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.
*Author for correspondence (e-mail: cathrine.carlson{at}basalmed.uio.no)
Accepted June 10, 2001
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SUMMARY |
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Key words: Centrosome, PKA, AKAP450, Mitosis, CDK1
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INTRODUCTION |
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Tethering of PKA to subcellular loci via A-kinase anchoring proteins (AKAPs) is important to mediate the effects of cAMP. AKAPs target PKA close to its substrates and in this way contribute specificity in the cAMP-PKA signaling system. Many AKAPs have been identified that are located in different cellular organelles and membranes (Colledge and Scott, 1999). Recently, two new AKAPs have been characterized that target PKA to centrosomes: AKAP350/AKAP450/CGNAP (Schmidt et al., 1999; Takahashi et al., 1999; Witczak et al., 1999) and pericentrin (Diviani et al., 2000). Whereas functions of AKAP450 are under investigation, pericentrin is a highly conserved component of the centrosomal matrix implicated in the organization of the mitotic spindle. Another AKAP (AKAP220) has also been localized to the centrosome area in developing germ cells and sperm (Reinton et al., 2000).
Centrosomes are major microtubule-organizing centers (MTOCs). During S phase, the cell duplicates its centrosome and, as prophase begins, the two daughter centrosomes separate and move to opposite positions in the cell. Each centrosome organizes its own array of microtubules. As the cell enters mitosis, the microtubule dynamics increase, enabling a rapid assembly and disassembly of the mitotic spindle. PKA modifies the microtubule dynamics and organization (Lamb et al., 1991), and it is anticipated that the centrosomal and microtubular localization of PKA are implicated in these functions. It has been shown that PKA switches off the effects of stathmin (Gradin et al., 1998), a centrosome- and microtubule-associated phosphoprotein involved in the regulation of microtubule dynamics.
Both RII and RIIß are found in the pericentriolar matrix of the centrosome during interphase (Keryer et al., 1999). At the onset of mitosis, the mitotic kinase CDK1 is associated with centrosomes (Bailly et al., 1989; Bailly et al., 1992) and RII
is phosphorylated by CDK1 on T54 and concomitantly dissociates from its centrosomal anchor (Keryer et al., 1998). Although RIIß contains a CDK1 phosphorylation site (T69) (Keryer et al., 1993), it does not detach from the centrosome at mitosis. Together with the observation that normal differentiated cells and cancer cells have centrosomal RIIß (whereas normal dividing cells only express RII
), cell cycle-dependent redistribution of RII
is interesting (Keryer et al., 1999). To study the mechanisms of redistribution of RII
and the functional implications of the detachment of RII
from centrosomes at mitosis, we made cell lines stably expressing wild-type and mutated RII
(T54E) on an RII
-deficient background (Reh cells; Taskén et al., 1993). Mutated RII
(T54E) was not phosphorylated by CDK1 and was retained at the mitotic centrosomes of the transfectants. CDK1 phosphorylation of wild-type RII
lowered the affinity for AKAP450 in vitro and dissociated RII
from purified centrosomes. This suggests that CDK1 phosphorylation serves as a molecular switch that regulates RII
association with centrosomal AKAPs.
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MATERIALS AND METHODS |
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Transfection and GFP constructs
Wild-type RII was mutated (Thr54 to Glu) as previously described (Keryer et al., 1998). The wild-type and mutated cDNAs were cloned into the expression vector pMEP4 (Invitrogen) as a KpnI/BamHI fragment and placed under the control of the human metallothionein IIa promotor. Reh cells (which are RII
deficient) were electroporated (320 V, 960 µF) with 15 µg of linearized constructs. Stably transfected cells were selected with hygromycin B (250 µg ml-1). RII
and RII
(T54E) with SacII/XhoI ends were amplified using the above constructs as templates and subcloned into pEGFP-N1 to yield constructs directing expression of RII
with green fluorescent protein (GFP) fused to the C terminus. Additional mutants, RII
(T54L)-GFP and RII
(T54V)-GFP were made by the mutation of Thr54 to Leu or Val, respectively. Reh cells (20x106) were transfected with 20 µg of DNA by electroporation (320 V, 960 µF), incubated for 24-48 hours and analysed for GFP fluorescence.
