From the Department of Pharmacology, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232-6600 and the
¶ Department of Medicine, University of British Columbia,
Vancouver, British Columbia V6T 1Z3, Canada
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ABSTRACT |
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A growing body of evidence indicates
that regulation of protein-serine/threonine phosphatase 2A (PP2A)
involves its association with other cellular and viral proteins in
multiprotein complexes. PP2A-containing protein complexes may exist
that contribute to PP2A's important regulatory role in many cellular
processes. To identify such protein complexes, PP2A was partially
purified from rat brain soluble extracts following treatment with a
reversible cross-linker to stabilize large molecular size forms of
PP2A. Compared with native (uncross-linked) PP2A, cross-linked PP2A revealed an enrichment of p70 S6 kinase and two p21-activated kinases
(PAK1 and PAK3) in the PP2A complex, indicating these kinases may
associate with PP2A. The existence of protein kinase-PP2A complexes in
rat brain soluble extracts was further substantiated by the following
results: 1) independent immunoprecipitation of the kinases revealed
that PP2A co-precipitated with p70 S6 kinase and the two PAK isoforms;
2) glutathione S-transferase fusion proteins of p70 S6
kinase and PAK3 each isolated PP2A; and 3) PAK3 and p70 S6 kinase bound
to microcystin-Sepharose (an affinity resin for PP2A-PP1).
Cumulatively, these findings provide evidence for association of PP2A
with p70 S6 kinase, PAK1, and PAK3 in the context of the cellular
environment. Moreover, together with the recent reports describing
associations of PP2A with
Ca2+/calmodulin-dependent protein kinase IV
(Westphal, R. S., Anderson, K. A., Means, A. R., and
Wadzinski, B. E. (1998) Science 280, 1258-1261) and
casein kinase II Reversible phosphorylation of proteins is a major mechanism for
the control of intracellular signaling cascades and maintenance of
cellular homeostasis (1). The phosphorylation state of proteins is
determined by the integrated actions of both protein kinases and
phosphatases, which are regulated at many levels through numerous mechanisms. While the substrate selectivity of protein kinases and
phosphatases is one determinant for the control of protein phosphorylation, additional mechanisms are needed to ensure specificity of these phosphorylation events. It is clear that regulation of enzyme
function through the assembly of multiprotein complexes is another
mechanism available for maintaining specificity in intracellular
signaling processes (2). For example, in processes involving
protein-serine/threonine phosphorylation, the A kinase-anchoring protein AKAP79 serves as a multivalent scaffold protein capable of
targeting three signaling molecules (e.g.
cAMP-dependent protein kinase, protein kinase C, and
calcineurin) to the post-synaptic density in neurons (3). In a similar
fashion, the yeast Ste5 scaffold protein coordinates the association of
at least three kinases within the mitogen-activated protein kinase
signaling cascade (4-6). Thus, these multiprotein complexes restrict
access of kinases and phosphatases to particular microenvironments
and/or specific proteins, and foster specificity of phosphorylation
events by optimally positioning these enzymes to respond rapidly to
cellular stimuli.
Many studies have implicated an important regulatory role for
protein-serine/threonine phosphatase 2A
(PP2A)1 in a wide variety of
cellular functions including metabolism, transcription and translation,
ion transport, development, cell growth, and differentiation (reviewed
in Refs. 7-10). The activity of an enzyme involved in so many cellular
processes is likely to be tightly controlled in vivo. For
example, the PP2A holoenzyme is a heterotrimer composed of catalytic
(C), structural (A), and regulatory (B) subunits (reviewed in Refs.
7-10). To date, over 15 regulatory B subunits have been identified for
PP2A; these subunits influence catalytic activity of PP2A and are
predicted to be involved in subcellular targeting of the holoenzyme. In addition to regulatory subunits, PP2A activity is influenced by subunit
phosphorylation (11-14) and carboxymethylation (15, 16). Thus, both
regulatory subunit binding and post-translational modifications are
important mechanisms controlling PP2A function.
