A mechanistic approach to understand the role of p90 ribosomal S6 kinases in Prostate Cancer

The RSKs are known to be involved in many cancers, including Prostate Cancer (PCa), however the representation of the Ribosomal S6 Kinase (RSK) isoforms is skewed and there is little investigation into the RSK regulation of the Androgen Receptor (AR). Here we show in model African Green Monkey Kidney Fibroblast (COS-1 cells) that all RSKs regulate the AR transcriptional activity and interact with the AR in proximity. Analysis of PCa cell lines highlighted that all RSKs exhibited higher expression than the control but most significantly RSK2. We then transitioned to lymph node carcinoma of the prostate (LNCaP) cells as they are more biologically relevant, siRNA knockdown coupled with qPCR showed that key AR target genes was differentially regulated by the RSKs; RSK1 and RSK4 knockdown caused increased expression of NDRG1; RSK1 and RSK2 knockdown induced a decrease in TMPRSS2; RSK2 knockdown produced a significant decrease in PSA, respectively. To understand if these effects would translate to a cellular change we investigated proliferation, siRNA knockdown of RSK2 and RSK4 caused a significant decrease in LNCaP cells. Interestingly, investigation of RSK4 is also underrepresented in structural biology despite having a different mechanism from other isoforms. Here, we attempted to express, purify, and crystalize different domains of RSK4 to understand its mechanism of activation. Crystallization attempts were unsuccessful, potentially due proteolysis and chaperone contaminations. Kinetic assays showed that phospho-mimetic mutations were successful in creating a constitutively active mutant construct. Pull down assays experimentally showed that the AGC region of RSK4 is essential for the PDK1 independent activation. The multiple techniques used in this study strongly suggest that RSK are in fact involved in PCa signalling and warrant further investigation and highlight the nonredundant functions of the RSK isoforms and a novel relationship between AR and RSK4. Additionally, the pull-down assay highlighted that the AGC region of RSK facilitates interaction between the N- And C-terminal domains which has not been previously experimentally demonstrated thus supporting previous speculation around its activation mechanism.