1 Institute of Medical Biochemistry, Faculty of Medicine, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway
2 Ruhr Universität Bochum, Institut für Physiologische Chemie, MA2 Nord Raum 40, 44780 Bochum, Germany
*Author for correspondence (e-mail: philippe.collas{at}basalmed.uio.no)
Accepted June 10, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Mitosis, Chromosome Condensation, Phosphorylation, PKA, AKAP95
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The specificity of responses to cAMP is mediated by targeting of the RII subunit of PKA to organelles, cytoskeleton or membranes through associations with A-kinase-anchoring proteins (AKAPs) (Colledge and Scott, 1999). AKAP95 is a 95 kDa AKAP that is exclusively intranuclear during interphase (Coghlan et al., 1994; Eide et al., 1998). AKAP95 co-fractionates primarily with the nuclear matrix (Collas et al., 1999), but as no RII has been detected in interphase nuclei (Eide et al., 1998), the role of AKAP95 in the nucleus remains elusive. Nevertheless, recent studies have shown that AKAP95 is required for chromosome condensation at mitosis (Collas et al., 1999) by acting as a receptor for the condensin complex (Steen et al., 2000). AKAP95 mostly co-fractionates with mitotic chromosomes and associates with RII
to form a complex necessary to maintain chromosomes in a condensed form throughout mitosis (Collas et al., 1999). These observations suggest that the interaction between AKAP95 and RII
is cell-cycle dependent, but what regulates this interaction is unknown.
The subcellular localization and activity of protein kinases is often altered by protein phosphorylation. In contrast to RI, RII can be autophosphorylated by the catalytic subunit of PKA (Erlichman et al., 1983). Human RIIß is also phosphorylated on threonine 69 (T69) by the mitotic kinase CDK1 (Keryer et al., 1993). Deletion and mutation analyses have shown that human RII is also autophosphorylated on S99 throughout the cell cycle, and is hyperphosphorylated on T54 at mitosis and by purified CDK1 in vitro (Keryer et al., 1998). Furthermore, whereas RII
is associated with the centrosome-Golgi area during interphase, it is dislocated from this region at metaphase (Keryer et al., 1998). RII
is also solubilized from a particulate fraction of interphase cell extracts upon exposure to CDK1, suggesting that CDK1 phosphorylation of RII
at mitosis alters its subcellular localization (Keryer et al., 1998).
We report here that, at mitosis and in a mitotic cell extract, association of human RII with chromosome-bound AKAP95 requires phosphorylation of RII
on T54. Disrupting AKAP95-RII
anchoring or depleting RII
elicits premature chromatin decondensation, suggesting that the AKAP95-RII
complex plays a role in maintaining chromosomes condensed during mitosis. Nuclear reconstitution in vitro is accompanied by threonine dephosphorylation and dissociation of RII
from decondensing chromatin prior to reassembly of nuclear membranes. Our results suggest that T54 phosphorylation of RII
constitutes a molecular switch controlling anchoring of RII
to chromatin-bound AKAP95 at mitosis and its dissociation at mitosis exit.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies and recombinant proteins
Affinity-purified rabbit polyclonal antibodies against rat AKAP95 (Upstate Biotechnology) and monoclonal antibodies (mAbs) against human AKAP95 (mAb47; Transduction Laboratories) were described previously (Coghlan et al., 1994; Collas et al., 1999). Anti-human RII and anti-human RIIß mAbs were from Transduction Laboratories (Eide et al., 1998) and cross-react with rat RII
. Polyclonal antibodies against human lamin B receptor (LBR; a gift from J.-C. Courvalin, Institut Jacques Monod, Paris, France) were described previously (Collas et al., 1996). The anti-phosphothreonine mAb (anti-pT) was from New England Biolabs and the rabbit anti-phosphoserine (anti-pS) antibody was from Zymed.