An emerging body of evidence indicates that regulation of PP2A also
involves association with other proteins in addition to the core
phosphatase subunits. For example, PP2A has been shown to bind to the
Materials--
Affinity-purified rabbit anti-peptide antibodies
to p70 S6 kinase, rabbit anti-peptide antisera to PAK1 and PAK3, and
GST-PAK3 and GST-p70 S6 kinase fusion proteins were from Kinetek
Pharmaceuticals, Inc. (Vancouver, B.C., Canada). Goat anti-PAK Partial Purification of Native and Cross-linked
PP2A--
Soluble extracts were prepared from rat brains by
homogenization on ice (two 15-s Polytron bursts) in buffer A (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1 mM
EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 20 µg/ml soybean trypsin inhibitor, 2 µg/ml pepstatin,
and 2 mM benzamidine) and centrifugation at 100,000 × g for 1 h. One rat brain was used for analytical
purifications, and 10-15 rat brains were used for preparative
purifications. Samples were maintained at 4 °C throughout the
purification. Soluble proteins precipitating between 25% and 50%
ammonium sulfate were fractionated using FPLC on preparative Superdex-200 gel filtration in buffer A. The fractions corresponding to
proteins of large molecular size that contained PP2A were treated with
buffer or 5 mM DTSSP for 15 min on ice, and excess reactive cross-linker was quenched by the addition of 0.1 M
Tris-HCl, pH 7.5. Noncross-linked and cross-linked PP2A samples were
then partially purified by sequential fractionation on phenyl-Sepharose
(linear gradient of 0.3-0 M ammonium sulfate), MonoQ
(linear gradient of 0-0.5 M NaCl), and
aminohexyl-Sepharose (step gradient of 0-1.0 M NaCl in 0.1 M increments); the buffer used for these chromatographic steps consisted of 25 mM Tris-HCl, pH 7.5, 1 mM
dithiothreitol, 1 mM EDTA, and 1 mM EGTA. For
some experiments, a final analytical gel filtration step (Superdex-200
HR10/30) was added. Column fractions were assayed for phosphatase
activity toward protein kinase C-phosphorylated histone; the peak of
activity was pooled and applied to the next column.
Glutathione S-Transferase Fusion Protein Affinity
Chromatography--
Rat brain soluble extracts (approximately 1 mg of
protein) were incubated for 4 h at 4 °C with 10 µg of
purified GST-p70 S6 kinase or GST-PAK3 fusion protein coupled to
glutathione-Sepharose. The beads were pelleted and washed six times (5 min/wash) with PBS. Bound proteins were eluted at room temperature with
20 mM glutathione in PBS and subjected to immunoblot analysis.
Immunoprecipitations--
Rat brain soluble extracts (1-4 mg of
protein) were incubated for 4-16 h at 4 °C with 5-20 µl of
either affinity-purified p70 S6 kinase antibody, PAK1 or PAK3 antisera,
or control rabbit antiserum. Protein G-Sepharose (40 µl of a 1:1
slurry) or protein A-Sepharose (30 µl of a 1:1 slurry) was added and
the incubation continued for 45 min at 4 °C. The beads were pelleted
and washed six times (5 min/wash) with PBS. Bound proteins were eluted
with Laemmli sample buffer and subjected to immunoblot analysis.
Microcystin-Sepharose Affinity Isolations--
Rat brain soluble
extracts (4 mg) were pre-incubated with Me2SO or 7.5 µM microcystin-LR for 30 min at 4 °C.
Microcystin-Sepharose (45 µl of a 1:1 slurry) was added to the
extracts, and the samples were incubated for 1-4 h at 4 °C. The
beads were washed six times (5 min/wash) with buffer A, and bound
proteins eluted with Laemmli sample buffer were subjected to immunoblot analysis.
Kinase Assays--
Rat brain soluble extracts were subjected to
immunopurification using control serum or anti-peptide antiserum to
PAK1 and PAK3 as described above. Immune complexes were resuspended in kinase assay buffer containing 20 mM HEPES, pH 7.5, 10 mM magnesium chloride, 1 mM dithiothreitol, 0.1 mM [ Phosphatase Assay--
Fractions from each step of the
purification protocol were diluted (typically between 10- and 100-fold)
and incubated 10 min at 30 °C with protein kinase C-phosphorylated
histone (31) in buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, and 20 µg of bovine serum albumin in a total reaction
volume of 100 µl. The reactions were terminated by the addition of
trichloroacetic acid (20% final concentration). Following incubation
for 10 min at Immunoblot Analysis--
Protein samples were separated on
SDS-polyacrylamide gels (10%) and transferred to nitrocellulose in 10 mM CAPS containing 10% methanol for 1 h at 1 Amp.
