YAF2 exerts anti-apoptotic effect in human tumor cells in a FANK1- and phosphorylation-dependent manner
Introduction
The polycomb group (PcG) proteins play crucial roles in epigenetic regulation by using its histone modifying activities. PcG proteins mainly fall into two multisubunit complexes, namely the Polycomb repressive complex 1 (PRC1) and 2 (PRC2), which act synergistically to control gene expressions during organism development [1]. PRC1 consists of two subtypes: canonical PRC1 (CBX–PRC1) and non-canonical PRC1 (nc-PRC1, including RYBP–PRC1 and YAF2–PRC1) [2]. The former is recruited to H3K27me3 by CBXs to further catalyze monoubiquitination of adjacent histone H2A at lysine 119 (H2AK119Ub1) and target gene repression, whereas nc-PRC1 itself exhibits strong catalytic activity toward H2AK119 monoubiquitination and this modification is further recognized by RYBP or YAF2 to enroll other members of the nc-PRC1 [3,4].
In addition to modulate gene expression as a critical nc-PRC1 member, YAF2 as a RYBP family member was also reported to interact with a number of transcriptional factors to modulate target gene transcriptions. As a result, YAF2 plays vital roles during embryonic development, organogenesis, stem cell maintenance, and so on [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14]].
Contradicting results have been reported regarding to apoptosis-inducing activity of YAF2. During early zebrafish embryo development and organogenesis, YAF2 was reported to inhibit caspase-8-mediated apoptosis [12]. However, evidence also showed that YAF2 activates p53 and induces cell apoptosis by stabilizing PDCD5 [15]. To date, in spite of some progresses, we still know little about YAF2, especially its posttranslational modifications and functions.
We previously found that FANK1 is a binding partner of RYBP, and RYBP up-regulates FANK1 protein stability, activates AP-1 signaling pathway and induces tumor cell apoptosis [16]. Since YAF2 has high protein sequence homology to RYBP, we wonder whether YAF2 also interacts with and regulates tumor cell apoptosis through targeting FANK1 expression. Here, we provide evidences to support that YAF2 was phosphorylated at serine 167 and its phosphorylated form induced the accumulation of FANK1 protein through acting on ubiquitin-proteasome system. We finally demonstrated that YAF2 inhibits FANK1-mediated tumor cell apoptosis in a S167 phosphorylation-dependent manner. Thus, our current findings provide the basis for the discovery of novel anti-cancer drugs which target the S167 phosphorylation of YAF2.
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Cell lines and cell culture
HEK293T, HeLa, HCT116 and MCF7 cells were from Cell Resource Center from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. YAF2-null HeLa cell line was constructed in our lab. All the cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in 5% CO2 incubators at 37 °C.
Vector constructions
pFlag-FANK1 expression vectors and its truncated mutants were described previously [16]. Full-length YAF2 isoform 2 (coding 180 amino acid
The identification of YAF2 phosphorylation site
In our preliminary experiments, we always noticed two closely spaced bands when detecting either endogenous or exogenous YAF2 protein level by Western blotting using anti-YAF2 antibody or its tag antibody in HeLa, HCT116, or HEK293T cells. However, when the cellular lysates were pre-treated with ALP before electrophoresis, the band with higher molecular weight disappeared, whereas the band intensity with lower weight increased (Fig. 1A). These results indicated that YAF2 undergoes
Discussion
In this study, we identified and confirmed that YAF2 was phosphorylated at S167 and this modification increased the stability of FANK1 protein by inhibiting the proteasomal degradation of polyubiquitinated FANK1. We confirmed that FANK1 was a novel interacting partner of YAF2 and meanwhile mapped their binding domains. Subsequent studies established that YAF2 inhibited tumor cell apoptosis in a phosphorylation-dependent manner and FANK1 is required during this process. Thus, this study finds a
Author contributions
HC, BRH and DC conceived the study. DC designed experiment and revised the manuscript. SQZ, XZ and XG performed the experiments. SQZ wrote the manuscript. XLM cultured cells and provided technical help.
Declaration of competing interest
The authors declare that they have no conflicts of interest.
Acknowledgements
This project was supported by National Natural Science Foundation of China (81772986) and the CAMS Initiative for Innovative Medicine (2017-I2M-3-004).
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