From the Howard Hughes Medical Institute, Vollum
Institute, Oregon Health Sciences University,
Portland, Oregon 97201-3098, ¶ Department of Pharmacology and
Cancer Biology, Duke University Medical Center, Durham, North Carolina
27710, and the ** Discipline of Medical Biochemistry, School
of Biomedical Sciences, Faculty of Medicine and Health Sciences,
University of Newcastle, NSW, 2308, Australia
Received for publication, November 16, 2000, and in revised form, December 22, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The phosphorylation status of cellular proteins
is controlled by the opposing actions of protein kinases and
phosphatases. Compartmentalization of these enzymes is critical for
spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the
cAMP-dependent protein kinase (PKA) and the type 1 protein
phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott,
J. D. (1999) Curr. Biol. 9, 321-324). We now
demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c
activity (Ki = 2.9 ± 0.7 µM).
Mapping studies and activity measurements indicate that several
protein-protein interactions act synergistically to inhibit PP1. A
consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas
determinants between residues 1711 and 1901 inhibit the phosphatase.
Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits
suggests that AKAP220 inhibits the phosphatase in a manner distinct
from all known PP1 inhibitors and toxins. Intermolecular interactions
within the AKAP220 signaling complex further contribute to PP1
inhibition as addition of the PKA regulatory subunit (RII) enhances
phosphatase inhibition. These experiments indicate that regulation of
PP1 activity by AKAP220 involves a complex network of intra- and
intermolecular interactions.
Extracellular signals conveyed by hormones and neurotransmitters
are often relayed to precise intracellular sites through the generation
of soluble second messengers such as calcium, phospholipid, or cAMP
(1-3). Frequently, the targets for these molecules are second
messenger-regulated protein kinases and phosphatases, which, in turn,
catalyze changes in the phosphorylation status of key cellular proteins
(4). Prototypic examples are the cAMP-dependent protein
kinase (PKA)1 and the type 1 protein phosphatase (PP1), both of which are broad specificity enzymes
with ubiquitous patterns of expression. Accumulating evidence now
suggests that subcellular location is a key factor in determining the
substrate specificity of both enzyme classes (5, 6). In fact, families
of anchoring and targeting proteins have been identified that tether
PKA or PP1 to precise intracellular sites (7, 8).
The tetrameric PKA holoenzyme consists of a regulatory subunit dimer
(R) and two inactive catalytic subunits (C) (9). Upon elevation of
intracellular cAMP, the C subunit is released and becomes free to
phosphorylate cellular proteins in that vicinity. The location of the
PKA holoenzyme within the cell is controlled by high affinity
protein-protein interactions between the R subunit dimer and A-kinase
anchoring proteins (AKAPs) (10, 11). Over 40 AKAPs have been identified
to date, which localize PKA and other enzymes to a variety of cellular
membranes and distinct intracellular compartments (6, 12).
Compartmentalized pools of kinase are maintained within the vicinity of
activating elements such as G proteins and transmembrane receptors and
in close proximity to selected substrates such as ion channels,
mitochondrial proteins, cytoskeletal components, and cytoplasmic
enzymes (13-19). This sophisticated level of molecular organization
ensures selectivity in cAMP-responsive events (20).
PP1 location is controlled in a similar manner. The catalytic subunit
of the phosphatase, PP1c, associates with numerous targeting subunits
(21-23). Early examples are the GM and liver
glycogen-targeting subunit proteins, which target PP1 to skeletal
muscle and glycogen particles, respectively. However, additional
targeting subunits have been identified such as NIPP-1, p53-binding
protein-2, PP1 nuclear-targeting subunit, which direct PP1 to the
nucleus, whereas spinophillin attaches the phosphatase to the actin
cytoskeleton at postsynaptic sites in neurons (22, 24-34). A common
characteristic of this diverse protein family is their modular design
that includes the presence of a consensus phosphatase-targeting motif
that recognizes PP1c (35). The PP1c-targeting motif contains the
sequence (Lys/Arg)-(Ile/Val)-Xaa-Phe (where Xaa is any amino acid) and
has been shown to interact directly with a binding pocket on the
surface of PP1c (35). This "KVXF" motif is often
considered a hallmark of PP1-targeting subunits, although several
recent studies have suggested that there can be considerable degeneracy
in the sequence (35-37). This has led to the proposal that other
binding surfaces on targeting subunits and phosphatase inhibitor
proteins contact PP1c (38-40).
Several multivalent anchoring proteins have been identified that can
simultaneously associate with kinases and phosphatases (41). The first
example was the neuronal anchoring protein AKAP79 which maintains a
signaling scaffold of PKA, protein kinase C, and the
calcium-calmodulin-dependent phosphatase PP-2B (41-43). However, certain PP1-targeting subunits serve this function also. For
example, the protein targeted to glycogen maintains a signaling scaffold of PP1 and several enzymes involved in glycogen metabolism (44). AKAP149 recruits the PKA holoenzyme and PP1c to the lamina of
nuclear membranes (45). Likewise, the NMDA receptor-associated protein
Yotiao maintains an anchored PKA holoenzyme and constitutively active PP1c to regulate tightly the phosphorylation status and activity
of the NMDA receptor ion channel (36). In both cases, these more
sophisticated mechanisms of phosphatase tethering sequester the PP1c
with physiologically relevant substrates (12). Another example is the
vesicular anchoring protein AKAP220, which we recently showed was
capable of anchoring PKA and tethering PP1c (37, 46).