Proteins on the membrane were visualized with Ponceau S, followed by
washing in TTBS (25 mM Tris-HCl, pH 7.4, 137 mM
NaCl, 3 mM KCl, and 0.2% Tween 20). The membrane was blocked in 2-5% nonfat milk/TTBS, followed by incubation with the
indicated primary antibody. Membranes were then incubated with alkaline
phosphatase- or horseradish peroxidase-conjugated secondary antibodies;
bound antibodies were visualized by colorimetric detection or chemiluminescence.
Stabilization of Large Molecular Size Forms of PP2A by
Cross-linking--
To test the hypothesis that PP2A associates with
other cellular proteins, the Superdex-200 gel filtration profile of
PP2A activity and immunoreactivity in rat brain soluble extracts were compared in buffers containing "physiological" (150 mM
KCl) or "high" (500 mM KCl) salt concentrations. PP2A
activity and immunoreactivity eluted from the gel filtration column as
a fairly broad peak (150-700 kDa) under conditions of physiological
salt (Fig. 1, A and
B). In the presence of high salt, PP2A eluted as a much
sharper peak (100-250 kDa; data not shown), which is very similar to
the elution of purified native PP2A holoenzyme (Fig. 1C).
When pooled gel filtration fractions corresponding to proteins of
300-700 kDa were refractionated under conditions of physiological
salt, a significant portion of PP2A activity and protein eluted in
approximately the same molecular size region (data not shown). However,
additional attempts to purify these potential PP2A complexes on
ion-exchange or aminohexyl-Sepharose columns (i.e. PP2A
was eluted from these columns at salt concentrations greater than 250 mM) resulted in the loss of many PP2A large molecular size
complexes, as determined by subsequent gel filtration chromatography.
Together, the data indicate that PP2A holoenzymes may associate with
additional cellular proteins and that these complexes are relatively
stable in physiological salt concentrations.
To stabilize possible PP2A multiprotein complexes for purification, the
PP2A-containing fractions of large molecular size (300-700 kDa) from
gel filtration chromatography of rat brain soluble extracts were
treated with the thiol-reversible cross-linking reagent DTSSP; for
comparison, control samples were treated with buffer. Both
noncross-linked (native) and cross-linked PP2A complexes were partially
purified by sequential fractionation on phenyl-Sepharose, MonoQ,
aminohexyl-Sepharose, and Superdex-200 columns. Analysis of PP2A
activity (Fig. 1C) and immunoreactivity (Fig. 1D)
following the final gel filtration step demonstrated that the majority
of PP2A large molecular size complexes cross-linked in the presence of
150 mM KCl were retained following partial purification of the cross-linked PP2A sample (cross-linked PP2A in Fig. 1C).
In contrast, PP2A purified in the absence of cross-linker (native PP2A
in Fig. 1C) eluted from the gel filtration column at the predicted size of heterotrimeric PP2A holoenzyme; immunoblot analysis (data not shown) of the gel filtration fractions revealed a peak of
AB Identification of Candidate PP2A Interacting
Proteins--
Stabilization of large molecular size forms of PP2A by
cross-linking, and subsequent purification of samples containing PP2A activity, provided a method for isolating potential PP2A interacting proteins. To identify proteins selectively enriched in cross-linked samples, the starting material (i.e. pooled gel filtration
fractions corresponding to proteins of large molecular size) and
partially purified native (noncross-linked) and cross-linked PP2A
samples were subjected to immunoblot analysis with antibodies to
selected cellular proteins. PAK1, PAK3, and p70 S6 kinase were
significantly enriched in the partially purified cross-linked sample
but not in the untreated sample (Fig.
2A). Several other protein
kinases (e.g. Raf, Erk1, Erk2, and
calcium/calmodulin-dependent protein kinase II; data not
shown) and protein phosphatases (e.g. PP1
To test the specificity and efficacy of the protein kinase antibodies,
immunoprecipitations were performed from rat brain soluble extracts.
Immunoblot analysis of both extracts and immune complexes revealed
proteins migrating at the predicted size of PAK1, PAK3, and p70 S6
(Fig. 2B). The PAK1 and PAK3 antibodies appeared to be
specific since PAK3 could not be detected in PAK1 immune complexes and
PAK1 could not be detected in PAK3 immune complexes (data not shown).
