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doi: 10.1242/10.1242/jcs.00102

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CK2 constitutively associates with and phosphorylates chicken erythroid ankyrin and regulates its ability to bind to spectrin

Sourav Ghosh^*, Frank C. Dorsey and John V. Cox

Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, Tennessee 38163, USA
^* Present address: MCBL, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, CA 92037-1099, USA

View larger version (46K): [in a new window]   Fig. 1. Phosphorylation of chicken erythroid ankyrin in vivo and in vitro. Ankyrin immunoprecipitates were prepared from the detergent-soluble (S) and -insoluble (I) fractions from erythroid cells labeled with 32P-orthophosphate for 4 hours (lanes 1-4) or from unlabeled cells (lanes 5-8). Some of the cells used for these analyses were incubated in the presence of 100 nM calyculin A for 4 hours prior to detergent lysis (lanes 3, 4, 7, and 8). Immunoprecipitates from unlabeled cells were incubated in kinase buffer containing [-32P]-ATP for 30 minutes at 37°C prior to gel analysis (lanes 5-8). Immune complexes were resolved on a 6% SDS polyacrylamide gel. Individual ankyrin isoforms were visualized by staining with GelCode Blue, and 32P-labeled species were detected by autoradiography. Lane 8* is a five-fold longer exposure of lane 8. The migrations of the 225 kDa, 220 kDa and 205 kDa ankyrin isoforms are indicated to the left of the figure. The migration of the hyperphosphorylated 225 kDa (225*) and 205 kDa (205*) isoforms are indicated to the right of the figure.

View larger version (60K): [in a new window]   Fig. 2. Characterization of the erythroid ankyrin-associated kinase using an in gel kinase assay. A whole cell lysate was prepared from erythroid cells isolated from 10-day-old chicken embryos. This lysate (lanes 1 and 2) and an ankyrin immunoprecipitate prepared from the lysate (lane 3) were electrophoresed on a 12.5% SDS polyacrylamide gel containing either 50 µg/ml myelin basic protein (lane 1) or 50 µg/ml of chicken red blood cell membranes (lanes 2 and 3). Following electrophoresis, the gels were processed as described in the Materials and Methods and incubated in kinase buffer containing 25 µM ATP and 100 µCi [-32P] ATP. The gel was then washed and dried, and 32P-labeled species were detected by autoradiography. Molecular weight markers are indicated to the left of the figure.

View larger version (32K): [in a new window]   Fig. 3. CK2 coprecipitates with chicken erythroid ankyrin. Whole cell lysate prepared from erythroid cells from 10-day-old chicken embryos (lane 1) or immunoprecipitates prepared from this lysate using ankyrin preimmune (lane 2) or immune sera (lane 3) were electrophoresed on a 12.5% SDS polyacrylamide gel and transferred to nitrocellulose. This filter was incubated with a rabbit antibody that recognizes the and ' catalytic subunits of CK2, followed by protein A conjugated to horseradish peroxidase. After washing, immunoreactive species were detected by enhanced chemiluminescence.

View larger version (43K): [in a new window]   Fig. 4. Biochemical properties of the ankyrin-associated kinase. Ankyrin immunoprecipitates prepared from a whole cell lysate from erythroid cells were incubated in kinase buffer containing [-32P]-ATP (A and B, lanes 1-3) or [-32P]-GTP (A, lane 4) for 30 minutes at 37°C. CK2 inhibitors, including 5 mM EDTA (A, lane 2), 50 µg of heparin (A, lane 3), 10 µM TBB (B, lane 2) and 10 µM emodin (B, lane 3), were added to some of the immunoprecipitates during the in vitro kinase reaction. Alternatively, ankyrin precipitates prepared from whole cell lysates from control (C, lanes 1, 3, 5) or calyculin-A-treated erythroid cells (C, lane 2, 4, 6) were either incubated in kinase buffer containing [-32P]-ATP for 30 minutes at 37°C (C, lanes 1 and 2) or boiled for 2 minutes before the kinase reaction (C, lanes 3 and 4). To some of the heat-treated precipitates, purified CK2 from rat liver was added prior to incubation at 37°C for 30 minutes (C, lanes 5 and 6). Following the kinase reactions, samples were analyzed on 6% SDS polyacrylamide gels. The migration of the 225 kDa, 220 kDa, and 205 kDa ankyrin isoforms are indicated by dashes to the left of each panel. Dashes to the right of the panel C indicate the hyperphosphorylated (225*) 225 kDa and (205*) 205 kDa ankyrin isoforms.