The Ht31 peptide derived from the AKAP Ht31 was used as a specific inhibitor of AKAP-RII interaction (Carr et al., 1991). Control Ht31-P peptides contained two isoleucines mutated to prolines, disrupting the amphipathic helix structure of Ht31. Recombinant human wild-type RII and the RII
(T54A), RII
(T54D), RII
(T54E), RII
(T54L) and RII
(T54V) mutants were expressed and purified as described (Keryer et al., 1998; Tasken et al., 1993).
Interphase nuclei and mitotic chromatin
To isolate interphase Reh nuclei, Reh cells (2x106 ml-1) were suspended in 1 ml of hypotonic buffer (10 mM Hepes, pH 7.5, 2 mM MgCl2, 25 mM NaCl, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail). NP-40 was added to 0.5% and nuclei sedimented at 1000 g for 5 minutes. Nuclei were resuspended in ice-cold buffer N (hypotonic buffer with 250 mM sucrose), sedimented at 1000 g and resuspended in buffer N to 2x107 nuclei ml-1. Nuclei were used fresh or frozen at -80°C in buffer N containing 70% glycerol.
A soluble mitotic chromatin fraction was prepared from mitotic Reh or PC12 cells essentially as described previously, by digestion of chromosomes with 5 U ml-1 of micrococcal nuclease (MNase; Sigma) in TKM buffer (Collas et al., 1999). Released solubilized chromatin was separated from MNase-insoluble material by sedimentation and held on ice until use. Chromatin masses obtained in mitotic extract were retrieved by sedimentation at 1000 g through 1 M sucrose, washed in TKM buffer and solubilized with MNase as above.
Mitotic extracts
Mitotic Reh, Reh-RII or RII
(T54E) cells, as indicated, were washed twice in ice-cold lysis buffer (20 mM Hepes, pH 8.2, 5 mM MgCl2, 10 mM EDTA, 1 mM DTT, 20 µg ml-1 cytochalasin B and protease inhibitors) and sedimented. The cell pellet was resuspended in 1 volume of lysis buffer and incubated for 30 minutes on ice before Dounce homogenization using a tightly fitting glass pestle. The lysate was centrifuged at 10,000 g and the supernatant cleared at 200,000 g to produce a mitotic cytosolic extract (Collas et al., 1999). Extracts were aliquoted, frozen in liquid nitrogen and stored at -80°C.
In vitro nuclear disassembly and reconstitution
A nuclear disassembly assay consisted of 20 µl mitotic Reh, Reh-RII or Reh-RII
(T54E) extract, as indicated, and 1 µl nuclear suspension (105 nuclei). Reactions were started by the addition of an ATP-generating system and allowed to proceed at 30°C for 2 hours (Collas et al., 1999). Mitotic extracts supported chromatin condensation without DNA degradation (Steen et al., 2000). Chromatin condensation was monitored by staining aliquots with 0.1 µg ml-1 Hoechst 33342 and assessed by irregular and compact morphology of the chromatin. For immunofluorescence analysis of chromatin, chromatin masses were washed at 1000 g through a 1 M sucrose cushion.
The in vitro nuclear reassembly assay was adapted from that of Burke and Gerace (Burke and Gerace, 1986). Mitotic Reh-RII cells were Dounce homogenized in an equal volume of KHM (78 mM KCl, 50 mM Hepes (pH 7.0), 4 mM MgCl2, 10 mM EGTA, 8.4 mM CaCl2, 1 mM DTT, 20 µM cytochalasin B and protease inhibitors). The lysate was supplemented with the ATP-generating system and incubated for up to 75 minutes at 30°C. Chromosome decondensation was assessed after DNA labeling by the expanded and nearly spherical morphology of the chromatin. Nuclear envelope reformation was monitored by immunofluorescence using anti-LBR antibodies.