The PAK1 and PAK3 immune complexes also were tested for kinase activity
using myelin basic protein (MBP) as a substrate (Fig. 2C).
Both PAK immune complexes exhibited kinase activity toward MBP; the
enhanced phosphorylation of MBP in the presence of cdc42
(i.e. an activator PAK) is consistent with PAK activity. In
addition, the p70 S6 kinase immune complex possessed kinase activity
toward a synthetic peptide (data not shown), which previously has been
shown to be an excellent substrate for p70 S6 kinases (30).
Co-immunoprecipitation of PP2A with Protein Kinase--
The
enrichment of PAK1, PAK3, and p70 S6 kinase in the cross-linked sample
provides evidence that these kinases may associate with PP2A in
preformed multiprotein complexes in cells. As another strategy to
address whether PP2A interacted with these kinases, the anti-peptide
antibodies were used to independently immunoprecipitate the kinases and
the immune complexes were analyzed for the presence of PP2A by
immunoblotting. PP2A and p70 S6 kinase co-immunoprecipitated from rat
brain soluble extracts using antibodies directed against two different
domains in p70 S6 kinase (i.e. the carboxyl terminus and
amino terminus) (Fig. 3A).
Immunoprecipitation with antibodies to PAK1 and PAK3 also resulted in
co-isolation of PP2A (Fig. 3A), providing additional
evidence for the existence of preformed PP2A-protein kinase signaling
complexes. Isolation of PP2A with antibodies to either PAK3 or p70 S6
kinase was unaffected by pretreatment with microcystin, a specific
inhibitor of PP2A, demonstrating that PP2A catalytic activity is not
required for the interactions (data not shown; see below).
Isolation of PP2A with GST-Protein Kinase Fusion Proteins--
To
determine if exogenous recombinant kinases could interact with PP2A,
GST-p70 S6 kinase and GST-PAK3 fusion proteins were incubated with rat
brain soluble extracts, purified with glutathione-Sepharose, and
evaluated for the presence of PP2A by immunoblot analysis. PP2A was
present in glutathione eluates from samples incubated with either of
the GST-kinase fusion proteins but not in samples incubated with GST
alone (Fig. 3B). No PP1 immunoreactivity was detected in any
of the GST isolations (data not shown). These data demonstrate that the
recombinant kinases either directly or indirectly interact with PP2A
and can be used to isolate the phosphatase from rat brain soluble extracts.
Isolation of PAK3 and p70 S6 Kinase with
Microcystin-Sepharose--
Microcystin is an inhibitor of PP2A (and
PP1) that binds to the substrate binding site of the phosphatase
catalytic subunit (32). Because of its high affinity for these
phosphatases, microcystin-Sepharose has been utilized as an affinity
resin for isolating PP2A holoenzymes (i.e. catalytic subunit
and associated regulatory subunits) and multiprotein complexes
containing PP2A (28, 29, 33-36). Both PAK3 and p70 S6 kinase could be
isolated from rat brain soluble extracts with microcystin-Sepharose
(Fig. 3C). The isolation of PAK3 and p70 S6 kinase with this
resin presumably was due to their interaction, either directly or
indirectly, with PP2A. The binding of PAK3, p70 S6 kinase, and PP2A to
microcystin-Sepharose was attenuated by pretreatment of extracts with
unconjugated microcystin, indicating that binding occurred in a
microcystin-dependent manner. Furthermore, since
microcystin is an inhibitor of PP2A, phosphatase activity did not
appear to be required for interaction of PP2A with PAK3 and p70 S6 kinase.
PP2A has a prominent role in many cellular processes. However, the
mechanisms regulating activity and function of this enzyme in
vivo are not well defined. Since the activity of PP2A in
vitro is influenced by post-translational modifications (11-15)
and the oligomeric composition of the holoenzyme (reviewed in Refs.
7-10), it is likely these mechanisms regulate PP2A activity in
vivo as well. Recent studies also have revealed that PP2A
associates with additional proteins (17-28) and indicate that
regulation of PP2A activity may involve colocalization with specific
interacting proteins. The assembly of kinase and phosphatase molecules
into multiprotein complexes provides an attractive mechanism for
regulating enzymatic activity toward defined substrates by limiting
access of the enzymes to specific populations of proteins. An
additional level of regulation provided by these complexes is the
potential for intracomplex interactions that regulate activity of the
associated enzymes, as has been shown for regulation of PP2A activity
by an associated casein kinase 2 In the present study, PP2A was found to interact with PAK1, PAK3, and
p70 S6 kinase. Several lines of evidence are in agreement with this
conclusion. 1) PAK1, PAK3, and p70 S6 kinase co-purified with PP2A
holoenzyme following stabilization by cross-linking (Fig.
