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First published online 13 July 2004
doi: 10.1242/jcs.01210

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cAMP-induced degradation of cyclin D3 through association with GSK-3ß

Soheil Naderi^1,*, Kristine B. Gutzkow^1,*, Hege U. Låhne¹, Siri Lefdal¹, W. Johnathan Ryves², Adrian J. Harwood² and Heidi K. Blomhoff^1,

¹ Department of Medical Biochemistry, University of Oslo, P.O. Box 1112 Blindern, Oslo, N-0317, Norway
² MRC Laboratory for Molecular Cell Biology and Department of Biology, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (33K): [in a new window]   Fig. 1. Regulation of cyclin D3 expression and phosphorylation by forskolin. (A) Reh cells were cultured in the presence or absence of forskolin (100 µM) and harvested at the indicated times. Total RNA was recovered, and 15 µg of the RNA from each sample was analyzed by northern blotting using 32P-labelled cyclin D3 probe as described in Materials and Methods. The ethidium bromide-stained 18S RNA was used as a loading control (lower panel). (B) Reh cells were treated as in A, lysates were prepared and then subjected to immunoblotting with antibodies against cyclin D3 and actin. The immunoblot shown is the representative of five independent experiments. (C) Reh cells were treated with or without forskolin (100 µM) for 1 hour. Cells were lysed in buffer A, and the cell lysates were then treated with CIP in the presence or absence of PPI for 15 minutes at 30°C as described in Materials and Methods. The lysates were then resolved on SDS-PAGE, and cyclin D3 was detected by immunoblotting. The immunoblot shown is the representative of four independent experiments.

View larger version (26K): [in a new window]   Fig. 2. Forskolin increases the rate of cyclin D3 turnover. (A) Reh cells were pretreated with cycloheximide (25 µg/ml) for 15 minutes followed by forskolin (100 µM) over an 80 minute time course. Cells were harvested at the indicated time points after addition of forskolin, lysates were prepared and equal amounts of protein were analyzed by immunoblotting with cyclin D3 and actin antibodies. The blot shown is the representative of five independent experiments. (B) The immunoblot in A was scanned and the intensity of the protein bands was quantitated and plotted on a semi-log graph with the value obtained for cells not treated with cycloheximide set as 100%. The values were normalized with those of actin. (C) Freshly isolated human B cells were stimulated with a combination of anti-µ and SAC for 32 hours as described in Materials and Methods. Cells were then treated as in A for the indicated times and analysed for the expression of cyclin D3 and actin by western blotting.

View larger version (15K): [in a new window]   Fig. 3. Forskolin-induced reduction of cyclin D3 levels is mediated via cAMP. Reh cells were treated with the indicated concentrations of forskolin, IBMX or PGE2 for 2 hours. Following treatment, lysates were prepared, resolved on SDS-PAGE, and then subjected to immunoblotting with cyclin D3 and actin antibodies. The blot shown is the representative of two independent experiments.

View larger version (18K): [in a new window]   Fig. 4. Forskolin induces degradation of cyclin D3 via the ubiquitin-proteasome pathway. (A) Reh cells were pretreated with the proteasome inhibitors MG-132 (10 µM) or LLnL (100 µM) for 30 minutes before addition of forskolin (100 µM). Cells were harvested at 2 hours after addition of forskolin and total lysates were analyzed with antibodies against cyclin D3 and actin. The blot shown is the representative of four independent experiments. Lower panel, data shown represent the mean ±s.e. of the four independent experiments. The results in each experiment were quantified using a densitometer and the densitometric values of cyclin D3 were normalized with those of actin. (B) Reh cells transfected with HA-Ub vector were exposed to MG-132 (10 µM) for 30 minutes before treatment with 100 µM forskolin for 2 hours. Cells were harvested, whole cell extracts prepared, and immunoprecipitated with anti-cyclin D3 antibodies. The recovered proteins were resolved on SDS-PAGE and then subjected to immunoblotting with anti-HA and anti-cyclin D3 antibodies consecutively. The blot shown is the representative of three independent experiments. IP, immunoprecipitation; NRS, non-immune rabbit serum.

View larger version (18K): [in a new window]   Fig. 5. Effect of GSK-3ß inhibition on forskolin-induced degradation of cyclin D3. Reh cells were pretreated with the GSK-3ß inhibitor LiCl (20 mM) for 30 minutes before addition of forskolin (100 µM). Cells were harvested at 2 hours after addition of forskolin and total lysates were analyzed with antibodies against cyclin D3 and actin. The blot shown is the representative of four independent experiments. Lower panel, data shown represent the mean ±s.e. of the four independent experiments. The results in each experiment were quantified using a densitometer and the densitometric values of cyclin D3 were normalized with those of actin.

