Primary structure of rat ANPK and sequence comparison. (A) Predicted amino acid sequence of ANPK (GenBank accession no. AF036959). The numbers on the left depict amino acid positions. The catalytic kinase domain and the AR interaction domain (AR ID) are boxed. (B) Sequence alignment of protein kinase domains of ANPK and Dyrk. Roman numerals on the top denote the conserved kinase subdomains according to Hanks and Quinn (1991). Invariant residues in all the protein kinases are depicted with asterisks below the residues.

Expression of ANPK. (A) Northern blot of RNA from rat tissues. Polyadenylated RNA samples (5 μg/lane) from testis (1), prostate (2), and brain (3). (B) Northern blot of poly(A)⁺ RNA (2 μg/lane) from various human tissues. Human multiple tissue Northern blot II (CLONTECH) contains RNA from spleen (1), thymus (2), prostate (3), testis (4), small intestine (5), colon (6), and peripheral blood leukocytes (7). Both blots were hybridized with ³²P-labeled ANPK cDNA as described in MATERIALS AND METHODS. To check the integrity of the RNA samples, the rat blot was reprobed with rat γ-actin and the human blot with human β-actin cDNA, respectively. (C) Expression of ANPK protein in various cell lines. Fifteen micrograms of whole cell extracts from PC12 (1), N2A (2), LNCaP (3), CHO (4, 5), PC-3 (6), CV-1 (7), and COS-1 cells transfected with an ANPK expression vector (8), and mock-transfected COS-1 cells (9), or 2 μl of ANPK(1–1191) (10) and ANPK(159–1191) (11) produced by translation in vitro using reticulocyte lysate system were separated on 7.5% polyacrylamide gel under denaturing conditions and immunoblotted using rabbit antiserum against GST-ANPK(766–920).

Localization of ANPK in cultured cells and prostate. (A and B) CV-1 cells seeded on glass coverslips on 10-cm plastic plates were transfected with 1 μg of FLAG-ANPK(2–1191) expression vector using DOTAP transfection reagent as described in MATERIALS AND METHODS. Cells were fixed in 4% (wt/vol) paraformaldehyde, permeabilized, and ANPK antigen was visualized using (A) affinity-purified rabbit antiserum raised against GST-ANPK(766–920) or (B) anti-FLAG M2 antibody. (C) The distribution of endogenous ANPK in S115 cells as determined by using anti-ANPK antiserum. (D) Immunofluorescence of S115 cells with anti-ANPK antiserum neutralized with purified GST-ANPK(766–920) fusion protein. (E and F) Distribution of ANPK in rat prostate. Immunoperoxidase technique was applied to visualize ANPK antigen using rabbit antiserum raised against GST-ANPK(766–920). (E) ANPK is localized in nuclei of epithelial cells. (F) A higher magnification showing granular clusters of ANPK immunoreactivity in epithelial cell nuclei. Original magnification; E, ×760; F, ×1,900.

Protein kinase activity of ANPK. CHO cells were transiently transfected with 10 μg of expression vectors for FLAG-tagged ANPK(159–1191) and ANPK(K226R), in which Lys²²⁶ in the ATP-binding site was converted to an Arg. Proteins were immunoprecipitated using anti-FLAG mAb and subjected to immune complex kinase assays. (A) Phosphorylation of myelin basic protein (MBP, 5 μM) by immunopurified ANPK(159–1191) (lane 1), but not by ANPK(K226R) (lane 3), ANPK(159–1191), and ANPK(K226R) in the absence of MBP (lanes 2 and 4, respectively). (B) Autophosphorylation by ANPK(159–1191) (lanes 2 and 3) and ANPK(K226R) (lanes 1 and 4). (C) Protein kinase activity of purified GST-ANPK(159–920) expressed in E. coli. GST-ANPK(159–920) with MBP (lane 1), autophosphorylation of GST-ANPK(159–920) in the absence of MBP (lane 3), and MBP in the absence of GST-ANPK(159–920) (lane 2). (D) Comparison of MBP (lane 1), c-Jun (lane 2), histone H3 (lane 3), histone H1 (lane 4), and GST-ANPK(802–920) (lane 5) as substrates for GST-ANPK(159–920). Reaction mixture (20 μl) contained 0.7 μg of GST-ANPK(159–920) and 5 μM substrate. After 30°C for 30 min, the reactions were terminated by addition of 20 μl of 2× Laemmli sample buffer, after which the products were subjected to electrophoresis under denaturing conditions on 15% (A, C, and D) or 7.5% polyacrylamide gels (B) and visualized by autoradiography. (F) Solid-phase phosphorylation of synthetic peptides by ANPK. PhosphoSpots test strip (Jerini Bio Tools) containing covalently bound substrate peptides for indicated kinases was phosphorylated by incubating with 4 μg of GST-ANPK(159–920) in 500 μl of 2× kinase buffer containing 100 μM [γ-³²P]ATP at 22°C for 20 min. The reaction was stopped and filter washed extensively according to manufacturer’s instructions and analyzed by autoradiography. (The peptide no. 19 contains both Ser and Tyr residues as potential phosphoacceptor sites.)