View larger version (89K): [in a new window]   Fig. 5. One-dimensional phosphopeptide mapping analysis of erythroid ankyrin isoforms. Ankyrin immunoprecipitates were prepared from the detergent soluble (lanes 1-3) and insoluble fractions (lanes 4-8) of control (lanes 5-8) or calyculin A treated erythroid cells (lanes 1-4). Some of the precipitates were prepared from cells labeled with 32P-orthophosphate (lanes 3-5). The remaining precipitates, which were prepared from unlabeled cells, were phosphorylated in vitro by the coprecipitating kinase (lanes 2, 6 and 7) or by purified CK2 (lanes 1 and 8). In each instance, the precipitates were resolved on a 6% SDS polyacrylamide gel, and the gel was stained with GelCode Blue. The indicated ankyrin isoforms were excised from the gel, electroeluted, and the eluted proteins were digested with V8 endoproteinase. The resulting phosphopeptides were analyzed on 18% SDS polyacrylamide gels. The gels were dried, and 32P-labeled peptides were detected by autoradiography. Molecular weight markers are indicated to the left of the figure.

View larger version (50K): [in a new window]   Fig. 6. TBB inhibits the basal phosphorylation and the hyperphosphorylation of chicken erythroid ankyrin in vivo. Erythroid cells from 10-day-old chicken embryos were incubated in DMEM containing 1 mCi/ml 32P-orthophosphate at 37°C for 2 hours in the absence (lanes 1-4) or presence (lanes 5-8) of 100 nm calyculin A. Some of the cells were also treated with 60 µM TBB (lanes 3, 4, 7, and 8) during the labeling period. After labeling, the cells were detergent-fractionated and ankyrin immunoprecipitates were prepared and analyzed on a 6% SDS polyacrylamide gel. Immunoprecipitated polypeptides were visualized by staining with GelCode Blue, and 32P-labeled species were detected by autoradiography. Dashes to the right of each panel indicate the basally phosphorylated (225) and hyperphosphorylated (225*) 225 kDa ankyrin isoform.

View larger version (36K): [in a new window]   Fig. 7. CK2-dependent phosphorylation of ankyrin regulates its ability to associate with spectrin in vitro. Erythroid cells were lysed in isotonic buffer containing 1% Triton X-100, and an -spectrin immunoprecipitate was prepared from the resulting detergent insoluble fraction and washed into low salt buffer. The immunoprecipitate was then incubated with low salt buffer. The immunoprecipitate was then incubated with low salt buffer (lane 1) or with ankyrin that had been immunopurified from control cells (lane 4), from cells treated with calyculin A (lane 2), from cells treated with calyculin A plus TBB (lane 3) or from cells treated with TBB alone (lane 5). Following extensive washing in low salt buffer, the immunoprecipitates were split in half and processed for immunoblotting with ankyrin-specific or -spectrin-specific antibodies. The dash to the left of the ankyrin immunoblot indicates the basally phosphorylated 225 kDa ankyrin isoform.

View larger version (55K): [in a new window]   Fig. 8. In vivo and in vitro phosphorylation of ank3 isoforms from MDCK cells. A whole cell lysate from MDCK cells (A, lane 1) was resolved on a 6% SDS polyacrylamide gel, transferred to nitrocellulose, and processed for immunoblotting using rabbit antibodies specific for ank3. Ank3 antibodies were also used to prepare immunoprecipitates from MDCK cells labeled with 32Porthophosphate at 37°C for 4 hours in the absence (A, lanes 2 and 4) or presence (A, lanes 3 and 5) of 120 µm TBB. In addition, immunoprecipitates prepared from unlabeled MDCK cells were incubated in kinase buffer containing [-32P]-ATP for 30 minutes at 37°C (B). In some instances, 50 µg of heparin was included during the in vitro kinase reaction (B, lane 3). Alternatively, the precipitate was boiled for 10 minutes, which eliminated the activity of the coprecipitating kinase (B, lane 4), and subsequently incubated with 15 mU of purified CK2 from rat liver (B, lane 5). Samples were analyzed on 6% SDS polyacrylamide gels. Immunoprecipitated polypeptides were visualized by staining with GelCode Blue, and 32P-labeled species were detected by autoradiography. The migration of the 215 kDa and 200 kDa ank3 isoforms is indicated to the left of each panel.

View larger version (33K): [in a new window]   Fig. 9. One-dimensional phosphopeptide mapping of the 215 kDa ank3 isoform from MDCK cells. An ank3 immunoprecipitate prepared from MDCK cells was incubated in kinase buffer containing [-32P]-ATP (lane 1), or was heat-treated prior to incubation with purified rat liver CK2 in kinase buffer containing [-32P]-ATP (lane 2). Following the kinase reactions, the precipitates were analyzed on a 6% SDS polyacrylamide gel, and the gel was stained with GelCode Blue. The 215 kDa ank3 isoform from each in vitro reaction was excised from the gel, electroeluted, and the eluted protein was digested with V8 endoproteinase. The resulting phosphopeptides were analyzed on an 18% SDS polyacrylamide gel. The gel was dried, and 32P-labeled peptides were detected by autoradiography. Molecular weight markers are indicated to the left of the figure.