Association of PP1 with Its Regulatory Subunit AKAP149 Is Regulated by Serine
Phosphorylation Flanking the RVXF Motif of AKAP149†
Thomas Ku ¨ntziger, Marie Rogne, Rikke L. S. Folstad, and Philippe Collas*
Institute of Basic Medical Sciences, Department of Biochemistry, UniVersity of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway
ReceiVed January 12, 2006; ReVised Manuscript ReceiVed March 6, 2006
ABSTRACT: Reformation of the nuclear envelope at the end of mitosis involves the recruitment of the
B-type lamin phosphatase PP1 to nuclear membranes by A-kinase anchoring protein AKAP149. PP1
remains associated to AKAP149 throughout G1 but dissociates from AKAP149 when AKAP149 is
phosphorylated at the G1/S transition. We examine here the role of phosphorylation of serines flanking
the RVXF PP1-binding motif of AKAP149, on PP1 anchoring. The use of AKAP149 peptides encompassing
the RVXF motif and five flanking serines, either wild type (wt) or bearing SfA or SfD mutations,
specifically shows that phosphorylation of S151 or S159 abolishes PP1 binding to immobilized AKAP149.
Peptides with S151 or S159 as the only wt serine residue trigger dissociation of PP1 from immunopre-
cipitated AKAP149, whereas S151/159D mutants are ineffective. Furthermore, immunoprecipitated
AKAP149 from purified G1-phase nuclear envelopes binds PKA and PKC in overlay assays. PKA binding
to AKAP149 in vitro is unaffected by the presence of PKC or PP1, and similarly, PKC binding is
independent of PKA or PP1. The immunoprecipitated AKAP149 complex contains PKA and PKC activities.
Both AKAP149-associated PKA and PKC serine-phosphorylate immunoprecipitated AKAP149 in vitro;
however, only PKC-mediated phosphorylation promotes dissociation of PP1 from the AKAP. The results
suggest a putative temporally and spatially controlled mechanism promoting release of PP1 from AKAP149.
AKAP149 emerges as a scaffolding protein for multiple protein kinases and phosphatases that may be
involved in the integration of intracellular signals that converge at the nuclear envelope.
The nuclear envelope segregates chromosomes from the
cytoplasm and contributes to nuclear functions such as
replication and transcription through interactions of proteins
of the inner nuclear membrane with A- and B-type lamins,
chromatin, DNA, and transcriptional regulators (1, 2). The
nuclear envelope breaks down upon entry into mitosis,
reassembles around each set of daughter chromosomes in
telophase, and expands to accommodate nuclear growth in
G1 and S phases (1, 2). Because hyperphosphorylation of
nuclear lamins is associated with lamina disassembly,
continuous polymerization of nuclear lamins during G1
necessitates the presence of a lamin dephosphorylase activity
at of the near the nuclear envelope (3).
Nuclear dynamics is controlled by an array of protein
phosphatases and kinases, suggesting that several signaling
pathways intersect at the nuclear envelope. One class of
adaptor proteins tethering multiple signaling molecules
includes A-kinase anchoring proteins, or AKAPs1(4).
AKAPs mediate intracellular compartmentalization and
temporal specificity of cAMP signaling by cAMP-dependent
protein kinase (PKA) (5). AKAPs bind a PKA regulatory
subunit dimer through a consensus sequence, while a
targeting domain specifies subcellular localization. AKAPs
also interact with other signaling molecules such as protein
kinase C (PKC), phosphodiesterases, and protein phos-
phatases in a space- and time-regulated fashion (4, 6).
Protein phosphatase 1 (PP1) is a Ser/Thr phosphatase
consisting of a catalytic and a regulatory subunit. The latter
serves as targeting module for the PP1 catalytic subunit to,
or in vicinity of, its substrate. Most PP1 regulators contain
a degenerate RVXF motif which interacts with the hydro-
phobic pocket of PP1; this, however, does not preclude an
interaction of PP1 with other binding motifs (7, 8). The
regulatory subunit may also modulate the phosphatase
activity of PP1 through inhibition or activation mechanisms.
Interestingly, some regulatory subunits act as substrate-
specifiers of PP1 and repress or stimulate PP1 activity in a
substrate-dependent manner (9-13).
AKAP149 (14), also designated AKAP1, is a human 149-
kDa anchoring protein homologous to mouse AKAP121
identified in mitochondria (15-17) and in the endoplasmic
reticulum-nuclear envelope network (18). AKAP149 also
harbors the hallmarks of a PP1 regulatory subunit (13, 18).
It contains an RVXF motif (K153GVLF157) that mediates
interaction with PP1. Disruption of this sequence completely
abolishes PP1 binding, indicating that the KGVLF motif is
a key determinant in tethering PP1 to AKAP149 (18).
AKAP149 targets a fraction of nuclear PP1 to the nuclear
†This work was supported by the Research Council of Norway and
the Norwegian Cancer Society.
* To whom correspondence should be addressed. Tel: 4722851066;
Fax: 4722851058; E-mail: email@example.com.
1Abbreviations: AKAP, A-kinase anchoring protein; R?γPKC, mix
of R, ?, and γ PKC isoforms; CDK, cyclin-dependent kinase; DTT,
dithiothreitol; IP, immune precipitate; MYPT1, myosin phosphatase
target subunit 1; n-OG, n-octyl glucoside; PKA, cAMP-dependent
protein kinase; PKA-C, PKA catalytic subunit; PKC, protein kinase
C.; PKI, inhibitor of PKA.; PP1, protein phosphatase 1; PDE,
phosphodiesterase; pSer, phosphoserine.