Premature chromatin decondensation assay
A condensed Reh-RII chromatin substrate prepared in mitotic Reh-RII
extract was recovered by sedimentation through sucrose, washed in lysis buffer and incubated for up to 3 hours in 20 µl of fresh Reh-RII
mitotic extract containing 500 nM Ht31. Alternatively, condensed Reh, Reh-RII
or Reh-RII
(T54E) chromatin were obtained during a 2 hour incubation in the relevant mitotic extracts, and incubated in the same extracts for another 2 hours. Chromatin morphology was examined by Hoechst staining. Premature chromatin decondensation (PCD) referred to swelling of chromatin into ovoid or spherical objects with no discernable chromosomes while the extract remained mitotic, as judged by elevated histone H1 kinase activity (Collas et al., 1999).
Alkaline phosphatase treatment
Mitotic Reh chromatin was solubilized with MNase and incubated for 45 minutes at room temperature with either 100 U ml-1 calf intestinal alkaline phosphatase (APase; Promega) or as control 100 U ml-1 APase plus 20 mM of the phosphatase inhibitor sodium vanadate (Sigma). APase- and APase+sodium-vanadate-treated chromatin fractions were used for immunoprecipitations of RII and AKAP95.
Immunological procedures
SDS-PAGE and immunoblotting were performed as described earlier (Collas et al., 1999) with 30 µg protein per lane and using the following antibodies: anti-AKAP95 mAb47 (1:250 dilution), anti-RII mAb (1:250), anti-pS (1:250) and anti-pT (1:5000). Blots were revealed by enhanced chemiluminescence. Immunoprecipitations were preformed as reported earlier (Collas et al., 1999). Whole cells or whole in vitro nuclear disassembly or reassembly reaction mixtures were sonicated in immunoprecipitation (IP) buffer (10 mM Hepes, pH 8.2, 10 mM KCl, 2 mM EDTA, 1% Triton X-100 and protease inhibitors) and the lysate centrifuged at 15,000 g. The supernatant was pre-cleared with protein A/G agarose and immunoprecipitations were carried out with relevant antibodies (1:50 dilutions). Protein of immune complexes were eluted in SDS sample buffer. RII
and AKAP95 were also immunoprecipitated from cytosolic fractions and mitotic chromatin solubilized with MNase. Immunofluorescence analysis of cells or chromatin masses was done as described (Collas et al., 1996). Antibodies were used at a 1:100 dilution and DNA was stained with 0.1 µg ml-1 Hoechst 33342. Observations were made and photographs taken as described previously (Collas et al., 1999).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The differential distribution of human RII and RII
(T54E) at mitosis was confirmed by immunoblotting analysis of fractionated cells. Mitotic Reh-RII
and Reh-RII
(T54E) cells were subjected to Dounce homogenization and centrifuged at 15,000 g. The 15,000 g pellet (P15) was extracted with 1% Triton X-100, sedimented, suspended, digested with MNase and sedimented again to produce a soluble chromatin fraction and a particulate fraction. The 15,000 g supernatant (S15) was fractionated at 200,000 g into soluble (S200) and particulate (P200; membrane-enriched) fractions. In agreement with immunofluorescence data, AKAP95 was detected in all fractions, except P200, of both cell types, albeit primarily in the MNase-soluble chromatin fraction (Fig. 2A, MNase[S]). Both RII
and RII
(T54E) were detected in particulate (P15) and soluble (S200) fractions. However, most of the particulate RII
co-fractionated with MNase-soluble chromatin, whereas essentially all particulate RII
(T54E) was found in MNase-insoluble sediments (Fig. 2A, MNase(P)). Fractionation of mitotic rat PC12 neuronal cells corroborated our immunofluorescence observations. Whereas both AKAP95 and RII
were detected in a P15 fraction, rat RII
did not co-fractionate with AKAP95 in the MNase-soluble mitotic chromatin fraction but remained in the MNase particulate fraction (Fig. 2B). RIIß did partly co-fractionate with the MNase-soluble chromatin fraction (Fig. 2B).
