Stabilization of the Prostate-Specific Tumor Suppressor NKX3.1 by the Oncogenic Protein Kinase Pim-1 in Prostate Cancer Cells Achuth Padmanabhan, 1 Eliza B. Gosc, 1 and Charles J. Bieberich 1,2 * 1 Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 2 Marlene and Stewart Greenbaum Cancer Center, University of Maryland, Baltimore, Maryland ABSTRACT Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post-translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells. Pharmacologic Pim-1 inhibition using the small molecule inhibitor CX-6258 decreased steady state levels and half-life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S-proteasome, demonstrating that Pim-1 protects NKX3.1 from proteasome-mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim-1 phospho-acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N-terminal PEST domain is essential for Pim-1-mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim-1. Pim-1-mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells. J. Cell. Biochem. 114: 1050–1057, 2013. ß 2012 Wiley Periodicals, Inc. KEY WORDS: PROSTATE CANCER; NKX3.1; PIM-1 KINASE; PHOSPHORYLATION N KX3.1 is a homeodomain protein and a member of the NK- family of transcription factors whose expression in adult animals is restricted primarily to the prostate [Bieberich et al., 1996; He et al., 1997; Bhatia-Gaur et al., 1999]. In addition to its role in prostate development, NKX3.1 functions as a prostate-specific tumor suppressor [He et al., 1997; Bhatia-Gaur et al., 1999]. Diminished NKX3.1 expression is a consistent feature in both human and mouse prostate cancers and is observed in prostatic intraepithelial neoplasia (PIN) lesions [Abdulkadir et al., 2002; Kim et al., 2002; Bethel and Bieberich, 2007]. The genomic region harboring NKX3.1 (8p21) undergoes frequent loss of heterozygosity in human prostate cancer [He et al., 1997; Magee et al., 2003]. Targeted Nkx3.1 disruption in mice leads to mouse-PIN, prostate epithelial dysplasia, and hyperplasia, whereas ectopic NKX3.1 expression in prostate cancer cells suppresses their growth and tumorigenic potential [Bhatia-Gaur et al., 1999; Abdulkadir et al., 2002; Kim et al., 2002; Bethel et al., 2006; Bethel and Bieberich, 2007]. Although these data establish the role of NKX3.1 as a prostate- specific tumor suppressor, the regulation of NKX3.1 in prostate epithelial cells is complex. Discordance between NKX3.1 mRNA and protein levels was reported in prostate cancer cases, suggesting that NKX3.1 is post-translationally regulated in prostate cancer cells [Bethel et al., 2006]. Reports have since emerged linking protein phosphorylation to NKX3.1 stability. Phosphorylation at Thr89 and Thr93 by CK2 was shown to stabilize NKX3.1 [Li et al., 2006]. In contrast, the pro-inflammatory cytokines TNF-a and IL1-ß induced phosphorylation in an NKX3.1 C-terminal domain, causing rapid NKX3.1 ubiquitination and degradation [Markowski et al., 2008]. TNF-a-mediated phosphorylation at Ser196 was shown to signifi- cantly decrease NKX3.1 half-life, and phosphorylation at Ser185 and Ser195 was also shown to be critical in maintaining NKX3.1 steady- state levels in prostate epithelial cells [Markowski et al., 2008]. Here, we report the stabilization of the tumor suppressor NKX3.1 by the protein kinase Pim-1, a serine/threonine protein kinase Journal of Cellular Biochemistry ARTICLE Journal of Cellular Biochemistry 114:1050–1057 (2013) 1050 The authors declare no conflict of interest. *Correspondence to: Dr. Charles J. Bieberich, PhD, Department of Biological Sciences, UMBC, 1000 Hilltop Circle, Baltimore, MD 21250. E-mail: bieberic@umbc.edu Manuscript Received: 24 August 2012; Manuscript Accepted: 24 October 2012 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 5 November 2012 DOI 10.1002/jcb.24444  ß 2012 Wiley Periodicals, Inc.