Antibodies and recombinant proteins
A mouse monoclonal antibody against human RII (developed by K. Taskén in collaboration with Transduction Laboratories) was used at 1 µg µl-1 for western blotting and 2.5 µg µl-1 for immunoprecipitation and immunofluorescence. In some western blotting experiments, a polyclonal antiserum against human RII
(Keryer et al., 1999) was used at 1:500 dilution. An affinity-purified polyclonal antibody raised against AKAP 450 (a gift from W. A. Kemmner (Max Planck Institute for Development Biology, Tuebingen, Germany)) was used at 25 µg µl-1 for immunofluorescence and 0.1 µg µl-1 for immunoprecipitation. In the microtubule repolymerization experiments, we used an affinity-purified anti-RII
polyclonal antibody (4 µg µl-1) and a monoclonal anti-
-tubulin at 0.1 µg µl-1 (Sigma, T-9026). A mouse monoclonal antibody against human RIIß (Transduction Laboratories) was used at a 1 µg µl-1 dilution for western blotting and immunofluorescence. HRP-conjugated anti-mouse IgGs (1:5000 dilution, Transduction Laboratories) and anti-mouse or anti-rabbit IgGs (1:10,000 dilution, Jackson Immunoresearch) were used as secondary antibodies. Recombinant human RII
wild-type was expressed as a glutathione-S-transferase (GST) fusion protein in the Escherichia coli strain BL21, purified and cleaved as described (Keryer et al., 1998). Two putative RII-binding domains in AKAP450 were expressed as fusion proteins referred to as GST-AKAP450 (amino acids 1390-1595) and GST-AKAP450 (amino acids 2327-2602). GST-AKAP79 (amino acids 178-427) and GST-AKAP149 (amino acids 285-387) were expressed as previously described (Herberg et al., 2000). For the surface plasmon resonance (SPR) experiments, wild-type and mutated RII
(Thr54 to Ala (T54A), Asp (T54D), Leu (T54L) or Val (T54V)) were cloned into pRSET, expressed in the E. coli strain BL21 and purified by using cAMP coated beads as described (Herberg et al., 2000).
Immunofluorescence
Immunofluorescence analysis of cells or nucleus-centrosome complexes was done as previously described (Collas et al., 1996). TRITC- or FITC-conjugated secondary antibodies were used at 1:100 dilution and DNA was stained with 0.1 µg ml-1 Hoechst 33342. Observations were made and photographs were taken as previously described (Collas et al., 1999).
Immunoprecipitation and phosphorylation
Whole cells were sonicated and extracted in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) and the lysate centrifuged at 13,000g. The supernatant was precleared with protein A/G agarose (1:25 dilution) and RII was immunoprecipitated using anti-RII
mAb (2.5 µg µl-1). To phosphorylate RII
, the beads containing the immune complexes were prewashed in EBS phosphorylation buffer (80 mM sodium ß-glycerophosphate, 20 mM EGTA, 15 mM MgCl2, 100 mM sucrose, 1 mM dithiothreitol, pH 7.2) before incubation with 100 µM ATP and purified starfish oocyte CDK1 (mitotic kinase) (Labbé et al., 1989) (9 pmol minute-1 µl-1) for 45 minutes at 22°C.
Electrophoresis and immunoblotting
Proteins were separated by 7.5% or 10% SDS-PAGE containing 16% glycerol in the separating gel or 4.5% PAGE containing 2 M urea and transferred by electroblotting to PVDF membranes. The filters were blocked in 5% non-fat dry milk in PBS for 1 hour, incubated overnight at 4°C with primary antibodies, washed for 1 hour in PBS with 0.1% Tween-20 and incubated with a horseradish-peroxidase-conjugated secondary antibody. Blots were developed by enhanced chemiluminescence (Amersham). For RII overlay, filters were blocked, incubated with [32P]-RII and washed in blotto/BSA (0.1% BSA, 0.02% Na-azide, 0.05% Tween-20, 5% nonfat dried milk in PBS) solution as described previously (Bregman et al., 1989).