|
RII, but not RII
(T54E), interacts with chromatin-associated AKAP95 at mitosis
We have previously reported the interaction of RII with AKAP95 in a mitotic HeLa cell chromatin fraction (Collas et al., 1999). Immunoprecipitations of AKAP95 or RII
from interphase and mitotic Reh-RII
or Reh-RII
(T54E) cells showed that RII
and AKAP95 co-precipitated only at mitosis (Fig. 3A). By contrast, RII
(T54E) and AKAP95 did not co-precipitate. Immunoprecipitations performed with fractionated cells revealed that AKAP95 and RII
co-precipitated from MNase-soluble mitotic chromatin and from cytosol (Fig. 3B). AKAP95 and RII
(T54E) did not co-precipitate from either fraction (Fig. 3B), despite the presence of both proteins in the cytosol of Reh-RII
(T54E) cells (Fig. 2). Therefore, only wild-type RII
is capable of interacting with AKAP95 in a mitotic chromatin fraction.
|
|
The dynamics of the chromatin-associated human AKAP95-RII complex was examined using a cell-free extract that mimics mitotic nuclear disassembly. Purified interphase Reh nuclei (devoid of RII
) were incubated in mitotic extracts derived from Reh, Reh-RII
or Reh-RII
(T54E) cells. Within 2 hours, the chromatin condensed into compact masses as judged by DNA staining (Fig. 5A). As expected from in vivo data, AKAP95 was detected on chromatin by immunofluorescence in all extracts (Fig. 5A). RII
, absent in Reh extract, was detected on chromatin condensed in Reh-RII
extract and co-localized with AKAP95 (Fig. 5A). However, no RII
labeling occurred on chromatin condensed in Reh-RII
(T54E) extract. The location of wild-type RII
on chromatin was verified by immunoblotting analysis of purified chromatin fractions (Fig. 5B). Moreover, AKAP95 and RII
co-immunoprecipitated from condensed chromatin fractions solubilized with MNase, suggesting that, as in vivo, AKAP95 and RII
were associated (data not shown). Finally, when threonine phosphorylation of chromatin-bound AKAP95 and RII
was examined, we found that as in vivo, AKAP95 was not phosphorylated, whereas RII
was threonine phosphorylated (Fig. 5C). As RII
(T54E) was not threonine phosphorylated, this implies that RII
was threonine phosphorylated solely on T54 in the mitotic extract. Collectively, these results indicate that in vitro, RII
is recruited on chromatin where it interacts with AKAP95, whereas RII
(T54E) remains soluble. As in vivo, RII
recruitment to chromatin correlates with T54 phosphorylation.
|
|
The PCD assay was subsequently used to determine whether the T54E mutation of RII affected chromatin structure in mitotic extract. Reh chromatin was condensed in mitotic Reh extract (devoid of RII
), recovered and incubated for another 2 hours in Reh, Reh-RII
or Reh-RII
(T54E) mitotic extract. Whereas chromatin remained condensed in Reh-RII
extract, it underwent PCD in Reh extract and Reh-RII
(T54E) extract (Fig. 6E). Thus, both absence of RII
in the extract and the T54E mutation of RII
correlate with PCD.
A role of wild-type RII in preventing PCD was shown by the induction of PCD in Reh-RII
extract immunodepleted of RII
(Fig. 6F). PCD was largely inhibited in Reh extract containing 11 nM RII
, whereas 11 mM of RII
(T54A, E, L or V) were ineffective (Fig. 6F). By contrast, RII
(T54D) was capable of inhibiting PCD (Fig. 6F), an observation consistent with our previous data. In any situation, H1 kinase activity remained at mitotic levels, indicating that none of the treatments promoted exit of the extracts from mitosis (data not shown). These results indicate that PKA type II plays an essential role in the regulation of chromatin dynamics during mitosis. The data also suggest that mitotic T54 phosphorylation and subsequent association of RII
with AKAP95 on chromosomes is critical to maintain chromosomes condensed during mitosis.
