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CDK1-mediated phosphorylation of the RII regulatory subunit of PKA works as a molecular switch that promotes dissociation of RII from centrosomes at mitosis

¹ Institute of Medical Biochemistry, University of Oslo, PO Box 1112 Blindern, N-0317 Oslo, Norway
² Ruhr-Universität Bochum, Institut für Physiologische Chemie, Abt. Für Biochemie Supramolekularer Systeme, 44801 Bochum, Germany
³ Centre de Recherches de Biochimie Macromoléculaire, CNRS, BP 5051, 1919 Route de Mende, 34033 Montpellier Cedex 1, France
⁴ Institute for Nutrition Research, University of Oslo, PO Box 1046 Blindern, N-0317 Oslo, Norway
⁵ Institut Curie, Biologie du Cycle Cellulaire et de la Motilité, 75248 Paris Cedex 05, France.

View larger version (47K): [in a new window]   Fig. 1. Subcellular distribution of RII in cell lines expressing wild-type or mutant (T54E) RII. (A) Analysis of RII expression in stably transfected cell lines. Interphase cells (2x106 cells per lane) were subjected to SDS-PAGE and immunoblotting with a polyclonal antibody against human RII. Lanes: 1, wild-type Reh (RII-deficient); 2, Reh-pMEP4 (vector transfected); 3, Reh-RII; 4, Reh-RII(T54E). (B) RII was immunoprecipitated from interphase lysates (4x107 cells per lane) of Reh-RII (lane 2) and Reh-RII(T54E) (lane 3) cells. Precipitation from Reh was performed as control (lane 1). Half of the immune precipitates were immunoblotted using a anti-RII polyclonal antibody (upper panel). The other half was incubated for 45 minutes at 22°C in EBS phosphorylation buffer with [-32P]ATP in the presence of CDK1 (9 pmol minute-1 µl-1), separated by SDS-PAGE, dried and subjected to autoradiography (lower panel). The positions of non-phosphorylated (51 kDa) and phosphorylated (53 kDa) RII are indicated. (C-E) Interphase (I) and mitotic (M) Reh cell lines (C), mouse A9 fibroblasts (D) and primary cultures of peritubular cells prepared from rat testes (fibroblast-like) (E) were analyzed by immunofluorescence using anti-RII (upper panel; red in C and D, green in E) or anti-RIIß mAbs (lower panel; green in E) and an affinity-purified polyclonal antibody against AKAP450 (green in C and D, red in E). DNA was stained with Hoechst 33342 (blue). Arrows indicate mitotic centrosomes. Bar: 10 µm. (F) CDK1 phosphorylation of purified recombinant human (lane 1) and bovine (lane 2) RII (150 ng of each). The positions of phosphorylated RII (lanes 1 and 2) and autophosphorylated cyclin B (49 kDa, lane 3) are indicated.

View larger version (28K): [in a new window]   Fig. 2. Different locations of wild-type and mutated RII-GFP in mitotic cells. Reh cells were transiently transfected with GFP (A,B), RII-GFP (C,D), RII(T54E)-GFP (E,F), RII(T54L)-GFP (G,H) or RII(T54V)-GFP (I,J), fixed and stained with affinity-purified anti-AKAP450 polyclonal antibodies to stain centrosomes (I,J) and with Hoechst 33342 to stain DNA (inserts). GFP fluorescence was examined in interphase (A,C,E,G,I) and mitotic (B,D,F,H,J) cells. Arrows indicate the centrosomal association of RII.

View larger version (20K): [in a new window]   Fig. 3. Displacement of RII from Reh-RII but not Reh-RII(T54E) centrosomes after CDK1 phosphorylation. (A) Proteins from the Triton-X-100-insoluble fractions of Reh-RII and Reh-RII(T54E) interphase cells were incubated (for 45 minutes at 22°C in EBS phosphorylation buffer with 100 µM ATP) in the presence (lanes 3,5,7,9) or absence (lanes 2,4,6,8) of CDK1. After incubation, pellet (P; lanes 2-5) and supernatant (S; lanes 6-9; lower panel shows longer exposure) were separated by centrifugation and subjected to SDS-PAGE, and RII was immunodetected with anti-hRII mAb. The level of immunoreactive RII in each lane (in upper panel) was evaluated by densitometric scanning and relative intensities are given in the lower panel. As a positive control, 100 ng of human recombinant RII was incubated with CDK1 before the immunodetection (lane 1). (B) The localization of RII was analyzed by immunofluorescence in centrosome-nucleus complexes of interphase Reh-RII and Reh-RII(T54E) cells before and after incubation with CDK1. The location of RII and centrosomes was determined using anti-RII mAb (red) and anti-affinity purified anti-AKAP450 polyclonal antibodies (green). Inserts: DNA was stained with Hoechst 33342 (blue). Bars: 10 µm. (C) The percentage of centrosomes staining for RII before (-) and after (+) CDK1 incubation is given (n=200; * means P<0.001). One representative experiment of two or more is shown for all panels.

