Abstract
PIM1 is a serine/threonine kinase that promotes and maintains prostate tumorigenesis. While PIM1 protein levels are elevated in prostate cancer relative to local disease, the mechanisms by which PIM1 contributes to oncogenesis have not been fully elucidated. Here, we performed a direct, unbiased chemical genetic screen to identify PIM1 substrates in prostate cancer cells. The PIM1 substrates we identified were involved in a variety of oncogenic processes, and included N-Myc Downstream-Regulated Gene 1 (NDRG1), which has reported roles in suppressing cancer cell invasion and metastasis. NDRG1 is phosphorylated by PIM1 at serine 330 (pS330), and the level of NDRG1 pS330 is associated higher grade prostate tumors. We have shown that PIM1 phosphorylation of NDRG1 at S330 reduced its stability, nuclear localization, and interaction with AR, resulting in enhanced cell migration and invasion.
Highlights
PIM1 is a serine/threonine kinase that promotes and maintains prostate tumorigenesis
We focused on characterizing the effect of PIM1-dependent serine 330 phosphorylation of N-Myc Downstream-Regulated Gene 1 (NDRG1), given that a phosphoproteomic analyses of prostate cancer tissues by Drake et al.[43] demonstrated that levels of NDRG1 PIM1 at serine 330 (pS330) were found to be 7.7 times higher in metastatic lesions than in localized prostate cancer (Supplementary Data 1)
VCaP cells, a prostate cancer cell line with androgen receptor (AR) amplification, displayed the highest level of total NDRG1 and NDRG1 pS330, consistent with our findings that NDRG1 expression and phosphorylation are androgen-regulated (Fig. 4D; Supplementary Fig. 1). These results demonstrate that NDRG1 pS330 is correlated with high-grade prostate cancers, and that NDRG1 pS330 is present in multiple prostate cancer cell lines
Summary
PIM1 is a serine/threonine kinase that promotes and maintains prostate tumorigenesis. We performed a direct, unbiased chemical genetic screen to identify PIM1 substrates in prostate cancer cells. The PIM1 substrates we identified were involved in a variety of oncogenic processes, and included N-Myc Downstream-Regulated Gene 1 (NDRG1), which has reported roles in suppressing cancer cell invasion and metastasis. A similar approach has revealed the direct substrates of AMPK, with unexpected roles in mitosis and cytokinesis[21,22]; CDK9, with functions in transcriptional termination through phosphorylation of the 5′–3′ exonuclease XRN2;23 and CDK2, with a role in the DNA damage response by phosphorylation of NBS1, a necessary protein for DNA damage repair[24]. This is a rigorous approach that has revealed new substrates of kinases to yield novel mechanistic insights
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