Eriodictyol Inhibits RSK2-ATF1 Signaling and Suppresses EGF-induced Neoplastic Cell Transformation

Abstract

RSK2 is a widely expressed serine/threonine kinase, and its activation enhances cell proliferation. Here, we report that ATF1 is a novel substrate of RSK2 and that RSK2-ATF1 signaling plays an important role in EGF-induced neoplastic cell transformation. RSK2 phosphorylated ATF1 at Ser-63 and enhanced ATF1 transcriptional activity. Docking experiments using the crystal structure of the RSK2 N-terminal kinase domain combined with in vitro pulldown assays demonstrated that eriodictyol, a flavanone found in fruits, bound with the N-terminal kinase domain of RSK2 to inhibit RSK2 N-terminal kinase activity. In cells, eriodictyol inhibited phosphorylation of ATF1 but had no effect on the phosphorylation of RSK, MEK1/2, ERK1/2, p38 or JNKs, indicating that eriodictyol specifically suppresses RSK2 signaling. Furthermore, eriodictyol inhibited RSK2-mediated ATF1 transactivation and tumor promoter-induced transformation of JB6 Cl41 cells. Eriodictyol or knockdown of RSK2 or ATF1 also suppressed Ras-mediated focus formation. Overall, these results indicate that RSK2-ATF1 signaling plays an important role in neoplastic cell transformation and that eriodictyol is a novel natural compound for suppressing RSK2 kinase activity. RSK2 (ribosomal S6 kinase 2) is a member of the p90 RSK protein family that is activated by ERK1/2 and PDK1 (phosphoinositide-dependent kinase 1) (1, 2). RSK2 translocates to the nucleus when activated by growth factors, peptide hormones, or neurotransmitters (3, 4). Numerous proteins, such as the cAMP response element (CRE) 3 -binding protein (CREB), Elk-1, histones (5-9), ATF4 (activating transcription factor 4) (10), p53 (11), and NFAT3 (12), are phosphorylated by active RSK2. Based on its broad substrate specificity, the RSK2 protein mediates many cellular processes, including proliferation and transformation, as well as the cell cycle. Our recent study provided evidence indicating that RSK2 plays an important role in cell transformation induced by tumor promoters such as EGF and 12-O-tetradecanoylphorbol-13-acetate (13). Furthermore, RSK2 knock-out mice display reduced c-Fos-dependent osteosarcoma formation through the regulation of c-Fos protein stability (14). Thus, RSK2 likely plays a key role in cell proliferation and transformation.ATF1 is a member of the CREB family, which includes ATF1, CREB1, and the CRE modulator (15). In response to growth factors, stress signals, neurotransmitters, or other agents that elevate intracellular cAMP or Ca 2ϩ levels, CREB family members are activated and promote the expression of numerous cellular target genes that contain CREs in their promoters (16), including proto-oncogenes such as c-fos and c-jun (17, 18) and cell cycle genes such as cyclins D and A and other genes related to cell growth, proliferation, and neuronal activities (19,20). Phosphorylation of ATF1 at Ser-63 in its kinase-inducible domain by serine/threonine kinases enhances its transactivation activity by promoting recruitment of the coactivator CREB-binding protein/p300 (21). ATF1 is overexpr...

Tumor Necrosis Factor Receptor-associated Factor Family Protein 2 Is a Key Mediator of the Epidermal Growth Factor-induced Ribosomal S6 Kinase 2/cAMP-responsive Element-binding Protein/Fos Protein Signaling Pathway

“…1D). In addition, phosphorylation of JNK was not detectable as reported previously (15). This indicated that TRAF2 regulates the EGF pathway through other molecules but not IKK␣/␤ or JNK.…”

“…1F). CREB (12) and ATF1 (15) are well known or novel substrates, respectively, for RSK2. Both CREB and ATF1 contribute to the induction of c-Fos expression (18).…”

“…EGF induces extracellular signal-regulated kinase 1/2 (ERK1/2) activation, which directly phosphorylate and activate ribosomal S6 kinase 2 (RSK2). RSK2 then phosphorylates substrates like CREB (cAMP-responsive elementbinding protein) (12), c-Fos (13), ATF4 (activating transcription factor 4) (14), and ATF1 (activating transcription factor 1) (15), which promotes their cellular functions.…”

Signal Transduction and Molecular Targets of Selected Flavonoids

“…Eriodictyol (Table 1) is a flavanone that is found in various fruits, and we discovered that it also binds to the NTD of RSK2 to inhibit RSK2 N-terminal kinase activity (79,85,98). In this same study, we found that ATF1 is a novel substrate of RSK2 and that RSK2-ATF1 signaling plays an important role in EGF-induced neoplastic cell transformation.…”

“…Eriodictyol had no effect on the phosphorylation of RSK, MEK1/2, ERK1/2, p38, or JNKs, indicating that this compound specifically suppresses RSK2 signaling. The phosphorylation of ATF1 by RSK2 and its susequent cellular effects, including Ras-mediated focus formation, were markedly attenuated with eriodictyol treatment (98). Besides the Ras/Raf/MEK/MAPK pathway, flavanoids also strongly interact with the Src and Fyn tryrosine kinases to interfere with their activity (Fig.…”