Nuclear reassembly correlates with threonine dephosphorylation of RII and dissociation of RII
from AKAP95 and chromosomes
Our results suggest that T54 phosphorylation of RII controls AKAP95-RII
association on chromatin, which in turn affects chromatin dynamics during mitosis. According to this hypothesis, the reformation of nuclei at the end of mitosis should correlate with threonine dephosphorylation of RII
and/or dissociation of RII
from its chromatin-bound anchor.
To test this possibility, we used a cell-free system reminiscent of that developed by Burke and Gerace (Burke and Gerace, 1986), which supports nuclear reconstitution, and determined whether the AKAP95-RII interaction was altered during nuclear reassembly. Crude lysates of mitotic Reh-RII
cells were supplemented with an ATP-generating system and incubated at 30°C. Chromosome decondensation and reformation of the nuclear envelope were monitored by DNA staining and by immunofluorescence labeling of lamin B receptor (LBR), a marker of the inner nuclear membrane (Worman et al., 1990). LBR assembles onto chromosomes as early as anaphase (Chaudhary and Courvalin, 1993) and thus constitutes a reliable marker of early nuclear membrane reassembly. Distribution of RII
was monitored simultaneously by immunofluorescence.
Decondensation of the mitotic chromosomes took place within 75 minutes (Fig. 7A). Remarkably, RII staining (green) disappeared as LBR (red) gradually assembled on the chromatin surface. By 30 minutes, most RII
was removed from chromatin, whereas nuclear membranes had not fully reformed. By 75 minutes, nuclear membranes were reassembled and no RII
was detected on chromatin. Immunoblotting analysis of soluble and sedimented chromatin fractions before and after nuclear reassembly showed that, whereas AKAP95 remained associated with insoluble material, all detectable RII
was solubilized (Fig. 7B). Moreover, within 10 minutes of nuclear reassembly, RII
was fully threonine dephosphorylated, whereas strong threonine phosphorylation was detected in a control incubation in extract for 75 minutes without the ATP-generating system (M, 75 min).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We propose a model in which threonine phosphorylation of RII constitutes a molecular switch controlling the association of RII
with AKAP95, which in turn affects chromosome dynamics during mitosis (Fig. 8). During interphase, AKAP95 and RII
are located in distinct compartments separated by the nuclear envelope. At mitosis entry, AKAP95 associates with chromatin (Steen et al., 2000) and RII
is phosphorylated on T54 by CDK1. This turns on a molecular switch eliciting binding of, presumably, the PKA holoenzyme to AKAP95 via RII
(switch ON). Anchoring of RII
to chromatin-bound AKAP95 is required to prevent premature chromatin decondensation during mitosis. At mitosis exit, dephosphorylation of RII
by a threonine phosphatase (switch OFF) elicits dissociation of RII
from AKAP95 before the nuclear envelope reforms. This relieves the inhibition of chromatin decondensation imposed by the RII
-AKAP95 complex (switch OFF), enabling chromosome decondensation as the nucleus reassembles. In vivo, released RII
is presumably targeted back to the centrosome-Golgi area, where it is predominantly anchored during interphase (Eide et al., 1998). Moreover, during decondensation, AKAP95 remains associated with chromatin until it is redistributed to the nuclear matrix upon nuclear reformation (P.C., unpublished). Thus, the release of RII
precedes that of AKAP95, arguing that the AKAP95-RII
complex is disrupted rather than being detached as a whole from the chromatin.
|
Modulation of RII-AKAP binding by RII phosphorylation is probably not specific for AKAP95. We show in another paper (Carlson et al., 2001) that the interaction of human or bovine PKA with the centrosomal AKAP450 is disrupted by T54 phosphorylation of RII, whereas T54 dephosphorylation at mitosis exit promotes its reassociation. Interestingly, RIIß phosphorylation also lowers its ability to anchor MAP-2 in neurons (Keryer et al., 1993).