In this report we present evidence to suggest that PP1 targeting by
AKAP220 involves a consensus KVXF motif and additional binding determinants located in the C-terminal region of the anchoring protein. We demonstrate that AKAP220 is a competitive inhibitor of PP1
activity. Analysis of a truncated and a chimeric PP1 enzyme suggests
that phosphatase inhibition occurs by a mechanism distinct from many
known PP1 inhibitors. Most remarkably, the ability of AKAP220 to
function as an inhibitor of PP1 was further enhanced by anchoring of
the R subunit of PKA. These experiments provide evidence for an
additional level of control whereby the RII-AKAP220 interaction
augments the down-regulation of PP1 activity.
Generation of AKAP220 Fragments--
Fragments of AKAP220
were constructed using restriction enzyme digest and PCR. Numbering of
the AKAP220 fragments is based upon the amino acid sequence of the
human ortholog (47). Fragment 910-1901 was subcloned using
EcoRI restriction sites into Pet 30c (Novagen) for bacterial
expression and purification. BamHI digest of the 910-1901
fragment was used to construct fragment 910-1228; SacI
digest of the 910-1901 fragment was used to generate 1182-1901; and
SacI/HindIII double digest of the 910-1901
fragment generated fragment 1182-1591. PCR using oligos 5'
CGAGCTCGAACCCAAGGTTAAAAACCCTTGC and 5'
CCGCTCGAGGAGCCATCTTGCCCCAAACCTTCTA facilitated generation of fragment
1228-1901 by adding SacI and XhoI sites. This
fragment was then digested with HindIII to make fragment
1228-1591. PCR was again used to generate fragments 1591-1901 oligos
5' CGAGCTCTACTGTGACCTTAAAGAACTCC and Pet 30 T7 terminator, 1591-1714
oligos 5' CGAGCTCTACTGTGACCTTAAAGAACTCC and 5'
CCCAAGCTTGACAGACTCAGTTGACTGAAAGT, and 1711-1901 oligos 5'
CGAGCTCGAAGACTTTCAGTCAACTGAGTC and Pet30 T7 terminator.
SacI/HindIII double digest was then used to clone
the PCR fragments into Pet 30. Point mutations were generated by quick
change PCR mutagenesis (Stratagene) using different oligos for the Phe
Phosphatase Assay--
Phosphorylase b was
phosphorylated by phosphorylase kinase (in 100 mM Tris, 100 mM glycerophosphate, pH 8.2, 10 mM
MgCl2, 1 mM DTT, 2 mM CaCl) using
[ PP1 Overlay Assay--
The PP1 overlay assay was performed
essentially as described (37). Briefly, fragments were subjected to
SDS-PAGE and transfer to Immobilon. The membranes were incubated with
0.5 µg of recombinant PP1c AKAP220 Is a Competitive Inhibitor of PP1--
We have previously
shown that AKAP220 interacts with the PKA holoenzyme and PP1 in
vitro and inside cells (37, 46). To investigate further these
events it was important to define the mechanism of AKAP220 interaction
with the phosphatase. A recombinant fragment encompassing residues
910-1901 of human AKAP220 inhibited PP1 activity with an
IC50 of 3.6 ± 0.7 µM (n = 4) when phosphorylase a was used as a substrate (Fig.
1A). Control experiments
confirmed that this AKAP220 fragment inhibited recombinant PP1
One implication of these findings is that binding determinants on the
anchoring protein must influence the active site of the phosphatase.
Within the AKAP220 peptide is a core sequence of Lys-Val-Gln-Phe which
is a recognizable characteristic of many PP1-targeting subunits (35).
We have previously demonstrated that an AKAP220 peptide encompassing
this sequence (residues 1185-1207) binds PP1 Multiple Sites of Interaction between AKAP220 and PP1--
Two
recombinant fragments of human AKAP220 were further characterized to
locate sites on the anchoring protein that inhibit the phosphatase
(Fig. 2A). A large C-terminal
fragment, AKAP220-(910-1901), inhibits the phosphatase with an
IC50 of 3.6 ± 0.7 µM (n = 4, Fig. 2B). Solid-phase binding experiments using an
overlay procedure confirmed that this AKAP220 fragment retained the
ability to bind PP-1 (Fig. 2D). Furthermore, mutation of the
KVQF motif does not significantly impair the inhibitory potency of the
AKAP220-(910-1901) fragment (Fig. 2B). Substitution of
Val-1196 Mapping a Second PP1-binding Site on AKAP220--
A family of
AKAP220 fragments spanning selected regions of the anchoring protein
were generated to identify further phosphatase-binding sites and PP1
inhibitory determinants (Fig.
3A). Each AKAP220 fragment was
expressed in E. coli as a His6 fusion protein
and affinity-purified using FPLC His tag technology. Approximately equal amounts of each purified protein fragment were used (Fig. 3B), and interaction with PP1 was assessed by the overlay
assay (Fig. 3C). All binding studies were performed at least
three times. As expected all AKAP220 fragments including the KVQF
sequence bound PP1 (Fig. 3C, lanes 1-4), although weaker
binding was observed with AKAP220-(910-1228), a fragment that
contained the KVQF sequence at the extreme C terminus (Fig.
3C, lane 2). Most importantly, certain fragments
such as AKAP220-(1228-1901) that lack the KVQF sequence also retained
the ability to interact with PP1 as assessed by the overlay assay (Fig.
3C, lane 5). This led to the analysis of additional
fragments spanning this region (Fig. 3A) which permitted the
mapping of supplementary PP1 binding determinants between residues 1711 and 1901 of AKAP220 (Fig. 3C, lanes 6-9). Thus, at least
two regions of AKAP220 interact with PP1 as follows: a conserved
targeting motif between residues 1195 and 1198, and site(s) located
between residues 1711 and 1901 of the anchoring protein (Fig.
4A).