Biochemistry 2006, 45, 5868-5877
10.1021/bi060066s CCC: $33.50© 2006 American Chemical Society
Published on Web 04/11/2006
envelope and enhances PP1 phosphatase activity toward
B-type lamins upon nuclear envelope reformation at mitosis
exit, to promote lamin dephosphorylation and polymerization
(13, 19). In contrast, AKAP149 inhibits PP1 activity toward
phosphorylase a in vitro; as such, AKAP149 qualifies as a
substrate-specifying PP1 subunit (13).
Association of PP1 with AKAP149 at the nuclear envelope
is maintained throughout G1 (13). The importance of this
interaction to preserve nuclear integrity during G1 has been
underscored by inhibition of cell cycle progression and
depolymerization of the nuclear lamina after disrupting this
association, thereby displacing a pool of nuclear PP1,
specifically in G1 (13). However, PP1 is released from
nuclear envelope-associated AKAP149 at the G1/S phase
transition, concomitantly with Ser-phosphorylation of the
AKAP (13). On the basis of these observations, we set out
to determine the extent to which association of PP1 with
nuclear envelope-bound AKAP149 was regulated by phos-
phorylation of AKAP149 in vitro. Using AKAP149-derived
peptides encompassing the PP1-binding motif and bearing
SfA or SfD (single letter amino acid code) substitutions,
we show that phosphorylation of S151 or S159, on either
side of the RVXF motif, is sufficient to inhibit PP1 binding
to AKAP149. In addition, nuclear envelope-bound AKAP149
coimmunoprecipitates PKA and PKC activities. Both
AKAP149-associated PKA and PKC can phosphorylate
AKAP149; however, only PKC-mediated phosphorylation
of AKAP149 promotes dissociation of PP1 from AKAP149
in vitro. Our results suggest a temporally and spatially
controlled mechanism promoting release of PP1 from
AKAP149 and provide insights on the nature of signaling
pathways integrated by AKAP149 and that converge to the
Reagents and Antibodies. Chelerythrine chloride, protein
kinase A inhibitor (PKI), n-octyl glucoside (n-OG), and
peroxidase-conjugated ExtrAvidin were from Sigma-Aldrich
(St. Louis, MO). The PKC pseudosubstrate peptide PKC-
(19-31), a cocktail of purified rat brain R, ?, and γ isoforms
of PKC (referred here to as R?γPKC), and recombinant PP1
were from Upstate Biotechnology (Lake Placid, NY). Puri-
fied human ?II PKC was a gift from A. Fields (Mayo Clinic
Comprehensive Cancer Center, Jacksonville, FL). Strepta-
vidin-agarose beads were from Promega (Madison, WI).
Autocamtide 3 was from Life Technologies (Bethesda, MD).
[γ-32P]ATP was from DuPont NEN (Berverly, MA). Cyclin-
dependent kinase (CDK) inhibitors olomoucine and rosco-
vitine were from L. Meijer (CNRS, Roscoff, France).
Synthetic AKAP149 peptides, wild type (wt) and with SfA
substitutions (Table 1), were biotinylated at the NH2-terminal
end. The AKAP149-derived RVXF peptide SSPKGVLFSS
was as described (18). The Ht31 peptide, which binds the
RII subunit of PKA and is commonly used as an AKAP-
RII disruptor, and the control nondisruptor Ht31-P mutant
peptide, were as described (20-22). Anti-γ-tubulin mono-
clonal antibody (mAb; clone GTU-88) was from Sigma-
Aldrich. Anti-AKAP149 and anti-RIIR mAbs, and antibodies
against various PKC isoforms (sampler kit), were from BD
Biosciences (Santa Fe, CA). Recombinant human RIIR,
rabbit polyclonal anti-RIIR antibodies and rabbit polyclonal
antibodies against the PKA catalytic subunit (PKA-C) were
gifts from K. Taske ´n (University of Oslo) (23, 24). Anti-
bodies against PKC (clone MC-5) and PP1 (rabbit polyclonal
FL-18, mAb E-19) were from Santa Cruz Biotechnology
(Santa Cruz, CA). The anti-PP1 mAb recognized all PP1
catalytic subunits. Anti-phosphoserine (anti-pSer) antibodies
were from Zymed (San Francisco, CA). Rabbit polyclonal
antibodies against B-type lamins were a gift from B. Buendia
(Institut J. Monod, Paris, France) (25).
Cells, Nuclei and Nuclear EnVelopes. HeLa cells were
grown adherent in DMEM (Life Technologies) with 10%
fetal calf serum. For G1 phase synchronization, cells were
first arrested in M phase with 1 µM nocodazole for 16 h.
To allow cell cycle re-entry, cells were washed and replated
at 2.5 × 106cells per 175 cm2flask. Cells in G1 phase were
harvested within 3 h of replating (13). Nuclei were isolated
from G1 cells (or from confluent cell cultures, as indicated)
by Dounce homogenization using a tight-fitting glass pestle,
and nuclear envelopes were prepared as described (18).
Purified nuclei and nuclear envelopes were either analyzed
by SDS-PAGE or solubilized as described below for
AKAP149 Peptide Phosphorylation. Phosphorylation of
biotinylated AKAP149 peptides was carried out using a PKC
and CDK1 protein kinase mix. Peptides (10 µM) were
phosphorylated by 5 ng/µL rat R?γPKC and 25 ng purified
CDK1 for 30 min in 200 mM NaCl, 50 mM Tris-HCl (pH
7.4), 80 µM ?-glycerophosphate, 10 mM MgSO4, 100 µM
CaCl2, 40 µg/mL phosphatidylserine, 20 µM diacyglycerol,
80 µM ?-glycerophosphate, 1 mM dithiothreitol (DTT), 100
µM ATP, 0.01% Tween 20, and phosphatase inhibitors (26).
[γ-32P]ATP (1 µCi/mL) was added for autoradiography
analyses, in which case 10 µM cold ATP was used.