What is the function of the chromatin-associated RII-AKAP95 complex? We have recently shown that AKAP95 is required for both assembly and structural maintenance of mitotic chromosomes (Collas et al., 1999; Steen et al., 2000). Curiously, PKA activity is dispensable for chromosome condensation per se, but is required for maintenance of chromosomes in a condensed form during mitosis. A putative target for PKA is AKAP95 (Eide et al., 1998) but we have not detected any serine or threonine phosphorylation of AKAP95 during interphase or mitosis. Another possibility is that PKA phosphorylates subunits of the condensin complex required for chromosome condensation (Hirano et al., 1997). Additional proteins implicated in chromosome condensation or structural chromosomal proteins could also serve as PKA substrates (Hirano and Mitchison, 1993; Van Hooser et al., 1998). Although we do not formally demonstrate the existence of the PKA catalytic subunit on mitotic chromosomes, our previous (Collas et al., 1999) and present data suggest that PKA activity must be concentrated around chromosomes for maintenance of condensation, as disruption of PKA-AKAP95 anchoring causes premature decondensation of chromosomes.
If the chromatin-bound AKAP95-RII complex is important for regulating mitotic chromosome dynamics then how do Reh cells proceed through mitosis? As in the rat, RIIß might substitute for RII
in Reh cells (Tasken et al., 1993). Moreover, as most cancer cell lines, the Reh cell line is aneuploid and chromosome segregation defects at mitosis might result, in part, from abnormal chromatin dynamics. Proliferation of Reh cells in the absence of RII
also suggests that the cell cycle control machinery of these cells might not be entirely functional. Finally, the incidence of PCD of Reh cell chromosomes in vitro suggests that this cell line might be prone to chromosome segregation defects at mitosis.
In summary, we demonstrate a cell-cycle-dependent interaction of RII with AKAP95 in human cells. The phosphorylation status of RII
on T54 appears to regulate this association. It would be of interest to determine whether PKA anchoring to other AKAPs is also regulated by phosphorylation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Burke, B. and Gerace, L. (1986). A cell-free system to study reassembly of the nuclear envelope at the end of mitosis. Cell 44, 639-652.[Medline]
Carlson, C. R., Witczak, O., Vossenbein, L., Labbé, J.-C., Skålhegg, B. S., Keryer, G., Herberg, F. W., Collas, P. and Tasken, K. (2001). CDK1-mediated phosphorylation of the RII regulatory subunit of PKA works as a molecular switch that promotes dissociation of RII
from centrosomes at mitosis. J. Cell Sci. 114, 3243-3254.
Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D., Bishop, S. M., Acott, T. S., Brennan, R. G. and Scott, J. D. (1991). Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J. Biol. Chem. 266, 14188-14192.
Chaudhary, N. and Courvalin, J.-C. (1993). Stepwise reassembly of the nuclear envelope at the end of mitosis. J. Cell Biol. 122, 295-306.[Abstract]
Coghlan, V. M., Langeberg, L. K., Fernandez, A., Lamb, N. J. and Scott, J. D. (1994). Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase. J. Biol. Chem. 269, 7658-7665.
Collas, P., Courvalin, J.-C. and Poccia, D. L. (1996). Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein. J. Cell Biol. 135, 1715-1725.[Abstract]
Collas, P., Le Guellec, K. and Tasken, K. (1999). The A-kinase anchoring protein, AKAP95, is a multivalent protein with a key role in chromatin condensation at mitosis. J. Cell Biol. 147, 1167-1180.
