Research Article: Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG)

Date Published: June 17, 2015

Publisher: Public Library of Science

Author(s): Hanyong Chen, Ke Yao, Xiaoyu Chang, Jung-Hyun Shim, Hong-Gyum Kim, Margarita Malakhova, Dong-Joon Kim, Ann M. Bode, Zigang Dong, A R M Ruhul Amin.


The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.

Partial Text

For thousands of years, tea has been the most widely consumed beverage in the world. Historically, tea has been credited with various beneficial health effects, including medicinal efficacy in the prevention and treatment of numerous diseases [1, 2]. A number of epidemiological studies have shown that tea exerts cancer preventive activity at a variety of organ sites, including skin, lung, esophagus, colon, and pancreas [3, 4]. The polyphenols from green and black tea, epigallocatechin-3-gallate (EGCG) and theaflavins, respectively, are generally considered to be the most active and effective components of tea for inhibiting carcinogenesis [5]. EGCG is the major polyphenol in green tea and may account for 50 to 80% of the total catechins. A cup of green tea contains 100–200 mg of EGCG [6]. Previous reports showed that tea polyphenols inhibited 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and epidermal growth factor (EGF)-induced cell transformation [7]. Several proteins that can directly bind with EGCG have been identified, including Bcl-2, AMP-activated protein kinase, the 67-kDa laminin receptor, Ras-GTPase-activating protein SH3 domain-binding protein 1, glucose-regulated protein 78 and vimentin [8–14]. Recently, the human peptidyl prolyl cis/trans isomerase (Pin1), a downstream effecter of oncogenic Neu/Ras signaling, was crystallized with EGCG at 1.9Å resolution [15]. Even though several protein targets have been identified, the precise mechanisms responsible for the reported health effects of EGCG are still not very well understood. Searching for the EGCG “receptor” or high affinity proteins that bind to EGCG is believed to be an important first step in understanding the molecular and biochemical mechanisms of the health effects of tea polyphenols. In our study, computational docking and shape screening methods were used with our in-house database of kinases to screen for targets of EGCG. Interestingly, the results indicated that both the C-terminal (CTD) and N-terminal (NTD) of ribosomal S6 kinase 2 (RSK2) were in the top 10 of the list as targets of EGCG. RSK2 is a member of the p90RSK protein family that is activated by ERK1/2 and PDK1 (phosphoinositide-dependent kinase 1) [16]. RSK2 translocates to the nucleus when activated by growth factors, peptide hormones, or neurotransmitters. Once in the nucleus, RSK2 can phosphorylate various nuclear proteins, including several histones, activating transcription factor 4, p53, and nuclear factor of activated T-cells [17]. Based on its broad substrate specificity, the RSK2 protein is likely to mediate a variety of cellular processes, including proliferation as well as transformation. Our recent study provided evidence indicating that RSK2 plays an important role in cell transformation induced by tumor promoters such as EGF and TPA [18]. Therefore, deciphering the molecular activation mechanism of RSK2 is extremely important for understanding how to control RSK2 activity. In this study, we demonstrated that EGCG directly targets RSK2 at both kinase catalytic domains and inhibits RSK2 kinase activity in a dose-dependent manner. Moreover, we suggest that the inhibition of cell growth and EGF-induced cell transformation requires RSK2. Overall, these results indicate that EGCG is a novel natural compound that effectively suppresses RSK2 activity.

Cellular kinase-signaling networks are a major regulator of cancer progression and are often involved in pathogenesis. Kinase mutations are relatively common and potent drivers of oncogenesis [34, 35]. Targeting a single kinase has proven successful in some cases, such as the inhibition of EGFR [36]. However, results of this approach have been mixed. Difficulties include rapidly emerging resistance as well as considerable toxicity that can limit dosing to levels that are insufficient for blocking tumor growth [37, 38]. The complexity of cancer has led to recent interest in poly-pharmacological approaches for developing drugs that inhibit kinase activity [23].