Phosphorylated peptides were sedimented using Streptavidin-
Table 1. Mutational Analysis of AKAP149 Peptide Binding to PP1
with or without a PKC/CDK1 Kinase Mixa
aBiotinylated AKAP149(144-165) peptides were used in overlays
of immobilized PP1 with or without a PKC/CDK1 protein kinase mix.
Binding was detected with peroxidase-conjugated streptavidin. Amino
acids in italics denote the PP1 binding (‘RVXF’) domain or AKAP149.
Underlinings denote mutated amino acids. Results from two to three
replicates. A corresponding autoradiogram and overlay is shown in
AKAP149 Anchors Multiple Signaling Complexes
Biochemistry, Vol. 45, No. 18, 2006 5869
agarose beads, washed in 200 mM NaCl, 50 mM Tris-HCl
(pH 7.4), 1 mM DTT, 0.01% Tween 20, and phosphatase
inhibitors, and analyzed by autoradiography after spotting
onto nitrocellulose or by scintillation counting.
N-Octyl Glucoside Extraction of Nuclear EnVelopes.
Nuclear envelopes isolated from 50 000 nuclei purified from
G1 phase HeLa cells were digested for 1 h with 100 µg/mL
proteinase K in 10 mM Tris-HCl (pH 7.2)/100 mM NaCl
containing 250 mM MgCl2, sedimented at 15 000g and
washed by suspension and sedimentation in 10 mM Tris/
100 mM NaCl (27). This removed proteins, including
endogenous PKC. Membrane lipids were extracted for 1 h
at room temperature with 2% n-OG in 250 µL of extraction
buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgSO4, 0.1%
?-mercaptoethanol) (27). The detergent was removed by
overnight dialysis against extraction buffer at 4 °C. One-
half of the dialyzed fraction (‘n-OG extract 1’) was used as
a competitor for PKC binding in a single PKC overlay. A
control, noncompeting fraction was also prepared by leaving
n-OG in the fraction, thus preventing lipid reconstitution and
PKC binding (‘n-OG extract 2’). This fraction was dialyzed
as above against extraction buffer containing 2% n-OG.
Protein Kinase and Phosphatase Assays. PKC activity
associated with AKAP149-IPs was assessed by phosphory-
lation of immunoprecipitated B-type lamins. AKAP149-IPs
from nuclear envelopes purified from 107HeLa cells in G1
phase were incubated for 30 min with immunoprecipitated
B-type lamins (see below) in PKC phosphorylation buffer
containing 5 ng/µL rat R?γPKC and 10 µCi/mL [γ-32P]ATP
(26), and lamin phosphorylation was assessed by auto-
radiography. In vitro phosphorylation of immunoprecipitated
AKAP149 (from ∼5 × 107nuclear envelopes) by 5 ng/µL
rat R?γPKC was performed in PKC phosphorylation buffer
containing 10 µCi/mL [γ-32P]ATP. Phosphorylated substrates
were washed in the presence of phosphatase inhibitors before
analysis by autoradiography. When indicated, 10 µM chel-
erythrine chloride was added to the reaction. Phosphatase
activity associated with AKAP149-IPs was determined using
as a substrate, immunoprecipitated and in vitro phosphory-
lated B-type lamins, as described previously (13). PKA
phosphorylation reactions were carried out using 1.5 ng/µL
recombinant PKA catalytic subunit in PKA phosphorylation
buffer containing 10 µCi/mL [γ-32P]ATP (26).
Immunological Procedures. SDS-PAGE and immuno-
blotting analysis were performed as described (18) using
indicated antibodies. AKAP149 was immunoprecipitated
from G1 phase purified HeLa cell nuclei or nuclear enve-
lopes, as indicated, after solubilization in immunoprecipita-
tion buffer (10 mM Hepes, pH 7.5, 10 mM KCl, 2 mM
MgCl2, 1% Triton X-100, 1 mM DTT, and protease
inhibitors) (18). B-type lamins were immunoprecipitated
from solubilized nuclear envelopes isolated from confluent
HeLa cell cultures (13) and used as substrate for phos-
phorylation studies. When required, phosphatase inhibitors
(50 mM NaF, 5 mM sodium pyrophosphate, 0.1 mM sodium
orthovanadate) were included in the immunoprecipitation
buffer. RIIR, PKC, and PP1 were immunoprecipitated from
nuclear envelopes purified from G1 phase nuclei as described
(18) using 1:40 antibody dilutions.
Microcystin-Sepharose Affinity Purification of PP1.
Purification of PP1 on microcystin conjugated to sepharose
beads was done as described (18, 28). In short, nuclei purified
from G1-phase HeLa cells were harvested and lysed in
immunoprecipitation buffer. Lysates (250 µL; 4 mg protein)
were cleared with protein A-sepharose beads for 30 min at
4 °C and incubated with 25 µL of a 50% slurry of
microcystin-sepharose beads (Upstate Biotechnology) for 1
h. Beads were washed with buffer M (50 mM triethanol-
amine, pH 7.5, 0.1 mM EGTA, 5% glycerol, 0.5 M NaCl,
0.1% ?-mercaptoethanol, and protease inhibitors) to remove
unbound proteins. Affinity-purified PP1 and bound proteins
were eluted with 3 M NaSCN in buffer M. Input nuclear
lysates, unbound fractions and bound eluates were subjected
to immunoblotting. Control nuclear lysates samples were
incubated with protein A-sepharose beads and processed as
In Vitro Binding Assays. For RIIR and PKC overlays,
AKAP149-IPs were resolved by SDS PAGE and im-
mobilized on nitrocellulose (18). RII binding was detected
using recombinant32P-labeled human RIIR as a probe as
described earlier (29). For PKC overlays, the nitrocellulose
was incubated with 100 µM rat R?γPKC or 100 µM human
?II-PKC for 1 h in Tris-buffered saline/0.01% Tween 20
(TBST) and washed in TBST, and PKC binding was detected
using anti-PKC mAbs (1:500 dilution) and peroxidase-
conjugated anti-mouse antibodies. For AKAP149 peptide
overlays, 5 U recombinant PP1 were spotted onto nitro-
cellulose and overlaid for 1 h with 10 µM biotinylated
AKAP149 peptides. Peptides were used following a 30-min
phosphorylation reaction with a PKC/CDK1 kinase mix as
described above. Peptide binding was detected using per-
oxidase-conjugated ExtrAvidin. Control peptides were in-
cubated in phosphorylation buffer without kinases. Mem-
branes were washed in 50 mM Tris-HCl (pH 7.4)/50 mM
NaCl/0.01% Tween 20 containing phosphatase inhibitors
and binding was detected using peroxidase-conjugated
We have previously shown that a fraction of nuclear PP1
is recruited to nuclear envelope-associated AKAP149 in late
mitosis-early G1, a step necessary for lamin polymerization
(19). This interaction is essential for maintaining nuclear
integrity during G1 (13). PP1 dissociates from AKAP149 at
the G1/S phase transition concomitantly with Ser-phos-
phorylation of AKAP149 (13). On the basis of these
observations, we addressed the extent to which interaction
of PP1 with nuclear envelope-bound AKAP149 is, in vitro,
regulated by phosphorylation of AKAP149.