Colledge, M. and Scott, J. D. (1999). AKAPs: from structure to function. Trends. Cell Biol. 9, 216-221.[Medline]
Eide, T., Coghlan, V., Orstavik, S., Holsve, C., Solberg, R., Skalhegg, B. S., Lamb, N. J., Langeberg, L., Fernandez, A., Scott, J. D. et al. (1998). Molecular cloning, chromosomal localization, and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95. Exp. Cell Res. 238, 305-316.[Medline]
Erlichman, J., Rangel-Aldao, R. and Rosen, O. M. (1983). Reversible autophosphorylation of type II cAMP-dependent protein kinase: distinction between intramolecular and intermolecular reactions. Methods Enzymol. 99, 176-186.[Medline]
Hausken, Z. E., Dell-Acqua, M. L., Coghlan, V. M. and Scott, J. D. (1996). Mutational analysis of the AKAP-binding site of RII: classification of side-chain determinants for anchoring and isoform selective association with AKAPs. J. Biol. Chem. 271, 29016-29022.
Herberg, F. W., Maleszka, A., Eide, T., Vossebein, L. and Tasken, K. (2000). Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding. J. Mol. Biol. 298, 329-339.[Medline]
Hirano, T. and Mitchison, T. J. (1993). Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts. J. Cell Biol. 120, 601-612.[Abstract]
Hirano, T., Kobayashi, R. and Hirano, M. (1997). Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89, 511-521.[Medline]
Keryer, G., Luo, Z., Cavadore, J. C., Erlichman, J. and Bornens, M. (1993). Phosphorylation of the regulatory subunit of type II beta cAMP-dependent protein kinase by cyclin B/p34cdc2 kinase impairs its binding to microtubule-associated protein 2. Proc. Natl. Acad. Sci. USA 90, 5418-5422.[Abstract]
Keryer, G., Yassenko, M., Labbe, J. C., Castro, A., Lohmann, S. M., Evain-Brion, D. and Tasken, K. (1998). Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase. J. Biol. Chem. 273, 34594-34602.
Riabowol, K. T., Fink, J. S., Gilman, M. Z., Walsh, D. A., Goodman, R. H. and Feramisco, J. R. (1988). The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements. Nature 336, 83-86.[Medline]
Steen, R. L., Cubizolles, F., Le Guellec, K. and Collas, P. (2000). A-kinase anchoring protein (AKAP)95 recruits human chromosome-associated protein (hCAP)-D2/Eg7 for chromosome condensation in mitotic extract. J. Cell Biol. 149, 531-536.
Tasken, K., Skålhegg, B. S., Solberg, R., Andersson, K. B., Taylor, S. S., Lea, T., Blomhoff, H. K., Jahnsen, T. and Hansson, V. (1993). Novel isozymes of cAMP-dependent protein kinase exist in human cells due to formation of RI alpha-RI beta heterodimeric complexes. J. Biol. Chem. 268, 21276-21283.
Tasken, K., Andersson, K. B., Erikstein, B. K., Hansson, V., Jahnsen, T. and Blomhoff, H. K. (1994). Regulation of growth in a neoplastic B cell line by transfected subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase. Endocrinology 135, 2109-2119.[Abstract]
Tasken, K., Skålhegg, B. S., Tasken, K. A., Solberg, R., Knutsen, H. K., Levy, F. O., Sandberg, M., Orstavik, S., Larsen, T., Johansen, A. K. et al. (1997). Structure, function, and regulation of human cAMP-dependent protein kinases. Adv. Sec. Mess. Phosph. Res. 31, 191-204.
Van Hooser, A., Goodrich, D. W., Allis, C. D., Brinkley, B. R. and Mancini, M. A. (1998). Histone H3 phosphorylation is required for the initiation, but not maintenance, of mammalian chromosome condensation. J. Cell Sci 111, 3497-3506.
Witczak, O., Skålhegg, B. S., Keryer, G., Bornens, M., Taskén, K., Jahnsen, T. and rstavik, S. (1999). Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome AKAP450. EMBO J. 18, 1858-1868.
Worman, H. J., Evans, C. and Blobel, G. (1990). The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains. J. Cell Biol. 111, 1535-1542.[Abstract]