A logical next step was to ascertain if the C-terminal binding region
of AKAP220 influenced PP1 activity. Inhibition profiles for a
representative selection of AKAP220 fragments are presented in Fig. 4,
B and C. Initial experiments showed that
AKAP220-(1228-1901) inhibited PP1 activity with an IC50 of
3 ± 0.3 µM (n = 3) (Fig. 4B,
closed squares), whereas AKAP220-(910-1228), an upstream fragment that encompasses the targeting motif, had little effect on phosphatase activity (Fig. 4B, open circles). Further analysis indicated
that PP1 inhibitory determinants were located in the last 300 residues of the anchoring protein (Fig. 4C). Interestingly, residues
1711-1901 inhibited PP1 Analysis of Truncated and Chimeric Phosphatase Catalytic
Subunits--
Shenolikar and colleagues (40, 55) have established the
importance of the RII Enhances Inhibition of PP1 by AKAP220--
Although PKA and
PP1 bind to distinct regions of AKAP220, it was of interest to
establish if there was any cooperative effect of PKA anchoring on
phosphatase activity. Earlier reports (56, 57) had suggested that RII
was a noncompetitive inhibitor of PP1 with phosphorylase a
as a substrate. Since the C terminus of AKAP220 binds both RII and PP1,
it was important to explore the potential influence of these protein
interactions on inhibition of the phosphatase. Recombinant RII
inhibited PP1 with an IC50 = 2.2 ± 0.7 µM (n = 3) (Fig.
6A, open circles), a similar
inhibitory potency to the AKAP220-(910-1901) fragment (Fig. 6A,
closed triangles). However, PP1 inhibition was enhanced 4-fold
(IC50 = 0.59 ± 0.2 µM
(n = 3)) when RII was added to the enzyme reaction
(Fig. 6A, open triangles). Since the AKAP220-(910-1901)
fragment contains determinants of PKA anchoring, we reasoned that RII
could enhance PP1 inhibition in either an additive or a cooperative
manner (Fig. 5A, inset). Additive effects would require that
both RII and AKAP220 inhibit PP1 by binding at different sites on the
phosphatase. Cooperative effects could occur if the RII-AKAP220
complex constrained a preferred conformation or exposed additional
determinants within the anchoring protein that enhanced PP1 inhibition.
In an attempt to delineate between these two possible mechanisms, we
used AKAP220-(1711-1901), a fragment which lacks the RII-binding site
(Fig. 3A). This fragment inhibited PP1 with an
IC50 = 34.5 ± 5.9 µM (n = 3) in the absence of RII (Fig. 6B, closed squares).
However, PP1 inhibition was enhanced ~28-fold in the presence of
equimolar concentrations of RII (IC50 = 1.2 ± 0.2 µM (n = 3) Fig. 6B, open
squares). Control binding experiments confirmed that the
AKAP220-(1711-1901) fragment does not bind RII in vitro
(data not shown). These results suggest that AKAP220 and RII are
more likely to work additively to inhibit PP1 by binding to separate
sites on the phosphatase (Fig. 6B, inset). The greater
inhibitory potency of the larger AKAP220 fragment implies that RII
association with the anchoring protein may orient the regulatory
subunit to permit optimal inhibitory contact with the phosphatase (Fig.
6A, inset).
Conclusion--
The substrate specificity of the type I protein
phosphatase catalytic subunit PP1c is thought to be influenced in large
part through association with targeting subunits (58). This growing family of proteins not only controls the subcellular location of PP1
leading to selective dephosphorylation of certain substrates but also
influences the catalytic efficiency of the enzyme (21, 22, 36, 48,
59-61). To perform both functions it has been proposed that
PP1-targeting subunits and inhibitor proteins interact with multiple
sites on the phosphatase (38, 55, 62). Subcellular targeting is
mediated in part through a loosely conserved tetrapeptide sequence
KVXF found in many PP1-regulatory proteins (35). Additional binding sites participate in allosteric interactions that tailor the
substrate specificity of PP1c (40, 63-65).
In this report we demonstrate that two or more binding surfaces on
AKAP220 act synergistically to target and inhibit the phosphatase. On
the basis of these observations, we now propose a more sophisticated model for AKAP220-mediated PP1 targeting. Our data suggest that a
consensus-targeting motif including residues 1195-1198 of the anchoring protein is responsible for localizing the phosphatase, whereas inhibitory sites between residues 1711 and 1901 maintain the
enzyme in an inactive state. Our kinetic evidence indicates that
AKAP220 is a competitive inhibitor of PP1c activity (Fig. 1C). These latter findings are consistent with accumulating
evidence that other multivalent anchoring proteins such as AKAP79/150
and gravin bind and inhibit their anchored enzymes (66-68). A
previously unappreciated level of phosphatase regulation appears to
involve interaction with other proteins in the AKAP220-signaling
complex. Although free RII has been reported to inhibit PP1 in a
noncompetitive manner (56, 57), its recruitment into the AKAP220
signaling scaffold enhances phosphatase inhibition (Fig. 6). This
represents a new concept in AKAP signaling in which intermolecular
interactions within the signaling complex influence the activity of
other anchored enzymes. Another tier of regulation may be
phosphorylation of the anchored signaling components by PKA. For
example, thiophosphorylated RII is a more potent inhibitor of PP1 (57),
and PKA phosphorylation of regulatory molecules such as inhibitor 1 or
DARPP-32 enhances phosphatase inhibition (50). In this regard our
preliminary studies suggest that AKAP220 is a PKA
substrate.2 Future studies
will focus on whether the anchoring protein is phosphorylated in
vivo and if there are effects on phosphatase inhibition. The
complexity of these interactions will be more fully apparent when a
three-dimensional structure of the PKA-AKAP220-PP1 complex is solved.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
Ala, Val
Ala, and FV
AA mutations. The Phe
Ala oligos
used were 5' GCACTCAGGGAAGAAGGTTCAGGCTGCAGAAGC and 5'
GCTTCTGCAGCCTGAACCTTCTTCCCTGAGTGC. The Val
Ala oligos used were 5'
GCACTCAGGGAAGAAGGCTCAGTTTGCAGAAGC and 5'
GCTTCTGCAAACTGAGCCTTCTTCCCTGAGTGC. The FV
AA oligos used were 5'
GCACTCAGGGAAGAAGGCTCAGGCTGCAGAAGC and 5'
GCTTCTGCAGCCTGAGCCTTCTTCCCTGAGTGC. Each construct was sequenced in both directions to confirm the presence of the PCR-induced mutation.