Phosphorylation of Serines Flanking the RVXF Motif of
AKAP149 Peptide Inhibits Interaction with PP1 In Vitro.
Many PP1 holoenzymes are controlled by reversible phos-
phorylation of their regulatory subunits (7). Most regulators
of PP1, including AKAP149, contain a degenerate RVXF
sequence that binds PP1 (7, 8). For a number of these
regulatory subunits, phosphorylation of serine residues within
or close to the RVXF motif disrupts binding of this motif to
PP1. The RVXF sequence of AKAP149 is flanked by five
serines (S150S151PKGVLFS158S159KS161) that qualify as po-
tential phosphorylation sites for PKC, caseine kinases, and
CDKs (Table 1).
To determine whether binding to PP1 of AKAP149
peptides harboring the RVXF motif and flanking residues
5870 Biochemistry, Vol. 45, No. 18, 2006
Ku ¨ntziger et al.
was affected by serine phosphorylation, biotinylated
AKAP149(144-165) peptides (designated peptides P1-P20)
were synthesized. The peptides were either wild type or
contained single or multiple combinations of SfA mutations
to destroy a phosphorylation site, or SfD mutations to
constitutively mimic a phosphorylated Ser (Table 1). Binding
to PP1 was examined in an overlay assay in which im-
mobilized recombinant PP1 was overlaid with biotinylated
AKAP149 peptides. Peptide binding was detected using
HRP-conjugated streptavidin (Figure 1A). AKAP149 pep-
tides were phosphorylated in vitro by a mix of purified PKC
and CDK1. Phosphorylation was assessed by scintillation
counting (data not shown) and demonstrated qualitatively
by autoradiography of dot-blotted peptides (most likely
representing a mixture of phosphopeptides and nonphospho-
peptides; Figure 1B, upper panel). Filters containing im-
mobilized PP1 were overlaid with each peptide following
preincubation of the peptides without or with the kinase mix
under phosphorylation conditions. Binding results are shown
in Figure 1B (lower panel) and summarized in Table 1.
Phosphorylation of wild type (wt) peptide P1 abolished
binding to PP1, whereas preincubation of P3 (in which all
five serines were mutated to alanines) with the kinase mix
did not affect its ability to bind PP1. Thus, binding of
AKAP149 peptides to immobilized PP1 is regulated by serine
phosphorylation near the RVXF motif. Figure 1B also shows
that S151, S159, and S161 were phosphorylated by the kinase
mix (peptides P3 to P14; Table 1). Of these three sites, only
S151 or S159 phosphorylation disrupted the peptide-PP1
interaction (Figure 1B). Phosphorylation of S161 did not
affect the interaction. Therefore, S151 or S159 is implicated
in protein kinase-mediated control of PP1 binding to
AKAP149 peptides. S150 and S158 are not phosphorylated
by the kinase mix (Figure 1B). Moreover, a kinase-mediated
modulation of binding could be confirmed by SfD muta-
tions on S151 or S159 (peptides P16, P17 and P15), which
mimics constitutive phosphorylation of these sites by intro-
ducing a negative charge while maintaining the mass of a
phosphorylated Ser (Figure 1B). Last, SfD substitutions on
S150, S158 and S161 did not abolish binding to PP1 in the
absence of protein kinase mix (Figure 1B; peptides P18-
P20), ruling out a role of phosphorylation of these residues
as a modulator of binding to PP1.
The role of S151 and S159 phosphorylation in the
disruption of the AKAP149-PP1 complex was examined in
a binding competition assay. Nuclear extracts prepared from
G1-phase HeLa cells (to ensure an association of PP1 with
nuclear envelope-bound AKAP149) (13) were incubated for
1 h with 10 µM AKAP149 peptides. Endogenous AKAP149
was immunoprecipitated and coprecipitation of PP1 was
monitored by immunoblotting. As expected, AKAP149
coimmunoprecipitated PP1 (Figure 2, lane 1). However, PP1
no longer coprecipitated with AKAP149 after preincubation
with P1 (wt peptide), P7 or P10 (containing only S151wt or
S159wt, respectively), and with peptides bearing the S151A
(P13) or S159A (P14) mutations in combination with S161A
(Figure 2). In contrast, peptides that harbored S151D (P16),
S159D (P17) or V155A (a mutation in the RVXF PP1-
binding motif that abolishes interaction with PP1; peptide
P2) mutations did not affect coprecipitation of PP1 with
AKAP149 (Figure 2). We concluded that binding of
AKAP149(144-165) peptides to PP1 is modulated by
phosphorylation of the peptide on S151 or S159.