Phosphorylation of AR in vitro and in vivo. (A) AR is phosphorylated in vitro by ERK2 and CDC2 kinase, but not by ANPK. Histidine tagged H₆-ZFR (residues 554–644 of hAR), H₆-N-TERM (residues 141–547), and GST-HLBD (residues 624–919) were expressed in bacteria, affinity purified, and phosphorylated using indicated protein kinases under the conditions described in Figure 4, except that the concentration of GST-HLBD was 1 μM. ERK2 and CDC2 were used at 5 ng/μl and 0.25 U/μl, respectively. PHAS-I and histone H1 served as established control substrates for ERK2 and CDC2, respectively. (B) Effect of cotransfected ANPK on phosphorylation of AR in vivo. CV-1 cells were cotransfected by the calcium phosphate method with expression vectors for AR and ANPK as indicated. After a 48-h culture in the presence of 100 nM testosterone, cells were metabolically labeled for 2.5 h with [³²P]orthophosphate, and AR was immunoprecipitated as described in MATERIALS AND METHODS. In vivo labeled AR was subjected to electrophoresis under denaturing conditions on 7.5% polyacrylamide gel and visualized by autoradiography. Lower panel shows immunoblot analysis of the same samples using rabbit K333 antiserum raised against full-length rAR.

Protein kinase activity and autophosphorylation of different ANPK mutants. (A) Structural features of ANPK mutants studied. (B) Protein kinase activity of ANPK mutants. FLAG-tagged ANPK and indicated mutant proteins were expressed in CHO cells, immunoprecipitated using anti-FLAG monoclonal antibody, and subjected to immune complex kinase assays for 30 min at 30°C in the absence (–) and presence (+) of 5 μM MBP as described in MATERIALS AND METHODS. (C) Immune complex kinase assays of various ANPK forms in the absence of added substrate. Different ANPK forms were expressed to similar levels as judged by immunoblotting analysis (our unpublished data). (D) Phosphoamino acid analysis of autophosphorylated ANPK and MBP and c-Jun phosphorylated by ANPK in vitro. After in vitro kinase assay using immunopurified ANPK (1) and ANPK(S357A/Y359A) (2) from CHO cells, or MBP (3) and c-Jun (4) as substrates in the GST-ANPK(159–920) catalyzed phosphorylation, proteins were transferred onto PVDF membrane, radioactive bands corresponding to phosphorylated proteins were cut out and subjected to acid hydrolysis as described in MATERIALS AND METHODS. Phosphoamino acids were resolved by one-dimensional electrophoresis at pH 2.5. Positions of unlabeled internal phosphoamino acid standards are depicted.

Interaction between AR and ANPK in yeast and mammalian cells and in vitro. (A) Plasmids expressing LexA or LexA fused to full-length AR (LexA-AR), AR ZFR including part of the hinge region (LexA-ZFR), AR ZFR without hinge region sequences (LexA-ZFR-s) or AR hinge-LBD fragment (LexA-HLBD) were introduced into L40 yeast cells together with expression plasmids for VP16 AD and VP16 AD fused to ANPK(766–920) (VP16-ANPK-ID). Transformants were grown in the presence (+) or absence (–) of 50 nM testosterone (Test). β-Gal activity in extracts from liquid yeast cultures are shown. Each bar depicts the average of three independent yeast transformants. Immunoblot and whole-cell ligand-binding assays indicated that the LexA-AR fusion proteins examined were expressed to comparable levels (our unpublished data). (B) Interaction of AR with ANPK in CV-1 cells. The ability of rAR and the DBD of Gal4 (Gal4-AR) as a fusion protein to interact with the residues 159–920 of ANPK fused to VP16 AD (VP16-ANPK) or with polyoma virus coat protein fused to VP16 AD (VP16-CP) was examined in CV-1 cells using the reporter plasmid pG5CAT. Cells (2.3 × 10⁵ cells/35-mm dish) were transfected with 1.5 μg of each chimeric expression vector and 3 μg of pG5CAT reporter using DOTAP transfection reagent. Eighteen hours after transfection, the medium was changed to one containing charcoal-stripped 2% (vol/vol) FBS in the presence (+) or absence (–) of 100 nM testosterone (T), and the cells were incubated for an additional 30 h. Transcriptional activation is expressed as the relative CAT activity corrected for protein concentration. Mean ± SE values for at least three separate experiments are shown. (C) Specific interaction of ANPK and AR ZFR in vitro. ³⁵S-Labeled full-length ANPK was synthesized by translation in vitro using reticulocyte lysate and incubated with GST alone (lane 2) or GST-AR ZFR (lane 3) adsorbed onto Glutathione Sepharose, after which the matrix was washed and bound proteins were analyzed as described in MATERIALS AND METHODS. Lane 1 represents 15% of the amount of labeled ANPK incubated with the matrix.