All fragments were expressed as N-terminal His6-tagged proteins in bacteria (BL21DE3) and purified via His tag purification using hi-trap chelating resin and FPLC (Amersham Pharmacia Biotech). Briefly, cells were pelleted by centrifugation at 5000 × g for 10 min and then sonicated in buffer A at 4 °C (20 mM HEPES, 500 mM NaCl, pH 7.9), and centrifuged
at 35,000 × g at 4 °C for 30 min. Fragment
910-1901 was predominantly an insoluble protein; therefore, 6 M urea was added to Buffer A and a second round of sonication and centrifugation was conducted. Supernatants were filtered
(0.2 µm) and then applied to hi-trap chelating resin (Amersham
Pharmacia Biotech) using FPLC. Bound proteins were eluted with a
stepwise gradient of imidazole (0-0.5 M) in buffer A. Fractions containing purified protein were identified by Coomassie
staining of SDS-PAGE gels. Stepwise dialysis removed imidazole and urea from the protein preparations.
-32P]ATP during a 3-h incubation at 30 °C
(phosphorylase b and phosphorylase kinase, Sigma). The
reaction was stopped by adding 50 mM NaF, 20 mM
EDTA (final concentrations) for an additional 15 min at 30 °C.
Phosphorylase a was then precipitated by incubation with an
equal volume of saturated ammonium sulfate solution on ice for 30 min
followed by a 10-min 14,000 × g spin. The pellet was resuspended in phosphorylase a solubilization buffer (50 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 1 mM DTT, 15 nM caffeine) and passed over a
Pierce desalting column to remove remaining free ATP. Phosphorylase a eluted from the column with solubilization buffer in the
first few fractions, whereas free nucleotide was retained on the
column. Phosphatase assay was performed in a total volume of 30 µl at 30 °C. Recombinant PP1 (generously provided by Dr. Ernest Lee) or
the native rabbit enzyme were diluted in phosphatase dilution buffer
(50 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.1%
bovine serum albumin, 1 mM DTT, 1 mM
MnCl2). AKAP220 fragments were diluted in phosphatase assay
buffer (50 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 3 mM EGTA, 1 mM DTT, 0.1% bovine serum albumin).
PP1 and inhibitors were incubated for 5 min at 30 °C and then
substrate was added. After 10 min at 30 °C, the reactions were
stopped with 30% trichloroacetic acid. Following a 10-min incubation
on ice and a 5-min, 14,000 × g spin, 100 µl of
supernatant was removed, and the amount of 32P
radioactivity released was measured by liquid scintillation counting.
Competitive inhibition assays were conducted over a 0.02-0.8
µM range of phosphorylase a and 0.01-10
µM AKAP220.
in TTBS (0.03% Tween,
Tris-buffered saline) for 2 h at room temperature. PP1 bound to
the membrane was detected by Western blot using a polyclonal
anti-PP1
antibody and chemiluminescence detection (Pierce).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
or
the purified rabbit enzyme to similar extent (Fig. 1B).
Double-reciprocal plots were used to calculate an inhibition constant
(Ki) of 2.9 ± 0.7 µM
(n = 3) demonstrating that the AKAP220-(910-1901) fragment was a competitive inhibitor of PP1
activity (Fig.
1C).
View larger version (26K):
[in a new window]
Fig. 1.
AKAP220 is a PP1 inhibitor. Purified
recombinant PP1 and native PP1c isolated from rabbit skeletal muscle
were incubated with the AKAP220-(910-1091) fragment over a
concentration range from 1 nM to 100 µM.
Phosphatase activity was measured as described under "Materials and
Methods" using phosphorylase a as a substrate. PP1
activity is presented as a percentage of phosphorylase a
dephosphorylation in the absence of AKAP fragment. A depicts
data collected from an average of four independent experiments.
B depicts a comparison of the inhibition profiles for
recombinant (open squares) and native (closed
squares) PP1 catalytic subunits over a similar concentration range
of AKAP220-(910-1901) fragments, with the data collected from three
independent experiments. C, detailed kinetic analysis of
AKAP220-(910-1901) fragment as a PP1
inhibitor was performed in the
presence of AKAP220-(910-1901) 3-10 µM.
Michaelis-Menten analysis was used to calculate the inhibition
constant.
with nanomolar
affinity (37). This sequence is unlikely to bind at the active site of
the enzyme as other investigators have demonstrated that related
"KVXF" peptides do not inhibit phosphatase activity (22,
26). More conclusive support for this view has been provided by
crystallographic analysis of PP1c complexed with a peptide derived from
the glycogen-targeting subunit GM. These elegant studies
show that the KVXF sequence binds to a hydrophobic
surface that is distal to the catalytic center of the phosphatase (35).
These protein-protein interactions must represent a principle targeting
interaction as delivery of KVXF peptides into tissue culture
cells and dissociated striatal neurons disrupts PP1 location (21, 22,
48-50). One relevant example is peptide-mediated disruption of PP1
from a phosphatase-kinase signaling complex maintained by
Yotiao, a scaffolding protein that binds to the cytoplasmic tail
of the NMDA-type glutamate receptor ion channel (36, 51). Interruption
of the Yotiao/PP1 interaction uncouples phosphatase regulation
of the ion channel and enhances cAMP-responsive currents (19, 36). In
some respects Yotiao and AKAP220 have related roles. Both
anchoring proteins maintain a PP1-PKA signaling complex and target the
enzymes to precise locations within cells. However, one important
distinction is that the 910-1901 fragment of AKAP220 is a reasonably
potent competitive inhibitor of PP1 activity. Thus, there must be
additional binding sites on AKAP220 that inhibit the phosphatase.
Ala, Phe-1198
Ala, or replacement of both residues
with alanine (Fig. 2C) within the context of the
AKAP220-(901-1901) fragment had no qualitative effect on PP1
interaction (Fig. 2D). However, phosphatase binding was
abolished when the same panel of mutants was screened for PP1
interaction within the context of a smaller fragment,
AKAP220-(1112-1258) (Fig. 2, E and F). Control
experiments confirmed that the wild-type AKAP220-(1112-1258) fragment
bound PP1 in the overlay assay (Fig. 2E) and activity
measurements confirmed that this 146-residue fragment which spans the
KVQF sequence does not inhibit the phosphatase (Fig. 2B).
Additional control experiments confirmed that equal amounts of each
AKAP220 fragment were used in the overlay blots (data not shown).
Collectively, these results allow us to conclude that inhibition of PP1
activity involves the C-terminal half of AKAP220 and does not require
binding through the KVQF sequence.
View larger version (20K):
[in a new window]
Fig. 2.
The consensus targeting domain in AKAP220 is
not required for inhibition of PP1. AKAP220 fragments were
analyzed for inhibition of recombinant PP1 activity using
phosphorylase a as a substrate. A, schematic
diagram depicts the size of each fragment in relation to the
full-length anchoring protein. The first and last residue of each
fragment is indicated, and the consensus PP1-targeting motif is
highlighted. B, the PP1
inhibitory properties
of three AKAP220 fragments were assayed as described under "Materials
and Methods." Dose-response curves for each fragment are presented:
residues 1112-1258 (black squares), residues 910-1901
(filled squares), and a mutant form of the
AKAP220-(910-1901) fragment with a disrupted targeting motif
(closed circles). Each curve represents averaged data from
four independent experiments. C, a schematic diagram showing
a series of mutations in the consensus targeting (KVXF)
motif of the AKAP220-(910-1901) fragment. Amino acids are shown in the
one-letter code. D, samples of each mutant
AKAP220-(910-1901) fragment (indicated above each lane)
were separated by SDS-polyacrylamide gel electrophoresis and
electrotransferred to a nitrocellulose filter. Solid-phase binding to
recombinant PP1
was assessed by an overlay assay. Molecular weight
markers are indicated. E, a schematic diagram showing a
series of mutations in the consensus targeting (KVXF) motif
of the AKAP220-(1112-1258) fragment. Amino acids are in the
one-letter code. F, samples of each mutant
AKAP220-(1112-1285) fragment (indicated above each lane) were
separated by SDS-polyacrylamide gel electrophoresis and
electrotransferred to a nitrocellulose filter. Solid-phase binding to
recombinant PP1c
was assessed by an overlay assay. Molecular weight
markers are indicated.
View larger version (37K):
[in a new window]
Fig. 3.
Mapping additional PP1 binding determinants
in AKAP220. AKAP220 fragments were screened for interaction with
PP1 using the overlay assay. A, schematic diagram depicts
the size of each fragment. AKAP220 fragments retaining the ability to
interact with PP1
are indicated in black. The first and
last residues of fragment are indicated; the PP1-targeting motif and
RII binding sites are boxed. Recombinant AKAP220 fragments
were separated by SDS-polyacrylamide gel electrophoresis and
electrotransferred to a nitrocellulose filter. B, protein
staining shows the relative purity of each AKAP220 fragment.
C, detection of solid-phase phosphatase binding used the
PP1
overlay. The first and last residues of each fragment are
indicated above and the lane numbers are indicated
below. The mobility of molecular weight markers is
indicated.
View larger version (21K):
[in a new window]
Fig. 4.
Mapping the PP1 inhibitory site on
AKAP220. The inhibitory properties of selected AKAP220 fragments
toward recombinant PP1 were measured using phosphorylase
a as a substrate. A, a schematic diagram depicts
the size of each fragment. Filled boxes represent AKAP220
fragments that inhibit PP1
activity. The first and last residues of
fragment are indicated; the PP1-targeting motif and PP1 inhibitory
region are highlighted. B, dose-response curves
comparing the inhibitory potency of AKAP220-(910-1228) (open
circles) and the AKAP220-(1228-1901) (closed boxes)
fragments. C, dose-response curves for the
AKAP220-(1228-1591) fragment (open circles),
AKAP220-(1591-1714) fragment (open diamond),
AKAP220-(1711-1901) fragment (closed squares), and
AKAP220-(1591-1901) fragment (closed triangles). All data
represent an average of three independent experiments.
with an IC50 of 48.9 ± 4.6 µM (n = 3) (Fig. 4C, closed
squares), whereas a larger fragment that included the RII binding
domain (AKAP220-(1591-1901)) inhibited the phosphatase to a 10-fold
greater extent (IC50 of 4.1 ± 1.1 µM
(n = 3); Fig. 4C, closed triangles). Thus it
would appear that multiple interactions including an inhibitory site
located between residues 1711 and 1901 promote tight binding and
inhibition of the phosphatase. This finding is consistent with
structure-function analysis on several protein inhibitors of PP1 such
as inhibitor-1 (I-1), inhibitor-2 (I-2), NIPP-1, and DARPP-32 (24, 25,
29, 38, 39, 49, 50, 52-54).
12-13 loop in PP1 as a region required for inhibition of PP1c by protein inhibitors such as I-1, I-2, and NIPP-1
and environmental toxins. To examine the mechanism of PP1 inhibition by
AKAP220, we first analyzed a chimeric PP1
catalytic subunit
containing C-terminal sequences from PP2A (55). This reengineered
phosphatase, termed CRHM2, is sensitive to toxins, such as microcystin,
tautomycin, and fostreicin, but is not inhibited by the phosphorylated
inhibitor proteins including I-1, I-2, or NIPP-1 (39, 55). When
incubated with AKAP220, the PP1/2A chimera (Fig.
5, open squares) is inhibited
to a similar extent as the wild-type PP1 (Fig. 5, closed
squares). In addition, we analyzed a truncated PP1
catalytic
subunit lacking the variable N- and C-terminal sequences, termed PP1
core, that was previously shown to be insensitive to both protein and
small molecule inhibitors. The PP1 core was also inhibited by AKAP220
in a manner similar to the wild-type phosphatase (Fig. 5, filled
triangles). These results indicate that AKAP220 inhibits
phosphatase activity by a mechanism distinct from known PP1 inhibitors.
To emphasize this point, fostreicin, an anti-cancer drug that inhibits
the PP1 catalytic core competed with AKAP220 for PP1 inhibition (data
not shown). Thus, the data suggest that the
12-13 loop in the PP1
catalytic subunit is not essential for its inhibition by AKAP220.
Furthermore, residues 1711-1901 of the anchoring protein bind the
phosphatase in a manner distinct from many known PP1 inhibitors,
proteins, and toxins.
View larger version (21K):
[in a new window]
Fig. 5.
The 12/13 loop is
not required for PP1 inhibition by AKAP220. The sensitivity of
PP1
(solid squares), CRHM2, the PP1/2A chimera
(open squares), and the PP1
core, residues 41-269
(filled triangles), to inhibition by the
AKAP220-(1711-1901) fragment was measured using phosphorylase
a as a substrate. Activity is presented as the percentage of
phosphatase activity measured in the absence of the AKAP220 inhibitory
fragment. Each experiment was performed three times, and the data are
shown with standard errors.
View larger version (24K):
[in a new window]
Fig. 6.
RII binding enhances PP1 inhibition by
AKAP220. The inhibitory properties of selected AKAP220 fragments
toward PP1 were measured in the presence of RII. A,
dose-response curves for AKAP220-(910-1901) fragment (closed
triangles), RII (open circles), and AKAP220-(910-1901)
fragment plus RII (open triangles) are presented.
Inset demonstrates the potential role of AKAP220 to
coordinate RII anchoring in a manner that optimizes inhibitory contact
with the phosphatase. B, dose-response curves for the
AKAP220-(1711-1901) fragment in the presence (open squares)
and absence of RII (closed squares). Inset
depicts how RII and the AKAP220 fragment act synergistically to inhibit
the phosphatase. All experiments were performed at least three
times.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank our colleagues at the Vollum
Institute for critical evaluation of this manuscript. We thank Dr.
Ernest Y. Lee for providing purified recombinant PP1, ICOS for the
910-1901 fragment of human AKAP220. We also thank Kimberly Sandstrom
for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK44239 (to J. D. S.) and DK52054 (to S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: RD8, VAMC, 3710 SW US Veterans Hospital Rd., Portland, OR 97201. Tel.: 503-220-8262 (ext. 54130); E-mail: schillac@ohsu.edu.
To whom correspondence should be addressed: Howard Hughes
Medical Institute, MRB 322 Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97201. Tel.:
503-494-4652; Fax: 503-494-0519; E-mail: scott@ohsu.edu.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M010398200
2 R. V. Schillace and J. D. Scott, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKA, cAMP-dependent protein kinase; AKAP, A-kinase anchoring protein; PP1, type 1 protein phosphatase; R, protein kinase A regulatory subunit; C, protein kinase A catalytic subunit; GM, muscle glycogen-targeting subunit; NIPP-1, nuclear inhibitor of PP1; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; CRHM2, a chimera of PP1-(1-273) and PP2A-(267-309); DARPP-32, dopamine- and cAMP-regulated phosphoprotein of apparent Mr 32,000; NMDA, N-methyl-D-aspartic acid; oligos, oligonucleotides; FPLC, fast protein liquid chromatography; PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Chin, D., and Means, A. R. (2000) Trends Cell Biol. 10, 322-328[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Nishizuka, Y.
(1995)
FASEB J.
9,
484-496 |
3. | Sutherland, E. W. (1972) Science 171, 401-408 |
4. | Krebs, E. G. (1985) Biochem. Soc. Trans. 13, 813-820[Medline] [Order article via Infotrieve] |
5. |
Cohen, P.,
and Cohen, T. W.
(1989)
J. Biol. Chem.
264,
21435-21438 |
6. | Colledge, M., and Scott, J. D. (1999) Trends Cell Biol. 9, 216-221[CrossRef][Medline] [Order article via Infotrieve] |
7. | Hubbard, M. J., and Cohen, P. (1993) Trends Biochem. Sci. 18, 172-177[CrossRef][Medline] [Order article via Infotrieve] |
8. | Faux, M. C., and Scott, J. D. (1996) Trends Biochem. Sci. 21, 312-315[CrossRef][Medline] [Order article via Infotrieve] |
9. | Taylor, S. S., Buechler, J. A., and Yonemoto, W. (1990) Annu. Rev. Biochem. 59, 971-1005[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Carr, D. W.,
Stofko-Hahn, R. E.,
Fraser, I. D. C.,
Bishop, S. M.,
Acott, T. S.,
Brennan, R. G.,
and Scott, J. D.