The AKAP149 Complex of the Nuclear EnVelope Contains
PKA ActiVity. In the identification of a putative kinase
associated with AKAP149, that might mediate AKAP149
phosphorylation, we first examined PKA. Indeed, AKAP149
has been shown to bind the RIIR subunit of PKA with a nM
FIGURE 1: Phosphorylation of AKAP149 peptides on S151 or S159 inhibits binding to PP1. (A) Principle of AKAP149 peptide overlay of
immobilized PP1. (B) Biotinylated AKAP149(144-165) peptides (see Table 1) were phosphorylated with a PKC/CDK1 protein kinase
mix and [γ-32P]ATP in vitro. Peptide phosphorylation was assessed by autoradiography of dot-blotted peptides (32P labeling). Note that a
mix of, presumably, phosphorylated and nonphosphorylated peptides was used here, as no separation of phosphorylated from potentially
unphosphorylated peptides was done. Immobilized recombinant PP1 was overlaid with AKAP149 peptides (P1-P20) after incubation of
the peptides with PKC/CDK1 kinase mix under phosphorylation conditions (+kinase) or in kinase buffer alone (-kinase). Peptide binding
was detected with peroxidase-conjugated streptavidin (overlay).
FIGURE 2: A G1-phase HeLa cell nuclear extract was incubated
for 1 h without peptide (lane 1) or with indicated AKAP149(144-
155) peptides (lanes 2-9). Nonphosphorylated peptides were used.
Endogenous AKAP149 was immunoprecipitated and association
of PP1 was monitored by immunoblotting of the immune precipi-
tates using rabbit polyclonal anti-PP1 antibodies. Mutations carried
by the peptides are shown below the blots. Wt, wild type.
AKAP149 Anchors Multiple Signaling Complexes
Biochemistry, Vol. 45, No. 18, 2006 5871
affinity in vitro (30). AKAP149 also binds RI isoforms (31).
Further evidence argues that AKAP149 targets at least some
RIIR to the nuclear envelope. First, AKAP149 and RIIR
coimmunoprecipitated from purified nuclear envelopes using
antibodies to either protein (Figure 3A). Blotting immune
precipitates with an antibody against γ-tubulin indicated that
nuclear envelope-associated RIIR did not originate from
centrosomes (32-34). Second, as expected, immunoprecipi-
tation of AKAP149 or RIIR coprecipitated the PKA catalytic
subunit PKA-C (Figure 3A). Third,32P-labeled RIIR bound
to immunoprecipitated AKAP149 in an overlay assay and
binding was inhibited by 10 µM of the AKAP-RII anchoring
competitor peptide Ht31, but not by the Ht31-P mutant
peptide (Figure 3B). Binding of RIIR was not affected by
addition of 10 µM of an AKAP149-derived PP1-binding
RVXF peptide (SSPKGVLFSS) or of 5 U recombinant PP1
in the assay (Figure 3B). Fourth, anti-AKAP149 antibodies
immunoprecipitated PKA activity, as shown by autophos-
phorylation of AKAP149-bound RIIR following stimulation
of the complex with cAMP (Figure 3C). Phosphorylation of
AKAP149 and RIIR was inhibited by 100 µM of the PKA-
specific inhibitor PKI (Figure 3C).
AKAP149-bound PKA did not affect the PP1 phosphatase
activity previously found to be associated with AKAP149
(18). AKAP149-IPs can dephosphorylate immunoprecipi-
tated, in vitro phosphorylated, B-type lamins, an activity
mediated by PP1 (13). This observation was confirmed here
(Figure 3D, lanes 1, 2). Furthermore, dephosphorylation of
B-type lamins by the AKAP149-PP1 holoenzyme was not
impaired by 10 µM of the AKAP-RII anchoring disruptor
peptide Ht31 and/or by 100 µM PKI in the assay (Figure
3D). Collectively, these results indicate that nuclear envelope-
associated AKAP149 anchors PKA, and this association is
compatible with the phosphatase activity of PP1 toward
Nuclear EnVelope-Associated AKAP149 Anchors PKC
ActiVity. PKC is also a known interphase B-type lamin kinase
(35), although the role of interphase lamin phosphorylation
remains unclear. We examined by immunoprecipitation
whether PKC was associated with the AKAP149 complex
of the nuclear envelope. Figure 4A shows that PKC cofrac-
tionated with nuclei and nuclear envelopes isolated from
confluent HeLa cells and coimmunoprecipitated with nuclear
envelope-associated AKAP149. Furthermore, an immunoblot
of purified HeLa cell nuclear envelopes using antibodies
against various PKC isoforms showed that PKCR and the
atypical PKC isoforms PKCι and PKCλ were detected at
the nuclear envelope (Figure 4B). In addition, PKCR
coimmunoprecipitated with AKAP149 from a purified
nuclear envelope preparation (Figure 4C). Whether PKC
directly bound to AKAP149 was determined in a PKC
overlay assay of AKAP149 using two forms of purified PKC.
Both rat R?γPKC and human ?II PKC were found to bind
to immunoprecipitated and immobilized AKAP149 (Figure
4D, upper and lower panels, respectively). Binding of these
PKC isoforms to AKAP149 was competed in the assay by
a 2% n-octyl glucoside (n-OG)-soluble and reconstituted
nuclear membrane lipid fraction previously shown to anchor
and bind PKC (36) (Figure 4D, n-OG extract 1). However,
a control (n-OG-soluble, nonreconstituted) fraction not
binding PKC had no effect (Figure 4D, n-OG extract 2). Last,
PKC binding was not affected by adding 10 µM RVXF
peptide, 10 µM Ht31 peptide, 10 µM recombinant RIIR, or
1 µM recombinant PP1 to the assay (Figure 4D).