Colocalization of AR and ANPK in CV-1 cell nuclei. CV-1 cells seeded on glass cover slips on 10-cm plastic plates were cotransfected with 2.5 μg of FLAG-ANPK(2–1191) expression vector and 1 μg of rAR vector using DOTAP transfection reagent. Double immunofluorescence labeling was performed using anti-FLAG M2 monoclonal antibody and rabbit K183 antiserum for rAR as described in MATERIALS AND METHODS and analyzed by using a Bio-Rad MRC-1024 confocal laser scanning system connected to a Zeiss axiovert 100/135 microscope. Both ANPK (A, red) and AR (B, green) are present in nuclear dots, and the majority of the two proteins colocalize as revealed by the resulting yellow image following superimposition of A and B.

ANPK enhances androgen-induced transcriptional activation. (A) CV-1 cells were transfected using the calcium phosphate method with 5 μg of pPB(-285/+32)-LUC reporter plasmid along with 0.5 μg of pSG5-rAR and indicated amounts (μg) of pFLAG-ANPK(159–1191) or kinase-defective pFLAG-ANPK(K226R) in the presence or absence of 100 nM testosterone (T) as depicted. Total amount of DNA was kept constant by adding empty pFLAG-CMV-2 expression vector as needed. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. Luciferase (LUC) activities were normalized using β-gal activity. LUC activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least six independent experiments are given. (B and C) ANPK does not modulate PR- and GR-dependent transcription. (B) CV-1 cells were transfected with 5 μg of pARE₂-E1b-CAT reporter containing two copies of the GRE/PRE/ARE motif of the rat tyrosine aminotransferase gene upstream of the adenovirus E1b TATA sequence along with 0.5 μg of pSG5-hGR, 5 μg of empty expression vector (pFLAG-CMV-2) (open bar) or pFLAG-ANPK(159–1191) (solid bar), and 2 μg of pCMVβ in the presence or absence of 100 nM dexamethasone (D). (C) CV-1 cells were transfected as in panel B, but using 0.5 μg of pSG5-hPR1 instead of pSG5-hGR in the presence or absence of 100 nM progesterone (P). CAT activities are normalized to β-gal activity and expressed relative to those achieved with pSG5-hGR or pSG5-hPR1 in the presence of P or D, respectively (= 100), and the mean ± SE values of at least three independent experiments are shown.

Influence of ANPK on the function of various AR mutants. (A) Structural features of AR mutants studied. (B) Effect of ANPK on AR mutants was examined in CV-1 cells by coexpressing rAR or the deletion mutants rAR▵40–147, rAR▵641–902, and rAR▵46–408/▵641–902 (0.5 μg of each pSG5 expression vector) in the presence of empty pFLAG-CMV2 expression vector (5 μg, open bars) or with pFLAG-ANPK(159–1191) (5 μg, solid bars) and 5 μg of pARE₂-E1b-CAT reporter in the presence of 100 nM testosterone. Cells were transiently transfected using the calcium phosphate method. β-Gal expression plasmid, pCMVβ (2 μg/10-cm plate), was used to control for transfection efficiency. CAT activities are expressed relative to that of pSG5-rAR in the presence of testosterone (= 100), and the mean ± SE values of at least three independent experiments are given.

Effect of ANPK on the amount of immunoreactive AR protein. CV-1 cells (2 × 10⁶ cells/10-cm dish) were transfected by the calcium phosphate method with 5 μg of pSG5-rAR or pSG5-rAR▵641–902 together with 15 μg of pFLAG-CMV2, 15 μg of pFLAG-ANPK(2–1191) or pFLAG-ANPK(K226R) and cultured for 30 h in the presence of 100 nM testosterone. Each lane contained 20 μg of whole cell protein from cells transfected with pSG5-rAR plus pFLAG-CMV2 (lane 1), pSG5-rAR plus pFLAG-ANPK(2–1191) (lane 2), pSG5-rAR plus pFLAG-ANPK(K226R) (lane 3), pSG5-rAR▵641–902 plus pFLAG-CMV-2 (lane 4) and pSG5-rAR▵641–902 plus pFLAG-ANPK(2–1191) (lane 5). Immunoblot analysis was performed using rabbit K183 antiserum raised against full-length rAR (Karvonen et al., 1997) and immunoblots were developed using Amersham ECL western blotting detection reagents. Equal loading was confirmed by reprobing the membranes with an anti-α-tubulin antibody.