(1991)
J. Biol. Chem.
266,
14188-14192 |
11. | Newlon, M. G., Roy, M., Morikis, D., Hausken, Z. E., Coghlan, V., Scott, J. D., and Jennings, P. A. (1999) Nat. Struct. Biol. 6, 222-227[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Schillace, R. V.,
and Scott, J. D.
(1999)
J. Clin. Invest.
103,
761-765 |
13. |
Fraser, I. D.,
Tavalin, S. J.,
Lester, L.,
Langeberg, L. K.,
Westphal, A. M.,
Dean, R. A.,
Marrion, N. V.,
and Scott, J. D.
(1998)
EMBO J.
17,
2261-2272 |
14. | Harada, H., Becknell, B., Wilm, M., Mann, M., Huang, L. J., Taylor, S. S., Scott, J. D., and Korsmeyer, S. J. (1999) Mol. Cell 3, 413-422[Medline] [Order article via Infotrieve] |
15. |
Huang, L. J.,
Wang, L.,
Ma, Y.,
Durick, K.,
Perkins, G.,
Deerinck, T. J.,
Ellisman, M. H.,
and Taylor, S. S.
(1999)
J. Cell Biol.
145,
951-959 |
16. | Colledge, M., Dean, R. A., Scott, G. K., Langeberg, L. K., Huganir, R. L., and Scott, J. D. (2000) Neuron 27, 107-119[Medline] [Order article via Infotrieve] |
17. | Fraser, I., Cong, M., Kim, J., Rollins, E., Daaka, Y., Lefkowitz, R., and Scott, J. (2000) Curr. Biol. 10, 409-412[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Westphal, R. S.,
Soderling, S. H.,
Alto, N. M.,
Langeberg, L. K.,
and Scott, J. D.
(2000)
EMBO J.
19,
4589-600 |
19. | Fraser, I. D., and Scott, J. D. (1999) Neuron 23, 423-426[Medline] [Order article via Infotrieve] |
20. | Edwards, A. S., and Scott, J. D. (2000) Curr. Opin. Cell Biol. 12, 217-221[CrossRef][Medline] [Order article via Infotrieve] |
21. | Hubbard, M. J., Dent, P., Smythe, C., and Cohen, P. (1990) Eur. J. Biochem. 189, 243-249[Abstract] |
22. | Johnson, D. F., Moorhead, G., Caudwell, F. B., Cohen, P., Chen, Y. H., Chen, M. X., and Cohen, P. T. W. (1996) Eur. J. Biochem. 239, 317-325[Abstract] |
23. |
Campos, M.,
Fadden, P.,
Alms, G.,
Qian, Z.,
and Haystead, T. A. J.
(1996)
J. Biol. Chem.
271,
28478-28484 |
24. |
Huang, H. B.,
Horiuchi, A.,
Watanabe, T.,
Shih, S. R.,
Tsay, H. J.,
Li, H. C.,
Greengard, P.,
and Nairn, A. C.
(1999)
J. Biol. Chem.
274,
7870-7878 |
25. |
Allen, P. B.,
Kwon, Y. G.,
Nairn, A. C.,
and Greengard, P.
(1998)
J. Biol. Chem.
273,
4089-4095 |
26. |
Allen, P. B.,
Ouimet, C. C.,
and Greengard, P.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
9956-9961 |
27. |
MacMillan, L. B.,
Bass, M. A.,
Cheng, N.,
Howard, E. F.,
Tamura, M.,
Strack, S.,
Wadzinski, B. E.,
and Colbran, R. J.
(1999)
J. Biol. Chem.
274,
35845-35854 |
28. | Colbran, R. J., Bass, M. A., McNeill, R. B., Bollen, M., Zhao, S., Wadzinski, B. E., and Strack, S. (1997) J. Neurochem. 69, 920-929[Medline] [Order article via Infotrieve] |
29. |
Van Eynde, A.,
Wera, S.,
Beullens, M.,
Torrekens, S.,
Van Leuven, F.,
Stalmans, W.,
and Bollen, M.
(1995)
J. Biol. Chem.
270,
28068-28074 |
30. | Bollen, M., DePaoli-Roach, A. A., and Stalmans, W. (1994) FEBS Lett. 344, 196-200[CrossRef][Medline] [Order article via Infotrieve] |
31. | Renouf, S., Beullens, M., Wera, S., Van Eynde, A., Sikela, J., Stalmans, W., and Bollen, M. (1995) FEBS Lett. 375, 75-78[CrossRef][Medline] [Order article via Infotrieve] |
32. | Helps, N. R., Barker, H. M., Elledge, S. J., and Cohen, P. T. W. (1995) FEBS Lett. 377, 395-300 |
33. | Caudwell, F. B., Hiraga, A., and Cohen, P. (1986) FEBS Lett. 194, 85-90[CrossRef][Medline] [Order article via Infotrieve] |
34. | Chen, Y. H., Chen, M. X., Alessi, D. R., Campbell, D. G., Shanahan, C., Cohen, P., and Cohen, P. T. W. (1994) FEBS Lett. 356, 51-55[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Egloff, M. P.,
Johnson, D. F.,
Moorhead, G.,
Cohen, P. T. W.,
Cohen, P.,
and Barford, D.
(1997)
EMBO J.
16,
1876-1887 |
36. |
Westphal, R. S.,
Tavalin, S. J.,
Lin, J. W.,
Alto, N. M.,
Fraser, I. D.,
Langeberg, L. K.,
Sheng, M.,
and Scott, J. D.
(1999)
Science
285,
93-96 |
37. | Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Park, I. K.,
and DePaoli-Roach, A. A.
