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

Chen H, Yao K, Chang X, Shim JH, Kim HG, Malakhova M, Kim DJ, Bode AM, Dong Z - PLoS ONE (2015)

Bottom Line: 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.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.In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN, 55912, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

EGCG inhibits RSK2 kinase activity.(A) Chemical structure of EGCG. (B) An immunoprecipitation/kinase assay was performed using different doses of EGCG (0, 1, 5, 10 μM) and 1 μg histone H3 protein as substrate. Results were analyzed by Western blot with antibodies to detect phosphorylated (p-) histone H3 (Ser10), total histone H3, p-RSK2 (Thr577) and His G, respectively. Each assay was performed 3 times and similar results were obtained. Representative blots are shown.
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pone.0130049.g001: EGCG inhibits RSK2 kinase activity.(A) Chemical structure of EGCG. (B) An immunoprecipitation/kinase assay was performed using different doses of EGCG (0, 1, 5, 10 μM) and 1 μg histone H3 protein as substrate. Results were analyzed by Western blot with antibodies to detect phosphorylated (p-) histone H3 (Ser10), total histone H3, p-RSK2 (Thr577) and His G, respectively. Each assay was performed 3 times and similar results were obtained. Representative blots are shown.

Mentions: EGCG (Fig 1A) is a major green tea catechin that has been reported to bind with and inhibit the activity of a number of different proteins. Its biological effects appear to be achieved through its modulation of multiple proteins rather than a single target, which supports the concept of poly-pharmacology [23]. To identify additional novel molecular targets of EGCG, a computational docking and screening method was performed using our in-house kinase database. The results indicated that both the C-terminal and N-terminal of RSK2 were in the top 10 list (Table 1). Additionally, our shape screening results showed that EGCG has a similarity score of 7.6 with two known RSK2 inhibitors, quercitrin [24] and afzelin [25]. Together, these results suggested that RSK2 is a potential target of EGCG.


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

Chen H, Yao K, Chang X, Shim JH, Kim HG, Malakhova M, Kim DJ, Bode AM, Dong Z - PLoS ONE (2015)

EGCG inhibits RSK2 kinase activity.(A) Chemical structure of EGCG. (B) An immunoprecipitation/kinase assay was performed using different doses of EGCG (0, 1, 5, 10 μM) and 1 μg histone H3 protein as substrate. Results were analyzed by Western blot with antibodies to detect phosphorylated (p-) histone H3 (Ser10), total histone H3, p-RSK2 (Thr577) and His G, respectively. Each assay was performed 3 times and similar results were obtained. Representative blots are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470687&req=5

pone.0130049.g001: EGCG inhibits RSK2 kinase activity.(A) Chemical structure of EGCG. (B) An immunoprecipitation/kinase assay was performed using different doses of EGCG (0, 1, 5, 10 μM) and 1 μg histone H3 protein as substrate. Results were analyzed by Western blot with antibodies to detect phosphorylated (p-) histone H3 (Ser10), total histone H3, p-RSK2 (Thr577) and His G, respectively. Each assay was performed 3 times and similar results were obtained. Representative blots are shown.
Mentions: EGCG (Fig 1A) is a major green tea catechin that has been reported to bind with and inhibit the activity of a number of different proteins. Its biological effects appear to be achieved through its modulation of multiple proteins rather than a single target, which supports the concept of poly-pharmacology [23]. To identify additional novel molecular targets of EGCG, a computational docking and screening method was performed using our in-house kinase database. The results indicated that both the C-terminal and N-terminal of RSK2 were in the top 10 list (Table 1). Additionally, our shape screening results showed that EGCG has a similarity score of 7.6 with two known RSK2 inhibitors, quercitrin [24] and afzelin [25]. Together, these results suggested that RSK2 is a potential target of EGCG.

Bottom Line: 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.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.In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN, 55912, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus