ma2, Lingang Zhuo2, Dong Guo2, Ke Zheng2, Chengzhi Zhou1, Shupei Wei1, Wenhua Huang1, Juhong Jiang1, Jing Liu1, Shiyue Li1, Jianxing He1,Yuhui Jiang2, and Nanshan Zhong1 Abstract The metabolic activity of fumarase (FH) participates in gene formation. Physiologically, FH Ser 46 phosphorylation pro- transcription linking to tumor cell growth. However, whether motes tumorigenesis through its suppressive effect on FH Thr this effect is implicated in lung cancer remains unclear. Here, 90 phosphorylation–mediated cell growth arrest in NSCLC we show TGFb induces p38-mediated FH phosphorylation at cells and correlates with poor prognosis in patients with lung Thr 90, which leads to a FH/CSL (also known as RBP-Jk)/p53 cancer. Our findings uncover an uncharacterized mechanism complex formation and FH accumulation at p21 promoter underlying the local effect of FH on TGFb-induced gene under concomitant activation of Notch signaling; in turn, transcription, on which the inhibitory effect from PAK4 pro- FH inhibits histone H3 Lys 36 demethylation and thereby motes tumorigenesis in lung cancer. promotes p21 transcription and cell growth arrest. In addition, FH is massively phosphorylated at the Ser 46 by PAK4 in Significance: Fumarase counteracts CSL via its meta- non–small cell lung cancer (NSCLC) cells, and PAK4- bolic activity to facilitate TGFb-induced cell growth phosphorylated FH binds to 14-3-3, resulting in cytosolic arrest, an effect largely blocked by PAK4-mediated phos- detention of FH and prohibition of FH/CSL/p53 complex phorylation of fumarase. Introduction factor 6 (TRAF6)-TGFb–associated kinase 1 (TAK1) is critical for p38 activation in response to TGFb (6, 9). Functionally, previous TGFb is a key regulator of cell fate during embryogenesis findings demonstrated p38 activation is essential for either TGFb- and has also been implicated in tumor metastasis as a potent induced cellular apoptosis or epithelial-to-mesenchymal transi- driver (1–3). Canonically, TGFb stimulation leads to activation of tion (EMT; refs. 10, 11), although how its multifaceted effects are signal transduction through transmembrane type I and type II coordinated remains elusive. serine/threonine kinase receptors (TbRI and TbRII, respectively), Crosstalk between Notch and TGFb signaling pathway has which phosphorylates Smad2/Smad3 and promotes their com- been implicated in various physiologic events. TGFb stimula- plex formation with Smad4 and launches their transcriptional tion is able to induce expression of Jagged1, which promotes activity (4, 5). In addition, TGFb is also able to initiate a number of EMT (12), and Notch activation is found to be indispensable non-Smad signaling pathways including p38 pathway, PI3K for TGFb-mediated EMT during cardiac development and pathway, and the Ras–ERK–MAPK pathway (6–8). It has been tumorigenesis (13). HDAC6 has been reported to be required known that the axis of ubiquitin ligase TNF-receptor-associated for activation of Notch signaling by TGFb1 during EMT in lung cancer (14). Referring to downstream transcriptional factor, 1State Key Laboratory of Respiratory Diseases; National Clinical Research Center NICD and Smad3 can form physical complex and cooperate to of Respiratory Diseases; Guangzhou Institute of Respiratory Health; First Affil- drive the activation of synthetic promoters containing multi- iated Hospital of Guangzhou Medical University, Guangzhou Medical University, merized CSL- or Smad3-binding sites (15). In terms of p21, Guangzhou, Guangdong, P.R. China. 2The Institute of Cell Metabolism and p53 plays a positive role in TGFb-induced p21 expression and Disease, Shanghai Key Laboratory of Pancreatic Disease, Shanghai General cell growth arrest at normal condition (16). A recent study Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, P.R. China. shows that CSL, which is the crucial transcriptional factor 3Department of Pharmacology, College of Basic Medical Sciences, Tianjin Medical University, Tianjin, China. under Notch signaling, blocks cell senescence through its inhibitory effects on p53-mediated p21 transcription at the Note: Supplementary data for this article are available at Cancer Research epigenetic level (17); this observation implies the regulatory Online (http://cancerres.aacrjournals.org/). effect of CSL on p53 is a potential linkage between TGFb and T. Chen, T. Wang, and W. Liang contributed equally to this article. Notch signaling. Corresponding Authors: Yuhui Jiang, The Institute of Cell Metabolism and Emerging evidence in recent years indicate that metabolic Disease, Shanghai Key Laboratory of Pancreatic