View larger version (33K): [in a new window]   Fig. 4. (A) Co-immunoprecipitation of AKAP450 with anti-RII from Reh-RII and Reh-RII(T54E) cells in interphase and mitosis (obtained by double thymidine block). The upper panel shows immunoprecipitation of RIPA-buffer extracts of 200x106 cells using anti-RII antibody. As a control, Reh cells were immunoprecipitated with anti-AKAP450 antibody. Samples were separated by 4.5% PAGE containing 2 M urea, transferred to nitrocellulose and detected using anti-AKAP450 antibodies. The AKAP450 band is indicated by an arrow. The lower panel shows the RII content in the precipitated samples. (B) Immunospecificity of the affinity-purified polyclonal AKAP450 antibodies. Lane 1 contains purified centrosomes (untreated); lanes 2 and 3 contain Triton-X-100 insoluble and -soluble fractions from the centrosomal preparation in lane 1, respectively; lanes 4 and 5 contain RIPA-insoluble and -soluble centrosomal fractions, respectively. The blot was analyzed by RII overlay (lower panel) and western blotting using the affinity-purified polyclonal AKAP450 antibodies (upper panel).

View larger version (16K): [in a new window]   Fig. 5. Phosphorylated RII binds with different affinity to the RII-binding motifs in AKAP450. Purified recombinant human RII was radiolabeled using the catalytic subunit of either PKA (A) or CDK1 (B) and used for overlays on blots with different AKAPs (100 ng). Lanes: 1, GST-AKAP79 (178-427); 2, GST-AKAP149 (285-387); 3, GST-AKAP450 (1390-1595); 4, GST-AKAP450 (2327-2602) (numbers indicate the amino acid extent of the recombinant fragments used). (C) Precipitation of RII isoforms with different GST-AKAPs (20 nM of both R and AKAP). In some experiments, RII was preincubated with CDK1 before the GST precipitation. Lanes: 1, RII (50 ng); 2, RII after CDK1 preincubation; 3, GST + RII (negative control); 4, GST-AKAP450 (1390-1595) + RII; 5, GST-AKAP450 (1390-1595) + RII-P; 6, GST-AKAP450 (2327-2602) + RII; 7, GST-AKAP450 (2327-2602) + RII-P; 8, GST-AKAP79 (178-427) + RII; 9, GST-AKAP149 (285-387) + RII. Immunodetection was with anti-hRII mAb.

View larger version (23K): [in a new window]   Fig. 6. Interaction of the AKAP450 N-terminal binding domain with wild-type hRII (A,B) and hRII(T54D) (C). A sensor chip with 60 RUs of 8-AHA-cAMP immobilized on each surface was used to capture (A) 120 RU and (B,C) 500 RU of the RII subunit to a separate flow cell. (A) AKAP450 (1390-1595) (100 nM) was run over immobilized CDK1-phosphorylated (dotted line) and unphosphorylated (solid line) RII for 180 seconds. The indicated concentrations of AKAP450 (1390-1595) were run over the immobilized wild-type hRII (B) and hRII(T54D) (C) subunits for 300 seconds and the association phases of AKAP450 (1390-1595) were monitored in 20 mM MOPS, pH 7.0, 150 mM NaCl, 1 mM DTT and 0.005% surfactant P20. The dissociation phase was monitored for another 300 seconds after omitting the AKAP from the running buffer. The immobilization of the R subunits and the regeneration are not shown. (D) Aligned sequences of the RII binding domains in AKAP450 along with the consensus binding sequence (Vijayaraghavan et al, 1999). * denotes residues that differ between the two AKAP450 domains and from the consensus.

View larger version (51K): [in a new window]   Fig. 7. Microtubuli repolymerization assays in mutant and wild-type RII-transfected cells. Reh, Reh-RII and Reh-RII(T54E) cells were incubated for 1 hour at 4°C to depolymerize microtubules. Repolymerization was started by heating to 37°C and samples were stopped at different time points (0-15 minutes) by methanol fixation. Aster formation was then analyzed by immunofluorescence using anti--tubulin mAb. Mitotic (A) and interphase (C) cells were examined. The right-hand columns show staining by anti-RIIß antibody in cells prior to depolymerization. Arrows indicate the centrosomal association of RIIß. (B) The number of mitotic cells that formed asters after depolymerization was scored. Reh vs RII, P<0.005; Reh vs RII(T54E), P<0.001.

View larger version (19K): [in a new window]   Fig. 8. CDK1 phosphorylation serves as a molecular switch that regulates the association of RII with centrosomal AKAP450. We propose this model for the regulation of RII association with AKAP450 by CDK1 phosphorylation. In interphase cells, unphosphorylated RII is tightly bound to the centrosomes through interaction with AKAP450. At mitosis entry, RII is phosphorylated at T54 by CDK1 and the centrosomal anchoring is disrupted. The binding is restored at mitosis exit by dephosphorylation of RII by a threonine phosphatase.