2A); 2) PP2A co-immunoprecipitated with the kinases using kinase-specific antibodies (Fig. 3A); 3) PP2A was isolated
by GST-p70 S6 and GST-PAK3 fusion proteins (Fig. 3B); and 4)
PAK3 and p70 S6 kinase were co-isolated with PP2A using
microcystin-Sepharose (Fig. 3C). The PP2A holoenzyme also
has been found to co-purify with the p70 S6 kinase from seastar
oocytes,2 providing
additional support for the existence of a p70 S6 kinase-PP2A complex.
These kinase-PP2A complexes likely represent independent complexes,
since the p70 S6 kinase immunoprecipitations and GST-p70 S6 kinase
fusion protein isolations, which were effective in isolating PP2A (Fig.
3, A and B, respectively), failed to isolate PAK3
(data not shown). Cumulatively, these data strongly support an
association of PP2A with PAK1, PAK3, and p70 S6 kinase in rat brain
soluble extracts either through a direct protein interaction or
indirectly through an intermediary protein. Although the exact nature
of these protein-protein interactions are not known, the following results indicate that phosphatase activity is not required for interaction of protein kinase with PP2A. 1) The isolation of PP2A with
PAK3 or p70 S6 kinase (i.e. either via immunoprecipitation or GST-protein kinase isolations) was unaffected by microcystin, an
inhibitor of PP2A that binds to phosphatase catalytic site, and 2) both
PAK3 and p70 S6 kinase could be isolated with microcystin-Sepharose (Fig. 3C).
An essential component of the present studies was the use of protein
cross-linking as a strategy to stabilize multiprotein complexes
containing PP2A for purification. This approach has been effectively
used to identify interacting proteins of the endoplasmic reticulum
involved in polypeptide translocation (37, 38). In the present studies,
candidate PP2A interacting proteins were identified through the
comparison of partially purified cross-linked and native samples of
PP2A. Evaluation of these samples by immunoblot analysis using several
antibodies to intracellular signaling molecules revealed selectivity of
this approach, as many other protein kinases and phosphatases did not
co-purify with PP2A (see "Results"). Each candidate protein
identified in the partially purified cross-linked sample was
subsequently shown, by independent experimental approaches, to interact
with PP2A, thus demonstrating the validity of the cross-linking
strategy. Furthermore, in similar experiments employing a different
purification scheme, the microtubule-associated protein tau was
identified as a protein enriched in cross-linked PP2A preparations
(data not shown). This observation is consistent with a recent report
showing a direct interaction between PP2A and tau (39), and lends
additional support to the use of cross-linking for identifying PP2A
interacting proteins. The differential isolation of several PP2A
interacting proteins (e.g. p70 S6 kinase and PAK versus tau) suggests that subpopulations of PP2A complexes
exist that can be isolated by cross-linking coupled with different
purification protocols.
