Stabilization of the prostate‐specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim‐1 in prostate cancer cells

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. © 2012 Wiley Periodicals, Inc.     

In silico studies of potential phosphoresidues in the human nucleophosmin/B23: its kinases and related biological processes

Human nucleophosmin/B23 is a phosphoprotein involved in ribosome biogenesis, centrosome duplication, cancer, and apoptosis. Its function, localization, and mobility within cells, are highly regulated by phosphorylation events. Up to 21 phosphosites of B23 have been experimentally verified even though the corresponding kinase is known only for seven of them. In this work, we predict the phosphorylation sites in human B23 using six kinase-specific servers (KinasePhos 2.0, PredPhospho, NetPhosK 1.0, PKC Scan, pkaPS, and MetaPredPS) plus DISPHOS 1.3, which is not kinase specific. The results were integrated with information regarding 3D structure and residue conservation of B23, as well as cellular localizations, cellular processes, signaling pathways and protein-protein interaction networks involving both B23 and each predicted kinase. Thus, all 40 potential phosphosites of B23 were predicted with significant score (>0.50) as substrates of at least one of 38 kinases. Thirteen of these residues are newly proposed showing high susceptibility of phosphorylation considering their solvent accessibility. Our results also suggest that the enzymes CDKs, PKC, CK2, PLK1, and PKA could phosphorylate B23 at higher number of sites than those previously reported. Furthermore, PDK, GSK3, ATM, MAPK, PKB, and CHK1 could mediate multisite phosphorylation of B23, although they have not been verified as kinases for this protein. Finally, we suggest that B23 phosphorylation is related to cellular processes such as apoptosis, cell survival, cell proliferation, and response to DNA damage stimulus, in which these kinases are involved. These predictions could contribute to a better understanding, as well as addressing further experimental studies, of B23 phosphorylation.     

Ganglioside GM3 inhibits hepatoma cell motility via down‐regulating activity of EGFR and PI3K/AKT signaling pathway

Two related sublines derived from murine ascites hepatoma cell lines Hca‐F25, which were selected for their markedly different metastatic potential to lymph nodes, were found to be distinct in their ganglioside patterns. The low metastatic cell line (HcaP) contained a major ganglioside GM3, whereas the high metastatic cell line (HcaF) contained a major ganglioside GM2. Suppression of GM3 by P4 enhanced the mobility and migration of the low metastatic HcaP cells in vitro. Increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 inhibited the mobility and migration. These results suggested that the differences in lymphatic metastasis potential between these two cell lines could be attributed to the differences in their ganglioside compositions, and GM3 could suppress the motility and migration of these cells. Further, we investigated the mechanism by which GM3 suppressed the cell mobility and migration. The results showed that suppression of GM3 synthesis by P4 in low metastatic HcaP cells promoted PKB/Akt phosphorylation at Ser473 and Thr308, and phosphorylation of EGFR at the Tyr1173. In contrast, increase in GM3 content in high metastatic HcaF cells by addition of exogenous GM3 into the culture medium suppressed phosphorylation of PKB/Akt and EGFR at the same residues. Taken together, these results suggested that the mechanism of GM3‐suppressed cell motility and migration may involve the inhibition of phosphorylation of EGFR and the activity of PI3K/AKT signaling pathway. J. Cell. Biochem. 114: 1616–1624, 2013. © 2013 Wiley Periodicals, Inc.