(1994)
J. Biol. Chem.
269,
28919-28928 |
39. |
Beullens, M.,
Van Eynde, A.,
Vulsteke, V.,
Connor, J.,
Shenolikar, S.,
Stalmans, W.,
and Bollen, M.
(1999)
J. Biol. Chem.
274,
14053-14061 |
40. |
Connor, J. H.,
Frederick, D.,
Huang, H.,
Yang, J.,
Helps, N. R.,
Cohen, P. T.,
Nairn, A. C.,
DePaoli-Roach, A.,
Tatchell, K.,
and Shenolikar, S.
(2000)
J. Biol. Chem.
275,
18670-18675 |
41. | Faux, M. C., and Scott, J. D. (1996) Cell 70, 8-12 |
42. | Coghlan, V. M., Hausken, Z. E., and Scott, J. D. (1995) Biochem. Soc. Trans. 23, 591-596 |
43. | Klauck, T. M., Faux, M. C., Labudda, K., Langeberg, L. K., Jaken, S., and Scott, J. D. (1996) Science 271, 1589-1592[Abstract] |
44. |
Printen, J. A.,
Brady, M. J.,
and Saltiel, A. R.
(1997)
Science
275,
1475-1478 |
45. |
Steen, R. L.,
Martins, S. B.,
Tasken, K.,
and Collas, P.
(2000)
J. Cell Biol.
150,
1251-1262 |
46. | Lester, L. B., Coghlan, V. M., Nauert, B., and Scott, J. D. (1996) J. Biol. Chem. 272, 9460-9465[CrossRef] |
47. | Reinton, N., Collas, P., Haugen, T. B., Skalhegg, B. S., Hansson, V., Jahnsen, T., and Tasken, K. (2000) Dev. Biol. 223, 194-204[CrossRef][Medline] [Order article via Infotrieve] |
48. | Dent, P., MacDougall, L. K., Mackintosh, C., Campbell, D. G., and Cohen, P. (1992) Eur. J. Biochem. 210, 1037-1044[Abstract] |
49. | Yan, Z., Hsieh-Wilson, L., Feng, J., Tomizawa, K., Allen, P. B., Fienberg, A. A., Nairn, A. C., and Greengard, P. (1999) Nat. Neurosci. 2, 13-17[CrossRef][Medline] [Order article via Infotrieve] |
50. | Greengard, P., Allen, P. B., and Nairn, A. C. (1999) Neuron 23, 435-447[Medline] [Order article via Infotrieve] |
51. |
Lin, J. W.,
Wyszynski, M.,
Madhavan, R.,
Sealock, R.,
Kim, J. U.,
and Sheng, M.
(1998)
J. Neurosci.
18,
2017-2027 |
52. |
Park, I. K.,
Roach, P.,
Bondor, J.,
Fox, S. P.,
and DePaoli-Roach, A. A.
(1994)
J. Biol. Chem.
269,
944-954 |
53. | Alessi, D. R., Street, A. J., Cohen, P., and Cohen, P. T. W. (1993) Eur. J. Biochem. 213, 1055-1066[Abstract] |
54. |
Snyder, G. L.,
Fienberg, A. A.,
Huganir, R. L.,
and Greengard, P.
(1998)
J. Neurosci.
18,
10297-10303 |
55. |
Connor, J. H.,
Kleeman, T.,
Barik, S.,
Honkanen, R. E.,
and Shenolikar, S.
(1999)
J. Biol. Chem.
274,
22366-22372 |
56. | Jurgensen, S. R., Chock, P. B., Taylor, S., Vandenheede, J. R., and Merlevede, W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7565-7569[Abstract] |
57. | Khatra, B. S., Printz, R., Cobb, C. E., and Corbin, J. D. (1985) Biochem. Biophys. Res. Commun. 130, 567-573[Medline] [Order article via Infotrieve] |
58. |
Zolnierowicz, S.,
and Bollen, M.
(2000)
EMBO J.
19,
483-488 |
59. |
Allen, P. B.,
Ouimet, C. C.,
and Greengard, P.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
9956-9961 |
60. |
Surks, H. K.,
Mochizuki, N.,
Kasai, Y.,
Georgescu, S. P.,
Tang, K. M.,
Ito, M.,
Lincoln, T. M.,
and Mendelsohn, M. E.
(1999)
Science
286,
1583-1587 |
61. | Alessi, D., Macdougall, L. K., Sola, M. M., Ikebe, M., and Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035[Abstract] |
62. |
Yang, J.,
Hurley, T. D.,
and DePaoli-Roach, A. A.
(2000)
J. Biol. Chem.
275,
22635-22634 |
63. | Liu, J., Wu, J., Oliver, C., Shenolikar, S., and Brautigan, D. L. (2000) Biochem. J. 346, 77-82[CrossRef][Medline] [Order article via Infotrieve] |
64. | Hsieh-Wilson, L. C., Allen, P. B., Watanabe, T., Nairn, A. C., and Greengard, P. (1999) Biochemistry 38, 4365-4373[CrossRef][Medline] [Order article via Infotrieve] |
65. |
Katayose, Y.,
Li, M.,
Al-Murrani, S. W.,
Shenolikar, S.,
and Damuni, Z.
(2000)
J. Biol. Chem.
275,
9209-9214 |
66. | Faux, M. C., Rollins, E. N., Edwards, A. S., Langeberg, L. K., Newton, A. C., and Scott, J. D. (1999) Biochem. J. 343, 443-452[CrossRef][Medline] [Order article via Infotrieve] |
67. |
Faux, M. C.,
and Scott, J. D.
(1997)
J. Biol. Chem.
272,
17038-17044 |
68. | Nauert, J. B., Klauck, T. M., Langeberg, L. K., and Scott, J. D. (1997) Curr. Biol. 7, 52-62[Medline] [Order article via Infotrieve] |