Abstract
PIM1 is a serine/threonine kinase over-expressed in prostate cancer. We have previously shown that PIM1 phosphorylates the androgen receptor (AR), the primary therapeutic target in prostate cancer, at serine 213 (pS213), which alters expression of select AR target genes. Therefore, we sought to investigate the mechanism whereby PIM1 phosphorylation of AR alters its transcriptional activity. We previously identified the AR co-activator, 14-3-3 ζ, as an endogenous PIM1 substrate in LNCaP cells. Here, we show that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, and that they extensively occupy the same sites on chromatin in an AR-dependent manner. Their occupancy at a number of genes involved in cell migration and invasion results in a PIM1-dependent increase in the expression of these genes. We also use rapid immunoprecipitation and mass spectrometry of endogenous proteins on chromatin (RIME), to find that select AR co-regulators, such as hnRNPK and TRIM28, interact with both AR and 14-3-3 ζ in PIM1 over-expressing cells. We conclude that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, which in turn recruits additional co-regulatory proteins to alter AR transcriptional activity.
Highlights
PIM1 is a serine/threonine kinase over-expressed in prostate cancer
We and others have previously shown that PIM1 phosphorylates androgen receptor (AR), the primary therapeutic target in prostate cancer, at S213, affecting the expression of select AR target genes[7,8]
We confirmed that in the presence of doxycycline, the cells over-express PIM1. This resulted in an increase in phosphorylation of AR at S213, as detected by the AR pS213 antibody generated in our laboratory as described previously (Fig. 1a)[18]
Summary
We have previously shown that PIM1 phosphorylates the androgen receptor (AR), the primary therapeutic target in prostate cancer, at serine 213 (pS213), which alters expression of select AR target genes. We and others have previously shown that PIM1 phosphorylates AR, the primary therapeutic target in prostate cancer, at S213, affecting the expression of select AR target genes[7,8] This phosphorylation of AR at S213 is associated with higher-grade tumors, castration-resistant disease, and altered AR transcriptional activity[7,9]. Noting that PIM1 has been shown to foster interactions between the [-3] proteins and its substrates, we hypothesized that PIM1 phosphorylation of AR mediates its interaction with the co-activator [-3] ζ15–17 Finding this to be the case, we used RNA seq and ChIP seq to further characterize the transcriptional changes and chromatin occupancy resulting from PIM1 phosphorylation and [-3] ζ coregulation of AR. The results indicate a coordinated response in which PIM1 phosphorylation of AR and [-3] ζ alters ARdependent gene expression
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