CDK1 phosphorylation of RII in Triton-X-100-insoluble fractions and purified nucleus-centrosome complexes
Interphase cells (20x106) were washed in PHEM buffer (45 mM Pipes, 45 mM Hepes, 10 mM EGTA, 5 mM MgCl2, pH 6.9) containing anti-proteases (10 µg ml-1 each of antipain, chymostatin, leupeptin and pepstatin A) and phosphatase inhibitors (1 µM okadaic acid and 0.1 mM sodium orthovanadate). The cells were pelleted (400g) and then extracted in the same buffer containing 0.5% Triton X-100; Triton-X-100-soluble and -insoluble fractions were separated by centrifugation. The Triton-X-100-insoluble fraction was sonicated twice for 10 seconds each, washed twice in EBS phosphorylation buffer (described above) before incubation for 30 minutes at 22°C with 100 µM ATP and CDK1 (30 pmol minute-1 µl-1). The pellet and supernatant were subsequently separated by centrifugation and both fractions were boiled in Laemmli buffer and analyzed for the amount of RII by western blotting. Nucleus-centrosome complexes were purified according to Maro and Bornens (Maro and Bornens, 1980) from cells by resuspension in cold buffer containing 0.25 M sucrose, 10 mM NaCl, 3 mM MgCl2, 0.5 mM PMSF, 1 mM dithiothreitol, 10 mM Tris-HCl, pH 7.4, with protease inhibitors. NP-40 was added to a final concentration of 0.5% and the cells were disrupted by vortexing for 15 seconds. Nucleus-centrosome complexes were pelleted at 200g for 10 minutes and washed in PHEM buffer containing 0.5% Triton X-100. After pelleting, the complexes were resuspended in EBS phosphorylation buffer and incubated in presence or absence of CDK1 and 100 µM ATP.
RII overlays
Purified recombinant human RII and purified bovine RII
were radiolabeled by purified CDK1 or catalytic subunit (C) of PKA and [
-32P] ATP. RII
(1.5 µg) was incubated with CDK1 (24 pmol minute-1 µl-1) in EBS buffer (described above) containing 0.7 µCi µl-1 of [
-32P] ATP for 1 hour at 22°C. Phosphorylation by PKA was done using 0.7 µCi µl-1 of [
-32P] ATP and 24 pmol minute-1 µl-1 of active C for 1 hour at 0°C. PKA- and CDK1-labeled RII
(6 nM final concentration each; 4.9x105 cpm µg-1 and 2.4x104 cpm µg-1, respectively) was purified by gel filtration (G-25 sepharose) and used in a modified western blot protocol as previously described (Bregman et al., 1989).
GST precipitation
R subunit (50 ng) was incubated in 20 µl EBS phosphorylation buffer (described above) containing 100 µM ATP for 1 hour at 22°C in presence or absence of purified CDK1 (10 pmol minute-1 µl-1). The R subunit was then diluted to 20 nM, mixed with 20 nM of different GST-AKAP fragments in a buffer containing 50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1 mM dithiothreitol, 1 mM PMSF, 0.1% Triton X-100, 20 mM EDTA (to inactivate CDK1), 5 mM benzamidine, protease inhibitors and incubated at room temperature for 30 minutes with rotation. Subsequently, 25 µl of glutathione-agarose beads were added and incubation continued further for 2 hours at 4°C with rotation, after which beads were pelleted by centrifugation at 1000g for 5 minutes and washed three times in 300 µl of the same buffer. Precipitates were eluted by boiling in SDS-sample buffer, subjected to SDS-PAGE and immunodetection of RII.
Surface plasmon resonance (SPR)
Recombinant RII was purified over a 8-amino-hexyl-amino-cAMP resin (Biolog, Bremen) and quality tested as described before (Herberg et al., 2000). To obtain cAMP-free RII
, RII
was unfolded with 6 M urea and refolded in buffer A (150 mM NaCl, 20 mM MOPS, pH 7.0, 0.005% surfactant P20 (Biacore AB)) (Buechler et al., 1993). Studies on the interaction between the RII
of PKA and AKAP protein were performed by SPR spectroscopy using a Biacore 2000 instrument (Biacore AB, Sweden) and a CM5 chip coated with 8-AHA cAMP as described before (Herberg et al., 2000). For review of the SPR technique, see Szabo et al. (Szabo et al., 1995). The surface concentration of wild-type and mutant RII
proteins was adjusted to 120 or 500 RU for analysis of interaction with GST-AKAP450 (1390-1595) and GST-AKAP450 (2327-2602) (fusions of GST with the two putative RII-binding domains or AKAP450 (Schmidt et al., 1999, Witczak et al., 1999)). A response (i.e. a change in the resonance signal) of 1000 Relative Units (RU) corresponds to a change in surface concentration on the sensor chip of about 1 (ng protein) mm-2 (Stenberg and Nygren, 1991).
Microtubule repolymerization assay
Cells were settled onto freshly L-lysin-coated coverslips for 30 minutes at 25°C. Microtubules were depolymerized at 4°C for 1 hour in medium. The polymerization was initiated by incubating the cells in RPMI medium at 37°C. Microtubule regrowth was stopped after 0-15 minutes by fixing with ice-cold 100% methanol. The cells were prepared for immunofluorescence as described above, except that a cytoskeleton stabilization buffer (PHEM buffer) was used instead of PBS.