We next determined whether AKAP149 immunoprecipi-
tated from nuclear envelopes harbored PKC activity. Incuba-
tion of AKAP149-IPs with Ca2+and diacylglycerol caused
phosphorylation of an immunoprecipitated B-type lamin
substrate (Figure 4E). Scanning densitometry of autoradio-
grams (Figure 4F) showed that this kinase activity was
inhibited by ∼80% with 10 µM chelerythrine chloride (a
specific PKC inhibitor) or 50 µM of the pseudosubstrate PKC
inhibitor peptide PKC(19-31) (Figure 4E,F). Under these
conditions, 1 mM of the CDK inhibitor roscovitine, 50 µM
FIGURE 3: The nuclear envelope-associated AKAP149 complex harbors PKA activity. (A) AKAP149 or PKA-RIIR was immunoprecipitated
(IP) from nuclear envelopes of G1-phase nuclei using anti-AKAP149 mAbs and anti-RIIR polyclonal antibodies, respectively. Immune
precipitates were immunoblotted using anti-AKAP149 mAbs, anti-RIIR mAbs, anti-PKA-C antibodies or anti-γ-tubulin antibodies. IgG,
control immunoprecipitation with nonimmune mouse IgGs. NE, nuclear envelopes. (B) AKAP149-IPs or control precipitates (IgG) immobilized
on nitrocellulose were incubated with32P-labeled RIIR alone or together with 10 µM Ht31, 10 µM Ht31-P, 10 µM AKAP149-derived
RVXF peptide, 5 U recombinant PP1 or 10 µM purified human ?II PKC. RII binding was detected by autoradiography. (C) An AKAP149-
IP was incubated with [γ-32P]ATP with or without 100 µM PKI under PKA phosphorylation conditions. RIIR autophosphorylation was
detected by autoradiography (32P labeling). RIIR was also detected by immunoblotting (anti-RIIR mAbs; Blot) after sedimentation of the
AKAP149-IP. (D) PKA does not affect the phosphatase activity of PP1 toward B-type lamins. An AKAP149-IP and immunoprecipitated
B-type lamins phosphorylated by purified PKC in vitro were mixed in buffer containing 10 µM chelerythrine chloride to inhibit AKAP149-
associated PKC, and 10 µM Ht31 or 100 µM PKI. Dephosphorylation of B-type lamins was visualized by autoradiography.
5872 Biochemistry, Vol. 45, No. 18, 2006
Ku ¨ntziger et al.
of the MEK inhibitor autocamtide 3, or 100 µM PKI had no
significant inhibitory effect on the B-type lamin kinase
activity of the AKAP149-IP (Figure 4E,F). We concluded
from these experiments that immunoprecipitated AKAP149
harbors PKC activity.
PKC and PKA Can Phosphorylate AKAP149 in Vitro but
Only PKC Abolishes Interaction with PP1. To determine
whether nuclear envelope-associated AKAP149 was a sub-
strate for PKA and PKC phosphorylation in vitro, AKAP149-
IPs from purified nuclear envelopes were incubated with
[γ-32P]ATP and rat R?γPKC under PKC phosphorylation
conditions. Alternatively, AKAP149-IPs were incubated with
[γ-32P]ATP and purified PKA catalytic subunit under PKA
phosphorylation conditions. Phosphorylation of AKAP149
was examined by autoradiography. AKAP149 was phos-
phorylated by both PKC and PKA, and this was inhibited
by 10 µM chelerythrine chloride or 100 µM PKI, respectively
(Figure 5A). Further, serine phosphorylation of AKAP149
by either kinase was shown by an anti-pSer immunoblot
(Figure 5A). Thus, immunoprecipitated AKAP149 is a
substrate for PKC or PKA phosphorylation in vitro.
Phosphorylation of immunoprecipitated AKAP149 by
exogenous PKC elicited the release of coimmunoprecipitated
PP1 from the complex, and this release was inhibited by 10
µM chelerythrine chloride (Figure 5B, lanes 1, 2). In contrast,
PKA-mediated phosphorylation of AKAP149 did not pro-
mote dissociation of PP1 (Figure 5B, lanes 3, 4). Relative
FIGURE 4: The AKAP149 complex contains PKC and harbors PKC activity. (A) AKAP149 was immunoprecipitated (IP) from nuclear
envelopes, and immune precipitates were immunoblotted using anti-AKAP149 or (pan) PKC antibodies. IgG, control immunoprecipitation
with nonimmune mouse IgGs. NE, nuclear envelopes. (B) Nuclear envelopes purified from HeLa cells in G1 phase were analyzed by
immunoblotting using a panel of antibodies against indicated isoforms of PKC. (C) Purified nuclear envelopes were solubilized (Input) and
AKAP149 immunoprecipitated. Immune precipitates were immunoblotted using anti-AKAP149 or anti-PKCR antibodies. IgG, control
immunoprecipitation with nonimmune IgGs. (D) PKC overlay of immobilized AKAP149-IPs. Immobilized AKAP149 and control (IgG)
IPs were incubated with 5 ng/µL rat R?γPKC. Where indicated, 10 µM of the AKAP149-derived RVXF peptide, 10 µM Ht31, 10 µM
RIIR, 5 U PP1, or competitor (n-OG extract 1) and noncompetitor (n-OG extract 2) n-OG extracts of nuclear envelopes were added.
PKC-binding to AKAP149 was detected using anti-PKC antibodies. (E) B-type lamin phosphorylation by AKAP149 complex-associated
PKC in vitro. Immunoprecipitated B-type lamins, as a substrate for PKC, were incubated with the AKAP149-IP in the presence of [γ-32P]-
ATP and indicated protein kinase inhibitors (Che, 10 µM; PKC(19-31), 50 µM; Ros, 1 mM; PKI, 100 µM; Aut3, 50 µM). Lamin B
phosphorylation was evaluated by autoradiography (32P labeling). B-type lamins were also immunoblotted to assess gel loading (Blot). (F)
Densitometric analysis of B-type lamin phosphorylation from duplicate autoradiograms such as that shown in (C) (mean(SD).