Although the physiological significance of p70 S6 kinase-PP2A and
PAK-PP2A complexes identified in the present report is currently unknown, these complexes are likely to be important regulatory components of the respective pathways. Activation of p70 S6 kinase by
mitogenic stimuli and various stress agents is associated with phosphorylation of the enzyme (reviewed in Refs. 40 and 41); dephosphorylation is thought to be mediated by PP2A (42, 43). The
immunosuppressive drug rapamycin has been shown to induce selective
dephosphorylation and inactivation of p70 S6 kinase (43), and to
disrupt the association of PP2A with the B cell receptor-associated
protein To date, six protein kinases have been shown to associate with PP2A
including casein kinase 2 (Heriche, J. K., Lebrin, F., Rabilloud, T.,
Leroy, D., Chambaz, E. M., and Goldberg, Y. (1997) Science 276, 952-955), the present data provide compelling
evidence for the existence of protein kinase-PP2A signaling modules as a new paradigm for the control of various intracellular signaling cascades.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2-adrenergic receptor (17), casein kinase 2
(18), HOX
11 (19), tau (20), translation termination eukaryotic release factor 1 (21), adenovirus E4orf4 protein (22),
4 protein (23), neurofilament
proteins (24, 25), and small t and middle t antigens encoded by DNA
tumor viruses (26, 27). Recently, we identified a CaMKIV-PP2A signaling
complex in which PP2A dephosphorylates the associated CaMKIV and
functions as a negative modulator of CaMKIV signaling (28). Thus, a
likely determinant for directing PP2A function is its association with other proteins in multiprotein signaling complexes. Based upon these
observations and the diverse roles for PP2A in cellular processes, we
postulated that additional PP2A protein complexes are formed in
vivo. In the present report, we demonstrate the existence of p70
S6 kinase-PP2A and PAK-PP2A complexes in rat brain. These findings
reveal two new PP2A interacting proteins and, together with our recent
demonstration of a CaMKIV-PP2A signaling complex, indicate that the
assembly of protein kinase-PP2A signaling modules is a general
mechanism for regulation of PP2A action in vivo.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(PAK3) IgG was obtained from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Monoclonal antibodies to PP2A catalytic subunit and CaMKIV
were purchased from Transduction Laboratories (Lexington, KY). The generation and characterization of rabbit anti-peptide antibodies directed against the A, B
, and C subunits of PP2A was described previously (25, 29). The cross-linking reagent DTSSP
(3,3'-dithiobis[sulfosuccinimidylpropionate]) was obtained from
Pierce. MonoQ HR5/5 and Superdex-200 HR10/30 columns, Superdex-200
preparative grade resin, phenyl-Sepharose, glutathione-Sepharose,
protein A-Sepharose, protein G-Sepharose (GammaBind Sepharose), and
aminohexyl-Sepharose were from Amersham Pharmacia Biotech.
Microcystin-Sepharose and microcystin-LR were obtained from Upstate
Biotechnology (Lake Placid, NY) and Alexis Biochemicals (San Diego,
CA), respectively. [
-32P]ATP was obtained from NEN
Life Science Products. Protein kinase C was a gift from Dr. Mike
Browning (University of Colorado Health Sciences Center, Denver, CO).
The cdc42 protein was a gift from Drs. G. Johnson and G. Fanger
(National Jewish Center, Denver, CO).
-32P]ATP (2000 cpm/pmol), and in the
presence and absence of 0.5 µg of GTP
S-treated cdc42. Bovine
myelin basic protein (2.5 µg) was added to the kinase reaction
mixture, and the samples were incubated at 30 °C for 15 min. The
reactions were stopped by addition of Laemmli sample buffer, and
the samples were analyzed by SDS-PAGE and autoradiography. The p70 S6
kinase immune complexes were assayed for kinase activity toward a
synthetic peptide (AKRRRLSSLRASTSKSESSQK) as described previously
(30).
20 °C, the samples were centrifuged at 10,000 × g, and [32P]Pi in the
supernatant was quantitated by liquid scintillation counting. Protein
kinase C-phosphorylated histone is a selective substrate for PP2A and
has been used to assay PP2A activity in the presence of PP1 (31).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Stabilization of PP2A high molecular weight
forms by cross-linking. A, rat brain soluble extracts
were fractionated on Superdex-200 using FPLC and the fractions were
assayed for phosphatase activity using protein kinase C-phosphorylated
histone as the substrate. Values are normalized to the peak of
phosphatase activity. B, aliquots of the gel filtration
fractions were analyzed by immunoblotting with antibodies to the
catalytic (C), structural (A), and regulatory
B subunits of PP2A. Presented is a representative of at least three
experiments. C and D, rat brain soluble extracts
were fractionated on Superdex-200 and assayed for phosphatase activity
toward protein kinase C-phosphorylated histone. Fractions in the large
molecular size range (fractions 18-23) containing phosphatase activity
were pooled, treated with buffer (native PP2A) or 5 mM
DTSSP (cross-linked PP2A), and purified as described under
"Experimental Procedures." The partially purified pools were then
analyzed by analytical Superdex-200 gel filtration and the fractions
were assayed for phosphatase activity (C) and PP2A subunit
immunoreactivity (D). To resolve individual proteins in the
cross-linked sample, the cross-links were reversed by addition of 0.1 M dithiothreitol prior to SDS-PAGE. In the absence of
reducing agent, cross-linked proteins were retained in large complexes as
demonstrated by the lack of individual protein bands on
SDS-polyacrylamide gels (data not shown). Values for phosphatase
activity were normalized to the peak of activity. Presented is a
representative experiment of at least two purifications.