View larger version (13K): [in a new window]   Fig. 6. Phosphorylation of cyclin D3 by GSK-3ß. (A) Bacterially produced His-cyclin D3 (wt) (4 µg) was incubated with increasing concentrations of purified GSK-3ß in the presence of [-32P]ATP for 30 minutes at 30°C. Following incubation, the samples were subjected to SDS-PAGE and autoradiography. One representative experiment of three is shown. (B) Bacterially produced His-cyclin D3 (wt) (4 µg), His-cyclin D3 (T283) (4 µg), and purified GSK-3ß (300 mU) were incubated in various combinations as indicated with [-32P]ATP in the presence or absence of 50 mM LiCl for 30 minutes at 30°C. The reactions were subjected to SDS-PAGE and autoradiography. One representative experiment of three is shown.

View larger version (24K): [in a new window]   Fig. 7. Regulation of cyclin D3 stability depends on the integrity of Thr-283. (A) Reh cells transfected with His-cyclin D3 (wt) or His-cyclin D3 (T283A) vectors were cultured in the presence or absence of forskolin (100 µM) and harvested at the indicated time points. Total cell lysates were prepared and then subjected to immunoblotting with antibodies against cyclin D3. The immunoblot shown is the representative of three independent experiments. (B) Reh cells engineered to express His-cyclin D3 (wt) or His-cyclin D3 (T283A) were pretreated with cycloheximide (25 µg/ml) for 15 minutes followed by forskolin (100 µM) over a 2-hour time course. Cells were harvested at the indicated time points after addition of forskolin, lysates were prepared and equal amounts of protein were analyzed by immunoblotting with cyclin D3 antibodies. The blot shown is the representative of four independent experiments. Lower panel, the immunoblots were exposed, scanned and the intensity of the protein bands was quantified and plotted on a semi-log graph with the value obtained for cells not treated with cycloheximide set as 100%. () Endogenous cyclin D3, cycloheximide; () endogenous cyclin D3, cycloheximide + forskolin; (µ) His-cyclin D3 (wt), cycloheximide; () His-cyclin D3 (wt), cycloheximide + forskolin; () His-cyclin D3 (T283A), cycloheximide; () His-cyclin D3 (T283A), cycloheximide + forskolin.

View larger version (21K): [in a new window]   Fig. 8. His-cyclin D3 (T283A) associates with CDK4 and CDK6. (A) Reh cells were transfected with expression vectors encoding His-cyclin D3 (wt) or His-cyclin D3 (T283A). Following transfection, whole cell extracts were prepared and immunoprecipitated (IP) with antibodies against CDK4, CDK6 or with non-immune rabbit serum (NRS). The recovered proteins were resolved on SDS-PAGE and then subjected to immunoblotting with anti-CDK4, CDK6, or cyclin D3 antibodies. As positive control for CDK4, CDK6, and cyclin D3, 50 µg total cell lysates prepared from Reh cells and cells expressing His-cyclin D3 (T283A) were analysed in parallel (total lysate). (B) Reh cells were mock-transfected or transfected with His-cyclin D3 (wt) or His-cyclin D3 (T283A) vectors. Following transfection, whole cell extracts were prepared and incubated with Ni-NTA agarose beads to isolate His-cyclin D3 and its associated proteins. The recovered proteins were then separated by SDS-PAGE and immunoblotted with anti-CDK4, CDK6, or cyclin D3 antibodies. As positive control for CDK4, CDK6, and cyclin D3, 50 µg total cell lysates were prepared from mock-transfected Reh cells, and cells expressing His-cyclin D3 (T283A) were analysed in parallel (total lysates).

View larger version (11K): [in a new window]   Fig. 9. His-cyclin D3 (T283A) expression abrogates the inhibitory effect of forskolin on CDK6 kinase activity. Mock-transfected Reh cells or cells that were transfected with His-cyclin D3 (wt) or His-cyclin D3 (T283A) vectors were treated with or without forskolin (100 µM). Two hours after treatment cells were harvested and lysed. One aliquot of each whole cell extract (600µg) was immunoprecipitated (IP) with 2 µg anti-CDK6 antibody or non-immune rabbit serum (NRS) and utilized for an in vitro CDK6 kinase assay (upper panel). A second aliquot of each whole cell extract was immunoprecipitated with anti-CDK6 antibody or NRS. The recovered proteins were separated on SDS-PAGE and then subjected to western blotting (W) with CDK6 (middle panel) or cyclin D3 (lower panel) antibodies. The results shown are representative of three independent experiments.