FIGURE 5: Exogenous PKC and PKA phosphorylate AKAP149 but
only PKC-mediated phosphorylation promotes dissociation of PP1
from the AKAP149 complex in vitro. (A) AKAP149 IPs were
incubated with 5 ng/µL rat R?γPKC with or without chelerythrine
chloride, or alternatively, with 1.5 ng/µL PKA catalytic subunit,
under PKC or PKA phosphorylation conditions, respectively.
AKAP149 phosphorylation was assessed by autoradiography (32P
labeling) and by anti-pSer immunoblotting. (B) Dissociation of PP1
from the AKAP149 complex was monitored by immunoblotting
of the AKAP149-IP (pellet) and reaction supernatant (sup) using
rabbit anti-PP1 antibodies. (C) Densitometric analysis of relative
PP1 release from the AKAP149-IP under conditions described
above. Duplicates of pellet and supernatant fractions were examined.
AKAP149 Anchors Multiple Signaling Complexes
Biochemistry, Vol. 45, No. 18, 2006 5873
amounts of PP1 released from AKAP149-IP are indicated
in Figure 5C. Moreover, PP1 release was abrogated by 10
µM of the competing AKAP149-derived peptide substrate
P10 (Table 1), which contained S159 as only serine residue
(data not shown). We concluded that both PKC and PKA
can phosphorylate immunoprecipitated AKAP149 in vitro.
However, phosphorylation by PKC, but not by PKA,
promotes dissociation of PP1 from the AKAP149 complex.
ActiVation of AKAP149-Associated PKC Phosphorylates
AKAP149 and Releases PP1 from the AKAP149 Complex.
The PKC activity found to be associated with immunopre-
cipitated AKAP149 prompted the question of whether
AKAP149-bound PKC would phosphorylate AKAP149.
AKAP149-IPs from nuclear envelopes purified from G1
phase cells were incubated with [γ-32P]ATP under PKC
phosphorylation conditions, without any exogenous kinase.
AKAP149 was phosphorylated and phosphorylation was
inhibited by 10 µM chelerythrine chloride (Figure 6A; Che)
or 10 µM AKAP149 competitor peptide P10 (data not
shown). An anti-pSer blot confirmed that AKAP149 was
serine-phosphorylated under these conditions (Figure 6A).
Immunoblotting of the AKAP149-IP with anti-PP1 antibodies
showed that AKAP149 phosphorylation by bound PKC in
vitro promoted the release of PP1 from the AKAP149
complex into a supernatant fraction (Figure 6B,D). PP1
dissociation was prevented with chelerythrine chloride and
peptide P10 (Figure 6B,D). The CDK inhibitors roscovitine
and olomoucine, as well as autocamtide 3, did not inhibit
PP1 release from AKAP149 (Figure 6D), arguing for a PKC-
We next determined whether PKA activity associated with
immunoprecipitated AKAP149 could phosphorylate the
AKAP. When PKA activity was stimulated with cAMP
(assayed by RIIR serine-autophosphorylation; Figure 6C,
lower two panels), AKAP149 phosphorylation was detected
(Figure 6C). However in this instance, PP1 remained
associated with the AKAP149-IP (Figure 6C) and PKI
logically did not affect PP1 release (Figure 6C,D). Taken
together, these results argue that, although both PKA and
PKC associated with the AKAP149 complex are able to
phosphorylate AKAP149 in vitro, dissociation of PP1 from
AKAP149 is elicited by PKC-mediated phosphorylation of
In most cases, the phosphatase activity of PP1 is regulated
through a controlled interaction of PP1 with its regulatory
subunit (7, 8). This interaction is often mediated by reversible
phosphorylation of the regulatory subunit on serine residues
flanking the RVXF motif, which may alter or disrupt binding
of the motif to PP1 (11, 37, 38). We show here that S151 or
S159 phosphorylation of AKAP149, on either side of the
PP1-binding RVXF motif, promotes release of PP1 from
AKAP149 in vitro. Dissociation is promoted by the activity
of AKAP149-associated PKC. Associated PKA does not
promote PP1 release although it is also able to phosphorylate
AKAP149. Interestingly, PKC-mediated phosphorylation of
the PP1 regulatory subunit myosin phosphatase target subunit
1 (MYPT1) on T34, which immediately precedes the KVKF
PP1-binding motif of MYPT1, does not seem to affect PP1
binding to MYPT1, although MYPT1 interaction with target
myosin is impaired (39). However, phosphorylation of a
second site of MYPT1 not only attenuates PP1 binding to
the regulatory subunit, but also affects the phosphatase
activity toward myosin light chain (39).