C protein that paralleled the peak of phosphatase activity (Fig.
1C). These findings demonstrate that the large molecular size forms of PP2A, potentially containing PP2A holoenzymes associated with interacting proteins, can be stabilized by cross-linking and
isolated by subsequent purification.
and PP4; Fig.
2A) present in the initial pool were not enriched in the
cross-linked sample. Interestingly, CaMKIV was detected in both native
and cross-linked samples, consistent with a more stable association of
this kinase with PP2A (see "Discussion"). These experiments also
demonstrated that the amount of PP2A catalytic subunit was comparable
in both samples (Fig. 2A), indicating the observed
enrichment of PAK1, PAK3, and p70 S6 kinase was not due to differences
in the amount of PP2A.
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Fig. 2.
Identification of proteins enriched in
cross-linked samples of PP2A. A, proteins of large
molecular size from the initial gel filtration column
(Starting Material; see Fig. 2) and partially
purified pools of native PP2A and cross-linked PP2A were resolved by
SDS-PAGE (in the presence of reducing agents) and subjected to
immunoblot analysis with the indicated antibodies. A shift in mobility
of cross-linked proteins was apparent in varying degrees, presumably
indicative of the amount of cross-linker incorporated into each
protein. Presented is a representative blot for at least two
independent purifications. B, PAK1, PAK3, and p70 S6 kinase
antibodies (top, middle, and bottom
panels, respectively) were incubated with buffer
(IgG) or 0.5 mg of rat brain soluble extract (IgG + extract). The isolated immune complexes and soluble
extracts (100 µg) were subjected to immunoblot analysis using PAK1,
PAK3, or p70 S6 kinase antibodies. The migrations of protein kinases
and IgG heavy chain are indicated by arrows. C,
immune complexes from control, PAK1, and PAK3 immunoprecipitations were
assayed for kinase activity in the absence ( ) and presence (+) of
GTP
S-activated cdc42 using MBP as a substrate. Kinase reactions were
terminated by the addition of Laemmli sample buffer, and
phosphorylated proteins were analyzed by SDS-PAGE and autoradiography.
Presented is a representative autoradiograph (n = 3).
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Fig. 3.
Co-isolation of PP2A with PAK1, PAK3, and p70
S6 kinase. A, rat brain soluble extracts were incubated
with control serum, PAK1 antiserum, PAK3 antiserum, or
affinity-purified rabbit polyclonal antibodies to the carboxyl terminus
(S6K-CT) and amino terminus (S6K-PNT) of p70 S6
kinase. Samples were incubated 4-18 h, isolated with protein
G-Sepharose, and bound proteins eluted with Laemmli sample buffer.
Eluates were analyzed for the presence of PP2A by immunoblotting using
a monoclonal antibody to the PP2A catalytic subunit. B, rat
brain soluble extracts were incubated with GST-PAK3 or GST-p70 S6
kinase immobilized on glutathione-Sepharose for 4 h.
Bound proteins were eluted with glutathione, resolved by SDS-PAGE, and
evaluated for the presence of PP2A catalytic subunit by immunoblotting.
C, rat brain soluble extracts were pre-incubated with
Me2SO ( ) or unconjugated microcystin-LR (+) and then
incubated with microcystin-Sepharose. Bound proteins were
eluted with SDS-PAGE sample buffer and subjected to immunoblot analysis
with the indicated antibodies (Ab). Results presented in
A-C are representative of at least three experiments
each.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(18) and regulation of CaMKIV activity by an associated PP2A (28).
