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First published online 15 April 2003
doi: 10.1242/jcs.00432

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AKAP149 is a novel PP1 specifier required to maintain nuclear envelope integrity in G1 phase

¹ Institute of Medical Biochemistry, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway
² Catholic University of Leuven, Division of Biochemistry, Campus Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium

View larger version (41K): [in a new window]   Fig. 1. PP1 transiently associates with the NE. (A) AKAP149 was immunoprecipitated from NEs after release of HeLa cells from mitotic arrest. Dissociation of PP1 from immunoprecipitated AKAP149 was monitored by immunoblotting. AKAP149 was also immunoblotted using anti-pS antibodies. Apparent Mr is shown in kDa. (B) Entry into the cell cycle after release from mitotic arrest was monitored by BrdU incorporation from time of release (arrow). Relative BrdU labeling intensity in two replicates is shown (n=40 cells/time point). (C) 293T, HUVEC, Jurkat and Bjab cells in G1 or S phase (2 and 15 hours post-release from mitotic arrest, respectively) were immunoblotted using anti-PP1 antibodies. NEs were prepared from the G1- and S-phase cells and immunoblotted.

View larger version (39K): [in a new window]   Fig. 2. AKAP149 does not co-precipitate with other known nuclear PP1 regulatory subunits. (A) Isolated nuclei and AKAP149-IPs from NEs were immunoblotted using antibodies against PP1, PNUTS, Sds22 and NIPP1. (B) 10 µM of AKAP149-derived RVXF or RAXF peptides were co-incubated with NIPP1-, PNUTS-, Sds22- or AKAP149-IPs for 1 hour, IPs were sedimented and dissociation of PP1 from the IPs was examined by western blotting of sedimented (P) and soluble (S) fractions.

View larger version (17K): [in a new window]   Fig. 3. AKAP149 is as a B-type lamin-specifier of PP1. (A) AKAP149-IPs were incubated with phosphorylase a and either 1 µM trypsin, 50 µM KNSRVTFSED (`RVXF') peptide, 50 µM KNSRATASED (`RAXA') peptide, or 1 µM I-2. Phosphorylase phosphatase activity was evaluated as the release of 32P measured by scintillation counting and expressed as a percentage (±s.d.) of activity after trypsinization in duplicate experiments. (B) AKAP149-IPs were incubated with immunoprecipitated B-type lamins phosphorylated in vitro with rat ßPKC and [32P]ATP. B-type lamin phosphatase activity was measured as in (A) in duplicate experiments. 50 µM SSPKGVLFSS (`RVXF') or SSPKGALFSS (`RAXF') peptides were used.

View larger version (36K): [in a new window]   Fig. 4. Targeting of active PP1 to NE-bound AKAP149 is required for B-type lamin assembly in vitro. (A) Nuclei were assembled in a nuclear reconstitution assay containing no peptide (–), 10 µM RVXF or 10 µM RAXF peptide. Where indicated, 1 µM I-2 or 100 nM okadaic acid (OA) were added one hour after initiation of nuclear reformation in the absence of peptide. PP1 distribution and B-type lamin assembly were analyzed by immunofluorescence two hours after initiation of the assembly reaction. Inset, nuclear membrane staining with 10 µg/ml DiOC6. Bar, 10 µm. (B) Percentages (±s.d.) of nuclei harboring perinuclear B-type lamin and perinuclear PP1 labeling under each condition (n300 nuclei/treatment in duplicate experiments).

View larger version (84K): [in a new window]   Fig. 5. AKAP149-derived RVXF peptides introduced into G1-phase nuclei inhibit DNA replication in vivo and in vitro. (A) Nuclei of early G1-phase HeLa cells were microinjected with peptide buffer (Mock), 10 nM RAXF or 10 nM RVXF peptide, together with a 150 kDa FITC-dextran (green label). Cells were cultured for eight hours with 100 µM BrdU. Mock-injected cells were also cultured with 100 µM BrdU and 50 µM aphidicolin (Aphid). BrdU incorporation was detected with anti-BrdU antibodies. Nuclei of S-phase cells were also injected with RVXF peptides, cultured for five hours and BrdU labeling monitored. Percentages (±s.d.) of cells labeled with BrdU are shown (n=40-45/treatment). Arrows point to non-injected cells. Bar, 10 µM. (B) Isolated G1-phase HeLa nuclei were loaded with RVXF or RAXF peptides or exposed to peptide buffer (Mock). Nuclei were incubated for three hours in S-phase extract containing [32P]dCTP, dNTPs and an ATP-regenerating system. [32P]dCTP incorporation was analyzed by autoradiography. G1-phase nuclei were also exposed to extract from G0- or S-phase cells under conditions promoting replication. (C) G1-phase nuclei were incubated in S-phase extract containing 0 mM or 1 mM olomoucine (Olo) and DNA synthesis was assessed as in A. (D) Nuclei purified from S-phase cells were loaded with peptides, incubated in S-phase extract and [32P]dCTP incorporation into replicated DNA was evaluated as in B.

View larger version (59K): [in a new window]   Fig. 6. AKAP149-derived RVXF peptides induce intranuclear lamina disassembly in G1 phase. (A) RVXF or RAXF peptides (10 nM) were injected into G1- or S-phase nuclei, as indicated, and 30 minutes later cells were fixed and analyzed by double immunofluorescence using anti-B-type lamin and anti-lamin A/C antibodies, and using anti-LBR and anti-nucleoporin (mAb414-reactive; Nups) antibodies. The injection solution contained phenol red (Ph, red). Between 45 and 55 cells were injected/treatment. Non-injected (input) G1 cells were also labeled. Bars, 10 µm. (B) Cells were metabolically labeled for 1.5 hours with 32P between 0.5 and 2 hours after release from mitotic arrest (G1) or while under mitotic block (M). B- or A-type lamins were immunoprecipitated and IPs analyzed by autoradiography and immunoblotting. A, B and C point to lamin A, B-type lamins and lamin C, respectively.

View larger version (32K): [in a new window]   Fig. 7. Transfection of AKAP149-derived RVXF peptides in G1 phase elicits lamin phosphorylation and disassembly. (A) RAXF or RVXF peptides were transfected for 1.5 hours using DOTAP in early G1-phase HeLa cells starting at 0.5 hour after release from mitotic arrest. Nuclei and NEs were purified and proteins immunoblotted using indicated antibodies. – indicates untreated cells; DOTAP indicates cells exposed to DOTAP alone. (B) Nuclei isolated from RVXF- or RAXF-transfected G1 cells were analyzed by immunofluorescence using anti-B-type lamin and anti-lamin A/C antibodies. Bar, 10 µm. (C) G1-phase cells were transfected with peptides as in A, while under metabolic 32P labeling. Nuclei were isolated, A- and B-type lamins were immunoprecipitated (IP) and analyzed by autoradiography (32P) and immunoblotting (Blot) using anti-lamin A/C and anti-B-type lamin antibodies. A, B and C point to lamin A, B-type lamins and lamin C, respectively.

View larger version (22K): [in a new window]   Fig. 8. RVXF peptides lead to apoptosis when introduced into G1-phase nuclei. (A) RAXF or RVXF peptides were transfected into G1-(left six panels) or S-phase (right four panels) cells as in Fig. 7 and cells were cultured for 12 hours. Cells were analyzed by TUNEL and DNA labeling using Hoechst 33342. Bars, 20 µm. (B) Proportions (±s.d.) of TUNEL-positive cells treated as indicated in G1 or S phase (n=300 cells/treatment in duplicate experiments).