Statistical analysis
For comparison of two groups, the Mann-Whitney U test was used. Statistical analysis was performed using Statistica (Statsoft, Tulsa, OK). P values are two-sided and are considered significant when <0.05.
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RESULTS |
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Subcellular location of RII-GFP and RII
(T54E, L or V)-GFP in Reh cells
We next examined the distribution of wild-type and mutant RII by GFP tagging in transiently transfected Reh cells. To avoid disruption of the dimerization and AKAP-binding domains of RII
, the GFP coding sequence was fused to the C terminus of the open reading frame of wild-type or mutated RII
in the vector pEGFP-N1 and transfected into Reh cells. As a negative control, we transfected Reh cells with vector expressing GFP only. Examination of transfected cells by fluorescence microscopy revealed that GFP alone was localized both in the cytoplasm and nucleus-chromatin of interphase and mitotic cells (Fig. 2A,B). RII
-GFP, RII
(T54E)-GFP and RII
(T54L)-GFP were localized to the Golgi/centrosome region in interphase cells (Fig. 2C,E,G). In mitotic cells, most of the RII
-GFP was localized to the cytoplasm but some weaker fluorescence also overlapped with chromatin (Fig. 2D). By contrast, RII
(T54E)-GFP and RII
(T54L)-GFP were still attached to the mitotic centrosomes (Fig. 2F,H). We conclude that the GFP fusion proteins are localized similarly to the proteins expressed in the stable transfectants. Cells transfected with RII
(T54V)-GFP did not display any centrosomal RII
-GFP staining (Fig. 2I,J, centrosomes evident from double staining with anti-AKAP450 antibody), although centrosomes were visualized by staining with anti-AKAP450 antibody (red). These observations are consistent with the low affinity of AKAP450 for this mutant protein as measured by SPR (Table 1).
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RII(T54E), but not RII
, immunoprecipitates with AKAP450 in mitotic cells
RII has been shown to bind the A-kinase anchoring protein AKAP450 in purified centrosomal fractions from KE37 lymphoblast cells (Witczak et al., 1999). We analyzed the association of the RII
with AKAP450 in Reh-RII
and Reh-RII
(T54E) cells by immunoprecipitation. Mitotic and interphase cells were lysed and the RII
-AKAP450 complex was immunoprecipitated using anti-RII
antibody. Precipitates were separated on a 4.5% acrylamide gel containing 2 M urea, and AKAP450 was detected by anti-AKAP450 antibody. As a control, we immunoprecipitated AKAP450 with anti-AKAP450 antibody from wild-type Reh cells. The presence of RII
and RII
(T54E) was analyzed by immunoblotting. AKAP450 immunoprecipitated with RII
from interphase lysates of both Reh-RII
and Reh-RII
(T54E) (Fig. 4A). In the mitotic lysates, AKAP450 was detected in the RII
-immunoprecipitates from Reh-RII
(T54E) but not in precipitates from Reh-RII
. This is consistent with the cell cycle redistribution of RII
to chromatin at mitosis (Fig. 1C,D). We conclude that the redistribution of RII
from centrosomes at mitosis occurs by dissociation from AKAP450. As a control, the immunospecificity of the affinity-purified polyclonal AKAP450 antibodies was assessed by immunoblot analysis of centrosomal preparations (Fig. 4B, upper panel, lane 1). This revealed a 450-kDa band that was resistant to detergent extraction (lanes 2 and 3) and partly solubilized by RIPA buffer extraction (lanes 4 and 5). RII overlay of the same blot to detect AKAPs (Fig. 4B, lower panel) demonstrated a band of the same mobility and distribution as that of the immunoreactive band (upper panel). We conclude that the immunoreactivity of the antibody is consistent with the characteristics of AKAP450.
