Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD, USA.
J Cell Biochem. 2013 May;114(5):1050-7. doi: 10.1002/jcb.24444.
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.
NKX3.1 的缺失是前列腺癌早期且一致的事件，并与前列腺上皮细胞增殖增加和预后不良相关。NKX3.1 的稳定性通过多个蛋白激酶在多个位点的磷酸化进行翻译后调节。在这里，我们报告了致癌蛋白激酶 Pim-1 对前列腺特异性肿瘤抑制因子 NKX3.1 的反常稳定作用。使用小分子抑制剂 CX-6258 进行药理学 Pim-1 抑制会降低 NKX3.1 蛋白的稳定状态水平和半衰期，但对 mRNA 没有影响。这种作用被 26S 蛋白酶体的抑制所逆转，表明 Pim-1 保护 NKX3.1 免受蛋白酶体介导的降解。质谱分析显示 Thr89、Ser185、Ser186、Ser195 和 Ser196 是 NKX3.1 上 Pim-1 的磷酸化接受位点。通过突变分析，我们确定 NKX3.1 在 Ser185、Ser186 和 N 端 PEST 结构域中的磷酸化对于 Pim-1 介导的稳定是必需的。此外，我们还确定了 Lys182 是 Pim-1 稳定 NKX3.1 的关键残基。Pim-1 介导的 NKX3.1 稳定可能对维持正常前列腺上皮细胞的正常细胞内稳态很重要，并可能维持前列腺癌细胞中基础 NKX3.1 蛋白水平。
