Identification of Kinase-Phosphatase Signaling Modules Composed of p70 S6 Kinase-Protein Phosphatase 2A (PP2A) and p21-activated Kinase-PP2A*

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 IIα (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.

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 postsynaptic 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][8][9][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)(12)(13)(14) and carboxymethylation (15,16). Thus, both regulatory subunit binding and post-transla-tional 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 ␤ 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.
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 g/ml leupeptin, 20 g/ml soybean trypsin inhibitor, 2 g/ml pepstatin, and 2 mM benzamidine) and centrifugation at 100,000 ϫ 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 g 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 l of either affinitypurified p70 S6 kinase antibody, PAK1 or PAK3 antisera, or control rabbit antiserum. Protein G-Sepharose (40 l of a 1:1 slurry) or protein A-Sepharose (30 l 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 Me 2 SO or 7.5 M microcystin-LR for 30 min at 4°C. Microcystin-Sepharose (45 l 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 [␥-32 P]ATP (2000 cpm/pmol), and in the presence and absence of 0.5 g of GTP␥S-treated cdc42. Bovine myelin basic protein (2.5 g) 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).
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 g of bovine serum albumin in a total reaction volume of 100 l. The reactions were terminated by the addition of trichloroacetic acid (20% final concentration). Following incubation for 10 min at Ϫ20°C, the samples were centrifuged at 10,000 ϫ g, and [ 32 P]P i 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).
Immunoblot Analysis-Protein samples were separated on SDSpolyacrylamide 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 crosslinking reagent DTSSP; for comparison, control samples were treated with buffer. Both noncross-linked (native) and crosslinked 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␣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.
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 crosslinked 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␣ 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.
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 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. 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 crosslinked 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)(34)(35)(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. 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 ϩ extract). The isolated immune complexes and soluble extracts (100 g) 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 (Ϫ) and presence (ϩ) 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 ϭ 3).

DISCUSSION
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)(12)(13)(14)(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)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(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␣ (18) and regulation of CaMKIV activity by an associated PP2A (28).
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-2 L. Charlton and S. Pelech, submitted for publication.
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 Me 2 SO (Ϫ) or unconjugated microcystin-LR (ϩ) 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.
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 ␣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 kinasephosphatase 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.
To date, six protein kinases have been shown to associate with PP2A including casein kinase 2␣ (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 elementbinding 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 loca-tion, and lend support for a general paradigm in cellular signaling whereby multiple kinase-PP2A complexes control diverse intracellular signaling cascades.