Myristoylation-mediated phase separation of EZH2 compartmentalizes STAT3 to promote lung cancer growth
Introduction
N-myristoylation is a lipid modification process through which a 14-carbon saturated myristic acid is covalently added to the amine group of the N-terminal glycine of different cellular proteins, such as oncoproteins [1]. In addition to the well-known membrane anchoring function, myristoylation mediates protein-protein or protein-lipid interactions, controls protein stability, and regulates transcription [[2], [3], [4], [5]]. In particular, myristoylation drives homo- or hetero-multimerization of modified proteins via hydrophobic interactions [[6], [7], [8]]. The importance of multiple myristoyl-oncoproteins in regulating tumorigenesis is well established [9,10]. The consensus sequence for myristoylation is assumed to be MGXXXSX, in which the G2 residue is essential for myristoylation, while the S6 residue is not a strict requirement [11]. The presence of a glutamine or an asparagine residue at the third position (Q3 or N3) is an alternate feature of myristoylated proteins lacking S6 [12,13]. In addition, a lysine residue at the seventh position (K7) also favors myristoylation [14,15]. Several studies based on large-scale and complementary structural approaches have provided a comprehensive list of myristoylated substrates in different species [11,16,17]. However, a large proportion of predicted myristoylated targets await experimental validation [18]. Therefore, unveiling novel myristoylated proteins is important for understanding their involvement in physiological and pathological processes.
A growing body of evidence points to a pivotal role of protein liquid-liquid phase separation (LLPS) in a wide range of biological processes, including carcinogenesis [[19], [20], [21], [22], [23]]. LLPS is driven by physical interactions between biomolecules. LLPS enables biomolecules to form a highly condensed liquid droplet-like phase, thereby providing an extremely efficient platform for the reaction. Hydrophobic interaction is one of the major factors driving LLPS, because a compound known to disrupt hydrophobic interactions, 1,6-hexanediol, is widely used to inhibit LLPS. Moreover, a number of proteins that form LLPS droplets are more efficient under high salt concentrations and high temperatures, which favor hydrophobic interactions [24,25]. LLPS is regulated through various modifications, such as lysine methylation and acetylation, arginine methylation, and serine and tyrosine phosphorylation [26]. However, little is known about the involvement of long fatty acylation in the formation of LLPS droplets. Based on its hydrophobic property and oligomerization capability, protein myristoylation could be a possible contributor to LLPS.
Enhancer of zeste homolog 2 (EZH2) is a lysine methyltransferase that targets a variety of substrates, including H3K27, AR and STAT3 [27,28]. EZH2 plays an oncogenic role in both histone methylation-dependent and independent manners and is an important anticancer drug target. Tazemetostat is a first-in-class EZH2 inhibitor that was recently approved for the treatment of sarcoma [29]. STAT3 is a bona fide substrate of EZH2 in oncogenic signaling. EZH2 interacts with STAT3 and methylates it at K180, enhancing STAT3 Y705 phosphorylation, a hallmark of STAT3 activation, resulting in elevated tumorigenicity of glioblastoma stem-like cells [28]. EZH2-mediated STAT3 K180 methylation has also been implicated in psoriasis, head and neck cancer, and lung cancer [[30], [31], [32]]. In addition, the K49 residue of STAT3 is methylated by EZH2, and this methylation is crucial for the transcriptional activity of STAT3 in response to IL-6 [33]. Interestingly, the interaction between EZH2 and STAT3 was largely enhanced by EZH2 S21 and T345 phosphorylation catalyzed by AKT and CDK4/6, respectively [28,31]. In contrast, AMPK catalyzes EZH2 T311 phosphorylation and negatively regulates methyltransferase activity as well as the oncogenic performance of EZH2 [34]. Besides phosphorylation, EZH2 is also regulated by methylation, acetylation, ubiquitination, and particularly, palmitoylation [[35], [36], [37]]. The presence of a G2 residue in EZH2 enables its myristoylation.
The N-terminus of EZH2 mediates protein-protein interactions and plays an important role in tumorigenesis [38]. The noncanonical H3K27 methylation-independent oncogenic function of EZH2 is likely to be achieved by N-terminal S21 phosphorylation and the subsequent EZH2-STAT3 interaction, as suggested by multiple studies [27,28,30]. Here, we provide evidence that EZH2 can be myristoylated at the N-terminal glycine in lung cancer cells. We show that myristoylation enables EZH2 to form phase-separated droplets in vitro and liquid-like nuclear puncta in lung cancer cells. Moreover, STAT3 is shown to co-localize with EZH2 in these puncta. The interaction between EZH2 and STAT3 is also enhanced by EZH2 myristolyation, leading to elevated STAT3 Y705 phosphorylation and increased STAT3 transcriptional activity. Furthermore, myristoylation of EZH2 promotes in vivo lung cancer cell growth. These findings provide a rationale for targeting EZH2 myristoylation in lung cancer intervention.
Section snippets
Reagents and cell culture
Reagents for click chemistry assay, including alkyne myristic acid, biotin picolyl azide, and Click-&-Go™ protein reaction buffer, were purchased from Click Chemistry Tools (Scottsdale, AZ, US). Streptavidin-horseradish peroxidase (HRP) was obtained from Beyotime (Shanghai, China). Anti-MYC magnetic beads were obtained from Bimake (Shanghai, China) and Anti-FLAG M2 affinity gels were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used for detection of the
EZH2 can be myristoylated at Gly2 in cells
The H3K27 methylation-independent oncogenic function of EZH2 has been documented in multiple cancers, including prostate cancer, glioblastoma, head and neck cancer, and lung cancer [27,28,30,32]. This H3K27 methylation-independent function of EZH2 appears to be specific to the N-terminal S21 phosphorylation, suggesting the potential importance of its N-terminal region. Moreover, the N-terminal sequence of EZH2 is not only evolutionarily conserved (Fig. 1A) but also matches two alternative
Discussion
Protein N-myristoylation is essential for cell signaling transduction by promoting protein-protein and protein-lipid interactions. Although proteins within the nuclear interior are integral parts of the cell signaling machinery, only a few myristoylated proteins have been identified in the nucleus [18]. In the present study, we demonstrated that EZH2, a nucleus-resident protein, can be modified by myristoylation at its G2 residue in lung cancer cells. Although the N-terminal sequence of EZH2
Authors' contributions
MW, YEC, JZ, and YZ conceived and designed the study; JZ, YZ, YX, XL, LZ, LH, and MW performed the experiments and analyzed the data; MW, YEC, LH, JZ, YZ, and YX wrote the paper. All authors read and approved the final manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions and by grants 31801058, 81802885, 81820108023, 31771608, 2018YFC1705505, and 2016YFC1302402 from the China Natural Science Foundation, grant BK20180839 from the Natural Science Foundation of Jiangsu Province, China. L.Z. was supported by Hui-Chun Chin and Tsung-Dao Lee Chinese Undergraduate Research Endowment (CURE).
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J. Zhang and Y. Zeng are co-first authors of this article.