Article Figures & Data
Figures
- FIG. 1.
CK2 phosphorylates NKX3.1 in vitro. (A) Two-dimensional PAGE analysis of recombinant NKX3.1 and CK2-phosphorylated NKX3.1. The gels were stained with Coomassie brilliant blue R-250. The NKX3.1 protein was shifted from pH ∼9 to ∼7 after the CK2 phosphorylation reaction. (B) MALDI spectra of the NKX3.1 protein in panel A acquired in linear positive mode using CHCA with ammonium phosphate as the matrix. Top panel: the spectrum for nonphosphorylated NKX3.1; bottom panel: the spectrum for CK2-phosphorylated NKX3.1. The m/z values of peaks corresponding to the tryptic peptides containing amino acids 69 to 81, 82 to 79, and 98 to 115 (theoretical masses: 1,314.64, 1,742.81, and 1,929.97 Da, respectively), as well as their phosphorylated forms (theoretical masses: 1,396.62, 1,902.77, and 2,009.95 Da, respectively) are in bold. The m/z values of the peptide corresponding to amino acids 148 to 154 (theoretical mass: 835.43 Da) that contains a consensus CK2 site is underlined. Tr denotes autotryptic fragments of the protease trypsin. Note the absence of peaks at 1,742.81 and 1,929.97 Da in the bottom spectrum. (C) MALDI-TOF PSD spectrum of the peptide of m/z 1,902.80 detected in panel B. The sequential losses of 80 Da from the parent ion indicate that the peptide of m/z 1,902.80 is diphosphorylated.
- FIG. 2.
CK2 blockade decreases the level of NKX3.1 in LNCaP cells. (A) Whole-cell lysates of LNCaP cells treated with apigenin were analyzed by Western blot analysis to detect NKX3.1 and β-tubulin. (B) Quantitative analysis of Western blots shown in panel A. NKX3.1 and tubulin bands were analyzed using the ImageQuant software. The relative NKX3.1 protein levels normalized to tubulin levels are graphed. Gray bar, dimethyl sulfoxide control; hashed bar, apigenin-treated samples. (C) Quantitative analysis of Northern blots to detect NKX3.1 mRNA in the same apigenin-treated LNCaP cells shown in panel A. Gray bar, dimethyl sulfoxide control; stippled bar, apigenin-treated samples.
- FIG. 3.
Replacement of CK2 phosphorylation sites Thr89 and Thr93 with alanines reduces the steady-state level of NKX3.1 in LNCaP cells. (A) LNCaP cells were transfected in triplicate with wild-type (WT) NKX3.1 and the T150A and T89A/T93A alanine substitution mutants and harvested after 24 h for Western blot analysis of NKX3.1 using an anti-HA antibody to detect the transfected forms of NKX3.1. (B) Northern blot analysis to detect NKX3.1 mRNA in the same transfected cells shown in panel A. NKX3.1END denotes the position of endogenous NKX3.1 mRNA; NKX3.1TF denotes the position of transfected NKX3.1 mRNA.
- FIG. 4.
Reduced half-life of the T89A/T93A mutant. (A) Western blot analysis of whole-cell extracts of LNCaP cells transfected with wild-type (WT) NKX3.1-HA or the T89A/T93A mutant. The cells were sampled at the indicated times after cycloheximide (CHX) treatment. The transfected forms of NKX3.1 were detected by an anti-HA antibody. (B) Quantification of Western blot data in panel A. The NKX3.1 levels were normalized to that of β-actin and graphed on a semilog plot. The calculated half-life values are indicated.
- FIG. 5.
NKX3.1 is degraded by the ubiquitin-proteasome system. (A) Western blot analysis to detect NKX3.1 in LNCaP cells treated with the proteasome inhibitor epoximicin (Epox). Two replicate wells were cultured in the presence or absence of epoximicin, and the whole-cell lysates were assayed with the antibodies indicated. (B) NKX3.1 is polyubiquitinated in vivo. NKX3.1 with empty vector and NKX3.1 with His-tagged ubiquitin (His-Ub) were cotransfected into LNCaP cells; 24 h after transfection, the cells were treated with MG132 for an additional 12 h and harvested in urea lysis buffer. The His-ubiquitin and ubiquitinated proteins were enriched by nickel resin chromatography and analyzed by Western blot using anti-NKX3.1 antibodies. L, lysate; F, flowthrough; E, eluate. Note that the discrete higher-molecular-weight forms of NKX3.1 are present in the lysate and eluate lanes. (C) Proteasome inhibition rescues the diminution of NKX3.1 induced by CK2 inhibition. Western blot analysis of NKX3.1 in LNCaP cells with or without DRB and MG132 treatment. DRB and/or MG132 was administered for 8 h and harvested for Western blot analysis using anti-NKX3.1 antibodies and antitubulin antibodies. (D) Proteasome inhibition increases the level of the T89A/T93A mutant. LNCaP cells in triplicate wells were transfected with the constructs indicated and exposed to MG132 for 12 h at 24 h posttransfection, followed by Western blot analysis using anti-NKX3.1 antibodies. The high-molecular-weight species that accumulate in the presence of MG132 represent polyubiquitinated isoforms of NKX3.1.
- FIG. 6.
NKX3.1 is phosphorylated by a 42-kDa kinase present in the LNCaP extract. (A) In-gel kinase assay of the Mono Q fractions of LNCaP lysate. Upper panel, human NKX3.1 as the substrate; middle panel, mouse Nkx3.1 as the substrate; lower panel, negative control with no protein cast in the gel. The arrowhead denotes the position of the 42-kDa kinase activity detected in gels with mouse and human NKX3.1 and absent in the control. The arrow denotes the position of the 44-kDa kinase activity detected only in the presence of human NKX3.1. Numbers indicate the fractions assayed. (B) Western blot analysis of fractions shown in panel A using the antibodies indicated. While using the anti-CK2β antibodies, cross-reacting species of other than 29 kDa were concentrated in fractions 9 to 13. M, molecular weight marker lane. (C) DRB selectively inhibits the 42-kDa kinase activity in an in-gel kinase assay. The arrowhead indicates the position of the 42-kDa kinase activity diminished in the presence of DRB. The arrow indicates the position of a 60-kDa kinase activity that is not inhibited by DRB and serves as a control for specificity.
- FIG. 7.
CK2α′ knockdown by siRNA decreases the steady-state level of NKX3.1. LNCaP cells were transfected with siRNA oligonucleotides targeting CK2α or CK2α′ or a negative control (Ctr) siRNA as indicated. Levels of NKX3.1, CK2α, and CK2α′ were analyzed by Western blot using specific antibodies. β-Actin served as a control.
