p90RSK Regulates p53 Pathway by MDM2 Phosphorylation in Thyroid Tumors

Simple Summary p90RSK is a downstream effector protein of the MAPK pathway. In many human cancers, the MAPK pathway is constitutively activated due to oncogenic mutations of its components. Consequently, p90RSK is hyperactive and capable of hyper phosphorylating substrates involved in tumorigenesis. p90RSK belongs to the AGC kinase family, which phosphorylates substrates with an RXRXXS/T consensus motif. MDM2 protein presents this consensus in its sequence at serine 166. MDM2 is a ubiquitin ligase that negatively regulates the protein stability of p53. The aim of this study has been to verify whether p90RSK is capable of phosphorylating MDM2 in serine 166 and to investigate the role of p90RSK in regulating the p53 pathway, especially in thyroid tumors in which the MAPK pathway is constitutively active and in which the development of specific drugs aimed at inhibiting the kinase activity of p90RSK could represent a new way of inhibiting tumorigenesis. The expression level of the tumor suppressor p53 is controlled by the E3 ubiquitin ligase MDM2 with a regulatory feedback loop, which allows p53 to upregulate its inhibitor MDM2. In this manuscript we demonstrated that p90RSK binds and phosphorylates MDM2 on serine 166 both in vitro and in vivo by kinase assay, immunoblot, and co-immunoprecipitation assay; this phosphorylation increases the stability of MDM2 which in turn binds p53, ubiquitinating it and promoting its degradation by proteasome. A pharmacological inhibitor of p90RSK, BI-D1870, decreases MDM2 phosphorylation, and restores p53 function, which in turn transcriptionally increases the expression of cell cycle inhibitor p21 and of pro-apoptotic protein Bax and downregulates the anti-apoptotic protein Bcl-2, causing a block of cell proliferation, measured by a BrdU assay and growth curve, and promoting apoptosis, measured by a TUNEL assay. Finally, an immunohistochemistry evaluation of primary thyroid tumors, in which p90RSK is very active, confirms MDM2 stabilization mediated by p90RSK phosphorylation.

Automated summary

This study aimed to verify whether p90RSK can phosphorylate MDM2 at serine 166 and investigate its role in regulating the p53 pathway, especially in thyroid tumors. It has been demonstrated that p90RSK can bind and phosphorylate MDM2 at serine 166, increasing the stability of MDM2 and promoting its degradation of p53. A pharmacological inhibitor of p90RSK, BI-D1870, can decrease MDM2 phosphorylation, restore p53 function, and inhibit cell proliferation while promoting apoptosis.