Disease, Shanghai 200080, enzymes, in addition to the basic role in metabolic pathways, China. Phone: 8602-1377-97983; E-mail: [email protected]; and Jianxing also vitally participate in the epigenetic regulation through related He, [email protected] metabolic activity (18). Fumarase (FH), which separately distri- doi: 10.1158/0008-5472.CAN-18-2575 butes in mitochondria and cytosol, is responsible for the revers- Ó2019 American Association for Cancer Research. ible hydration and dehydration of fumarate to malate in the www.aacrjournals.org 1383 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst January 25, 2019; DOI: 10.1158/0008-5472.CAN-18-2575 Chen et al. tricarboxylic acid cycle (19). Fumarate has been known to com- Materials pete with a-ketoglutarate to bind to a-ketoglutarate– Antibodies that recognize FH (ab110286), PAK4 (ab62509), dependent dioxygenases that are involved in histone and DNA CSL (ab25949), KDM2A (ab31739), H3 (ab1791), H3K36me2 demethylation, and thus exerts inhibitory effects (20). FH defi- (ab9049), Lamin B1 (ab65986), Smad3 (ab28379), 14-3-3 zeta ciency causes elevation of cellular fumarate level, which has been were purchased from Abcam. Antibodies that recognize p53 implicated in the development of renal cancer due to its inhibitory (sc-126) were obtained from Santa Cruz Biotechnology. Antibo- effect on TET-regulated DNA demethylation (21). FH is also dies against FH (#32975), Flag (#35535), His (#T505), and GST reported to be importantly involved in DNA repair (22, 23) or (#T509) were obtained from Signalway Antibody. Antibodies gene transcription in glucose-deficient conditions (24), in which against Flag (F3165), GST (G1160), and His (SAB4301134) were chromatin-associated FH precisely regulates H3K36me2 at the purchased from Sigma. Antibodies against Phospho-MAPKAPK-2 region of DNA breaks or gene promoter by antagonizing a-KG– (#3044), Phospho-JunB (D3C6, #8053), p38 (D13E1, #8690), dependent demethylase KDM2A/B. Given the tight relationship Phospho-p38 (Thr180/Tyr182; D3F9, #4511), Phospho-Akt between the functional status of FH and epigenetic regulation, it (Ser473; D9E, #4060), Phospho-SMAD2 (Ser465/Ser467; E8F3R, would be meaningful to extensively understand the concrete role #18338), Cleaved Notch1 (D3B8, #4147), SMRT (D8D2L, of FH in this regard under distinct genetic contexts. #62370), b-actin (#4970) were purchased from Cell Signaling In this study, we first found that TGFb induces the interaction Technology. FITC-labeled antibody against BrdU (11-5071) was between FH and CSL in a manner dependent on Notch signaling purchased from eBioscience. activation and p38-mediated phosphorylation of FH. This inter- Rabbit polyclonal FH pThr-90 and FH pSer-46 antibodies was action results in the formation of FH–CSL–p53 complex, and generated by Signalway Antibody. A peptide containing pThr-90 facilitates the recruitment of FH at the promoter of p53-targeted or FH pSer-46 was injected into rabbits. The rabbit serum was gene p21. Through inhibition of KDM2A activity, fumarate locally collected and purified using an affinity column with nonpho- produced from promoter-associated FH leads to the increase of sphorylated FH Thr-90 and Ser-46 peptide to exclude the anti- H3K36me2 and hence p21 transcription, which eventually relives bodies for nonphosphorylated FH, followed by an affinity col- the negative effects of CSL on p53-mediated cell growth arrest. umn with phosphorylated FH Thr-90 and Ser-46 peptide to bind Moreover, we found that FH is largely phosphorylated by PAK4 at to and purify the FH pThr-90 and FH pSer-46 antibody. The FH Ser 46 in lung cancer cells, and 14-3-3 binds to PAK4 phosphor- pThr-90 and FH pSer-46 antibodies were then eluted and ylated-FH, thus retaining FH in the cytosol and blocking its effect concentrated. on CSL–p53 axis. SP600125 and SB203580 were purchased from Cell Signal- ing Technology. AKT inhibitor and U0126 were purchased from EMD Biosciences. PAK4 inhibitor PF-3758309 was obtained Materials and Methods from Selleck Chemicals. Anti-FLAG M2 beads, alkaline phos- Cell culture phatase, and purified His-PAK4 were purchased from Abcam. Human normal bronchial epithelial cell line HBE and human TGFb was obtained from R&D Systems. Compound C, mono- NSCLC cell lines A549, NCI-H520, HCC-827, and NCI-H358, ethyl fumarate, diethyl-malate, BrdU, EDTA-free Protease were obtained from Shanghai Cell Bank (Shanghai Biological Inhibitor Cocktail, and PhosSTOP were purchased from Sigma. Sciences, Chinese Academy of Sciences, Shanghai, China) that Hygromycin,
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