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DISCUSSION |
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The interaction of RII with the N-terminal PKA binding domain in AKAP450 (1390-1595) was sensitive to CDK1 phosphorylation in filter overlay and GST precipitations. Furthermore, detailed analysis by SPR of the interaction of RII
with AKAP450 when RII
was immobilized on a cAMP chip showed clearly that binding to AKAP450 (1390-1595) was reduced when RII
was phosphorylated by CDK1 or when threonine was mutated to aspartic acid, mimicking a phospho-threonine. By contrast, binding to AKAP 450 (2327-2602) was not affected by any of the T54 mutations. The fact that mutagenesis did not affect binding to the C-terminal binding site could indicate that this site is not exposed on the surface of the native protein. Alternatively, this site might be occupied by RIIß. Preliminary data on affinity of RIIß to the N- and C-terminal binding sites of AKAP450 (not shown), indicate that the C-terminal site might be occupied by RIIß. Comparison of the two binding domains of AKAP450 might give some indication of determinants for sensitivity to CDK1 phosphorylation in the AKAP, but such determinants might also reside outside the consensus binding domain. The observation that mutation of T54 to large residues such as V and D (loss of binding) and E (strong binding) distinctly affect interaction with AKAP450 as well as with AKAP95 (Landsverk et al., 2001), could indicate that T54, in addition to the well-characterized dimerization- and AKAP-binding motif in RII
(Newlon et al, 1999), might be an important determinant for interaction with specific AKAPs. Although amino acids 45-75 in RII
is the region of RII
with the lowest conservation between species (Foss et al., 1997;
yen et al., 1989), we found potential CDK1 phosphorylation sites in mouse (S49) and bovine (T53) RII
by homology alignment. Thus, CDK1 phosphorylation of T54 is not a mechanism specific for human RII
and cell-cycle-dependent redistribution of RII
subunit also appears to operate in other species. In the rat, however, RII
was not redistributed, which is consistent with the lack of an N-terminal CDK1 phosphorylation site in the rat sequence. Interestingly, RIIß seemed to associate more with mitotic chromatin in the rat (this study) (Landsverk et al., 2001). To our knowledge, distribution of RII
and RIIß during the cell cycle has not been examined in vivo in the rat. The observations made here might suggest some redistribution of RIIß instead of RII
in rat, which could be pursued in future studies.
PKA has been shown to be involved in maintenance of the interphase microtubule network (Fernandez et al., 1995). PKA also switches off the effect of the destabilizing factor stathmin, which in turn promotes increased tubulin polymerization (Gradin et al., 1998). Overexpression of stathmin mutants that cannot be phosphorylated prevents the assembly of the mitotic spindle (Gradin et al., 1998). To address the function of the PKA-AKAP450 complex associated with the centrosome, we analyzed the microtubule nucleating activity of the transfected cell lines and showed that absence or presence of PKA-RII at mitosis had distinct effects on nucleation from mitotic centrosomes. This suggests that dissociation of PKA from the centrosome at mitosis is important for spindle formation. Based on our previous observations, centrosomal RIIß is present in differentiated non-dividing cells and in all cancer cell lines examined, but not in normal cycling cells (Keryer et al., 1999). For this reason, we examined levels of RIIß and found that Reh and Reh-RII
cells both had centrosomal RIIß. In the cell lines studied here, this might rescue effects that would come out more strongly in primary cycling cells. Also, requirements for accurate and timely progression through mitosis and segregation of chromosomes might be greater in the body than in cultured cancer cells and requirements for RII
redistribution correspondingly stronger. Another possibility is that not all of PKA should be dissociated from centrosomes. This notion would be consistent with observations made with mutation of the PKA phosphorylation site in stathmin, which also produces aberrant spindle formation, indicating a PKA requirement either just before entry into mitosis or during mitosis.
In summary, we propose a model in which CDK1 phosphorylation of T54 in RII acts as a molecular switch that regulates the association and dissociation of PKA with centrosomal AKAP450 (Fig. 8); preliminary data indicate a similar distribution of C subunit (data not shown). During interphase, PKA is localized to the centrosome through interaction of RII
with AKAP450. Upon mitosis entry, CDK1 is accumulated at the centrosome (Bailly et al., 1989) and RII
is phosphorylated on T54 by CDK1. The switch is turned off and PKA dissociates from the centrosomal AKAP450. At mitosis exit, a yet-unknown threonine phosphatase dephosphorylates RII
and turns the switch back. The affinity of RII
for AKAP450 is now higher and the PKA-RII
-AKAP450 complex reforms. In a parallel with this, we show an opposite situation with RII
interaction with AKAP95. CDK1 phosphorylation of RII
on T54 also seems to constitute a molecular switch controlling the association of PKA with chromosome-bound AKAP95 at mitosis (Landsverk et al., 2001; Collas et al., 1999). Anchoring of phosphorylated PKA to chromatin-associated AKAP95 is required to prevent premature decondensation of chromatin at mitosis. The two models fit together into a conceptual framework in which the association of RII
to the centrosome or chromatin is determined by the phosphorylation state of RII
, which is regulated by the mitotic kinase CDK1 and a threonine phosphatase. The threonine phosphatase, however, remains to be identified.
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ACKNOWLEDGMENTS |
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