4 (23). Thus, PP2A-containing complexes are likely to be
important in the regulation of p70 S6 kinase signaling pathway. With
respect to a role for the PAK-PP2A complexes in MAP kinase signaling,
many stimuli activate this cascade but comparatively little is known
regarding the mechanism(s) of inactivation. However, since PP2A appears
to be an important modulator of the MAP kinase pathway (39) and PAKs
lie upstream of MAP kinases (44), the identification of PAK-PP2A
complexes indicates that the MAP kinase pathway may be subject to some
form of regulation by kinase-phosphatase signaling modules. While the
interactions of p70 S6 kinase and PAK with PP2A did not require
phosphatase activity (see Results), the kinase and PP2A potentially
could have activity toward each other. For example, one might predict enhanced kinase activity in these immune complexes in the presence of a
phosphatase inhibitor if PP2A dephosphorylates and inactivates the
kinases. However, attempts to determine the affects of okadaic acid, a
PP2A inhibitor, on kinase activity and protein phosphorylation in the
p70 S6 kinase and PAK immune complexes did not yield definitive results. Therefore, it is conceivable that the physiological substrate for PP2A in these kinase-phosphatase complexes may be another component
of the respective signaling cascade (e.g. MAP kinase kinase
4 or Jun N-terminal kinase, Ref. 44; S6, Refs. 40 and 41), and not the
kinase present in the complex. An alternative explanation for the
inconclusive results is antibody inhibition of kinase and/or
phosphatase activity. Additional studies will be needed to address
these issues and to elucidate the function and regulation of these
protein kinase-PP2A complexes.
(18), CaMKIV (28), Janus kinase 2 (45),
and in the present studies PAK1, PAK3, and p70 S6 kinase. Pre-existing
complexes containing both kinase and phosphatase activities are likely
to be important in maintaining the appropriate phosphorylation state of
intracellular substrates necessary for the coordinated control of
protein phosphorylation. For example, PP2A activity was found to be
negatively regulated by an associated casein kinase 2
in a complex
that appears to play a role in regulation of cell growth (18). In
addition, we recently reported that a stable CaMKIV-PP2A complex can be
isolated from rat brain and Jurkat T cell extracts (28). Moreover, PP2A
was shown to dephosphorylate associated CaMKIV and function as a
negative regulator of CaMKIV to modulate the activity of
cAMP-responsive element-binding protein-mediated transcription in
Jurkat T cells. Numerous studies also have established that PP2A
regulates other protein kinase signaling cascades, including the p70 S6
kinase and MAP kinase pathways (reviewed in Refs. 8, 40, and 46). By
analogy to the casein kinase 2
-PP2A and CaMKIV-PP2A signaling
modules, the p70 S6 kinase-PP2A and PAK-PP2A complexes also may
represent examples of contiguous signaling modules. Identification of
interactions between PP2A and protein kinases provide compelling evidence that one cellular mechanism for regulation of specific phosphorylation events involves association of PP2A with protein kinases, presumably in a regulated fashion and in a specific cellular location, and lend support for a general paradigm in cellular signaling
whereby multiple kinase-PP2A complexes control diverse intracellular
signaling cascades.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Roger J. Colbran, Lee E. Limbird, and laboratory colleagues for critical discussions of the data and manuscript. We also acknowledge the Vanderbilt University Medical Center Cell Imaging Resource (supported by National Institutes of Health Grants CA68485 and DK20593).
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM51366 (to B. E. W.), by a grant from the National Cancer Institute of Canada (to S. L. P.), and by National Institutes of Health Grants DK20593 to the Vanderbilt Diabetes Research and Training Center, CA68485 to the Cancer Center, and MH19732 to the Center for Molecular Neurosciences.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.
§ Present address: Howard Hughes Medical Institute, Vollum Institute, OHSU, Portland OR 97201.
Recipient of a Faculty Development Award from the
Pharmaceutical Research and Manufacturers of America Foundation. To
whom correspondence should be addressed: Dept. of Pharmacology,
Vanderbilt University Medical Center, 424 MRB1, Nashville, TN
37232-6600. Tel.: 615-343-2080; Fax: 615-343-6532; E-mail:
brian.wadzinski{at}mcmail.vanderbilt.edu.
The abbreviations used are:
PP2A, protein-serine/threonine phosphatase 2A; PP1, protein-serine/threonine
phosphatase 1; PP4 (also known as PPX), protein-serine/threonine
phosphatase 4; PAK, p21-activated kinase; p70 S6 kinase, 70-kDa S6
protein kinase; CaMKIV, calcium/calmodulin-dependent
protein kinase IV; MAP, mitogen-activated protein; MBP, myelin basic
protein; GST, glutathione S-transferase; DTSSP, 3,3'-dithiobis[sulfosuccinimidylpropionate]; PAGE, polyacrylamide gel
electrophoresis; PBS, phosphate-buffered saline; TTBS, 0.1% Tween 20 in Tris-buffered saline; GTPS, guanosine
5'-3-O-(thio)triphosphate; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
2 L. Charlton and S. Pelech, submitted for publication.
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