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.
Highlights
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]
We found that the luciferase activity representing the binding of RSK2 and ATF1, a member of the CREB/ATF transcription factor family, was ϳ7-fold higher than that of the mock control
When cells are stimulated with tumor promoters, EGF or 12-O-tetradecanoylphorbol-13-acetate, phosphorylation of RSK is increased within 5 min, and stable expression of RSK2 in JB6 C141 cells significantly enhances colony formation in
Summary
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]. These results demonstrate that the inhibition of RSK2 activity by eriodictyol modulates RSK2-ATF1 signaling in cell proliferation and transformation. To determine whether RSK2 phosphorylates ATF1, we conducted an in vitro kinase assay with a commercially available active RSK2 protein and purified GST-ATF1 [35] and [32P]ATP.
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