View larger version (46K): [in a new window]   Fig. 10. Analysis of subcellular localization of cyclin D3 and GSK-3ß. (A) Reh cells were pretreated with the proteasome inhibitor MG-132 (10 µM) for 30 minutes before addition of forskolin (100 µM). Cells were cytospun onto coverslips at 2 hours after addition of forskolin, fixed in paraformaldehyde, and subjected to immunofluorescence microscopy after staining with cyclin D3 or GSK-3ß antibodies. The nuclear and cellular morphologies were visualized by Hoechst staining and phase contrast microscopy, respectively. One representative experiment of four is shown. (B) Reh cells were cultured in the presence or absence of 100 µM forskolin and harvested at the indicated time points. Subcellular fractions were prepared as described in Materials and Methods. Equal amounts of protein were resolved by SDS-PAGE and detected by immunoblotting with antibodies against GSK-3ß, actin and AKAP95. The blot shown is the representative of three independent experiments.

View larger version (25K): [in a new window]   Fig. 11. Association between cyclin D3 and GSK-3ß. (A) In vivo. Reh cells were treated with or without forskolin (100 µM) and harvested at the indicated times. Whole cell extracts were prepared and immunoprecipitated (IP) with anti-cyclin D3 antibodies. The immunoprecipitates were then subjected to western blot analysis (W) with the indicated antibodies. The right-most lane shows the background where cell extracts obtained from control cells were immunoprecipitated with antibodies against cyclin A. The blot shown in the upper panel is the representative of four independent experiments. Lower panel, the results in each experiment were quantified using a densitometer and the densitometric values of GSK-3ß were normalized with those of cyclin D3. The values obtained were then plotted with the value for cells not treated with forskolin set as 100%. Vertical bars indicate the mean ±s.e. (B) In vitro. 2 µg of bacterially expressed His-cyclin D3 (wt) or His-cyclin D3 (T283A) proteins bound to Ni-NTA agarose beads or Ni-NTA agarose beads alone (upper panel) were incubated with whole cell extracts (600 µg) from Reh cells stimulated with forskolin (100 µM) for 2 hours or cells left unstimulated. The bound proteins were resolved on SDS-PAGE and analyzed by immunoblotting with anti-GSK-3ß antibodies (middle panel). The blot shown is the representative of four independent experiments. The results in each experiment were quantified and plotted as described in A (lower panel). (C) 2 µg His-cyclin D3 (wt) protein bound to Ni-NTA agarose beads or Ni-NTA agarose beads alone (upper panel) were incubated with 0.6 µg purified GSK-3ß protein for 2 hours. The beads were then subjected to western blot analysis with anti-GSK-3ß antibody (lower panel). One representative experiment of three is shown. (D) Reh cells transfected with His-cyclin D3 (T283A) expression vector were treated with 100 µM forskolin or left untreated. His-cyclin D3 (T283A) was precipitated by incubating the cell extracts with Ni-NTA agarose beads and then analyzed by western blot analysis (W) with the indicated antibodies. The right-most lane (control) shows the background where cell extracts obtained from mock-transfected Reh cells were precipitated with Ni-NTA agarose beads. The blot shown is the representative of four independent experiments. The results in each experiment were quantified and plotted as described in A (lower panel).

View larger version (22K): [in a new window]   Fig. 12. Regulation of GSK-3ß activity by forskolin. (A) Reh cells were treated with or without forskolin (100 µM) and harvested at the indicated times. Whole cell extracts were prepared, immunoprecipitated with GSK-3ß antibodies and assayed for activity towards Tau. The specificity of the kinase reactions was examined by assaying the GSK-3ß activity recovered from control cells in the presence of 50 µM LiCl, or by immunoprecipitation of control cell extracts with non-immune mouse serum (NMS) (upper panel). The activities were then expressed as a percentage of the activity of GSK-3ß recovered from untreated cells (100%) and are mean ±s.e. of five independent experiments (lower panel). (B) Reh cells were treated as in A. Whole cell extracts were prepared, immunoprecipitated with GSK-3ß antibodies and assayed for activity towards GST-cyclin D1. The specificity of the kinase reactions was examined as described in A (upper panel). The activities were then plotted with the activity of GSK-3ß recovered from control cell set as 100% (lower panel). (C) Reh cells were treated as in A, total cell lysates were prepared and then subjected to immunoblotting with antibodies against GSK-3ß, GSK-3ß (Ser-9) and actin. The immunoblotblot shown is the representative of four independent experiments.

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