FIGURE 6: AKAP149-bound PKC phosphorylates AKAP149 and causes release of PP1 from the AKAP149 complex. (A) An AKAP149-IP
from purified nuclear envelopes was incubated for 30 min in PKC phosphorylation buffer containing phosphatase inhibitors, [γ-32P]ATP,
and either 0 (-) or 10 µM chelerythrine chloride (Che). AKAP149 phosphorylation was assessed by autoradiography (32P labeling). The
AKAP149-IP was immunoblotted with anti-AKAP149 and anti-pSer antibodies. (B) The AKAP149-IP and the phosphorylation reaction
supernatant (sup) were also immunoblotted using rabbit anti-PP1 antibodies to monitor PP1 dissociation from the AKAP149 complex. (C)
AKAP149-IPs from purified nuclear envelopes were incubated for 30 min in PKA phosphorylation buffer containing [γ-32P]ATP and either
0 (-) or 100 µM PKI. AKAP149 phosphorylation was assessed by autoradiography (32P labeling), and AKAP149-IPs were immunoblotted
with anti-AKAP149, anti-pSer and rabbit anti-PP1 antibodies as in A and B. The reaction supernatant (sup) was also probed with anti-PP1
antibodies as in (B). Lower two panels, autophosphorylation of PKA-RIIR bound to AKAP149 under PKA phosphorylation conditions,
monitored by autoradiography and electrophoretic shift by SDS-PAGE and immunoblotting. (D) Relative release of PP1 from the AKAP149
complex was assessed by densitometric analysis of triplicate blots of reaction supernatants. The phosphorylation reaction contained chelerythrine
chloride (10 µM), PKI (100 µM), as shown in (B) and (C), but also AKAP149 peptide P10, and the protein kinase inhibitors roscovitine
(1 mM), olomoucine (1 mM), and autocamtide 3 (50 µM).
5874 Biochemistry, Vol. 45, No. 18, 2006
Ku ¨ntziger et al.
AKAP149 is only one of several AKAPs that bind and
regulate protein phosphatases. The neuronal AKAP79 has
been shown to anchor PKA, PKC and PP2B/calcineurin
modules in membranes (40), and acts as an inhibitor of
calcineurin (41). AKAP220 has also been shown to bind and
regulate PP1 (42). Interestingly, the ability of AKAP220 to
inhibit PP1 activity toward glycogen phosphorylase a is
enhanced in vitro by anchoring of the RII subunit of PKA
(42). Other phosphatase-interacting AKAPs are the centroso-
mal/Golgi AKAP350/AKAP450 (43) and the NMDA recep-
tor-associated protein Yotiao, which regulates NMDA re-
ceptor phosphorylation by targeting of PKA and PP1 (44).
However, in contrast to AKAP220 and AKAP149, Yotiao
anchors PP1 in a constitutively active state toward its known
substrate (44). Last, mAKAP anchors PKA, PP1 and PP2A,
together with phosphodiesterase (PDE) PDE4D3 at the
nuclear envelope of cardiomyocytes (45-47). PKA may
phosphorylate the ryanodine receptor RyR2 and PDE4D3
at the nuclear envelope to promote ion channel activity,
whereas PP2A reverses RyR2 phosphorylation (45).
It has recently been reported that AKAP149 coimmuno-
precipitates with PDE4A in Jurkat T cell lysates (48); thus,
it would be of interest to determine whether nuclear
envelope-associated AKAP149 also harbors a phospho-
diesterase, and if so, which isoform. PDEs control cAMP
concentrations by hydrolyzing cAMP formed by adenylyl
cyclases and are key players in the formation of local cyclic
nucleotides gradient, thereby contributing to the spatio-
temporal regulation of cyclic nucleotide signaling (6).
We have shown here that AKAP149 also associates with
PKA and PKC. The role of PKA at the nuclear envelope
remains unclear at present. A possibility is that a local
elevated concentration of PKA at the nuclear envelope is
important for eliciting CREB signaling upon a rise in cAMP
(49). One function of the AKAP149-associated PKC might
be to down-regulate PP1 activity toward B-type lamins by
phosphorylating AKAP149, thus dissociating PP1 from
AKAP149 and removing it from the vicinity of the nuclear
lamina. This role would be particularly interesting in light
of results showing that PKC is also implicated in the
interphase phosphorylation of B-type lamins (35). PKC could
therefore control the phosphorylation state of B-type lamins
in interphase, both directly and by modulating the presence
and activity of PP1 in the vicinity of the lamina through
phosphorylation of AKAP149. To support this hypothesis,
AKAP149 has been reported to coimmunoprecipitate with
B-type lamins in HeLa cells (19) and to directly bind lamins
in myotubes (50). Despite its anchoring of PKC, AKAP149
is not phosphorylated until the end of G1, suggesting that
PKC activity during G1 is specific for, at least, B-type
lamins. This implies the existence of additional spatial
regulators of PKC activity at or near the nuclear envelope
during interphase, or the presence of active phosphatases
opposing PKC activity toward AKAP149.
Other roles for AKAP149 may include enhancement of
PKC targeting to the nuclear envelope upon mitogenic
stimulation, perhaps to facilitate the activation of PKC at
the nuclear membrane (36). It is known that the nucleus-
targeted ?II isoform of PKC is required for the G2-M phase
transition (51) and has been identified as a mitotic nuclear
lamin kinase (52) promoting, together with other kinases,
nuclear lamina disassembly. Another role for AKAP149-
bound PKC at the nuclear envelope might be in apoptosis,
as human leukemia HL60 cells undergoing apoptosis display
lamin B phosphorylation by PKCR and proteolysis before
DNA fragmentation (53).
AKAP149 emerges from our studies as a scaffolding
protein for PKA, PKC, and PP1. It would be interesting to
determine whether all these signaling molecules are anchored
at the nuclear envelope by AKAP149 at the same time and
in the same complex. Given the highly dynamic picture that
has emerged from the studies of the assembly of AKAP
complexes, it is very tempting to speculate that the nuclear
envelope-associated AKAP149 complex shows great diver-
sity in its composition. This diversity might result from
differences in tissue, bound substrate and in response to cell
signaling events. We suggest that the AKAP149-associated
complex is involved in the integration of signaling pathways
that converge at or pass through the nuclear envelope, and
that a tight spatio-temporal control of the composition of
the AKAP149-associated complex in the nuclear envelope
allows for a dynamic and flexible response to extracellular
and intracellular signals.
The authors are grateful to Drs. Brigitte Buendia, Laurent
Meijer, Alan P. Fields, and Kjetil Taske ´n for generous gifts
of reagents. Dr. Mathieu Bollen (University of Leuven,
Belgium) is thanked for many valuable discussions.
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