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J. Biol. Chem., Vol. 279, Issue 53, 54983-54986, December 31, 2004
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12 Directly Interacts with PP2A
12-STIMULATED PP2A PHOSPHATASE ACTIVITY AND DEPHOSPHORYLATION OF MICROTUBULE-ASSOCIATED PROTEIN, Tau*
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From the
Renal Division, Department of Medicine and Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the ¶Department of Biology, University of North Carolina, Asheville, North Carolina 28804
Received for publication, October 26, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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12/13 family of heterotrimeric G proteins modulate multiple cellular processes including regulation of the actin cytoskeleton. G12/13 interact with several cytoskeletal/scaffolding proteins, and in a yeast two-hybrid screen with G12, we detected an interaction with the scaffolding subunit (A) of the Ser/Thr phosphatase, protein phosphatase 2A (PP2A). PP2A dephosphorylates multiple substrates including tau, a microtubule-associated protein that is hyperphosphorylated in neurofibrillary tangles. The interaction of A and G12 was confirmed by coimmunoprecipitation studies in transfected COS cells and by glutathione S-transferase (GST)-G12 pull-downs from cell lysates of primary neurons. The interaction was specific for A and G12 and was independent of G12 conformation. Endogenous A and G12 colocalized by immunofluorescent microscopy in Caco-2 cells and in neurons. In vitro reconstitution of GST-G12 or recombinant G12 with PP2A core enzyme resulted in 
300% stimulation of PP2A activity that was not detected with other G subunits and was similar with GTP
S- and GDP-liganded G12. When tau and active kinase (Cdk5 and p25) were cotransfected in to COS cells, there was robust tau phosphorylation. Co-expression of wild type or QL12 with tau and the active kinase resulted in 60 ± 15% reductions in tau phosphorylation. In primary cortical neurons stimulated with lysophosphatitic acid, a 50% decrease in tau phosphorylation was observed. The G12 effect on tau phosphorylation was inhibited by the PP2A inhibitor, okadaic acid (50 nM), in COS cells and neurons. Taken together, these findings reveal novel, direct regulation of PP2A activity by G12 and potential in vivo modulation of PP2A target proteins including tau. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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GTPase activity (reviewed in Ref. 1), and the GPR (G protein regulatory) protein family that share a conserved motif that inhibits GDP release from the Gi/o families of G subunits (2–4). G12/13 have multiple cellular functions including regulation of the actin cytoskeleton (5) and many functions are shared by both G12 and G13 subunits. G12/13 regulation of the actin cytoskeleton and stress fiber formation occurs through direct interactions with Rho regulatory proteins (6, 7), and other aspects of G12/13 signaling are regulated through specific interactions with membrane or scaffolding proteins. For example, the binding of G12 and G13 to the C terminus of E-cadherin displaces 
-catenin permitting it to signal (8, 9). We identified direct binding of both wild type and constitutively active (QL)12 to ZO-1 and regulation of paracellular permeability of Madin-Darby canine kidney cells (10, 11). In addition, G12 interacts with AKAP-lbc, a scaffolding molecule that organizes components of cAMP signaling and Rho signaling machinery (12). Here, we have identified the A subunit of Ser/Thr phosphatase PP2A as another "scaffolding" protein that selectively interacts with G12.
Tau is a microtubule-associated protein that is predominantly expressed in neurons and functions to stabilize the cytoskeleton. Tau phosphorylation is necessary for microtubule binding and other protein interactions, but hyperphosphorylated tau is the predominant component of neurofibrillary tangles, a pathologic hallmark finding found in several neurodegenerative disorders including Alzheimer disease and FTDP-17 (frontotemporal dementia and Parkinson's disease linked to chromosome 17; reviewed in Ref. 13). Several kinases phosphorylate tau including mitogen-activated protein kinase (MAP), glycogen synthase 3 (GSK-3), tau-tubulin kinase, and cyclin-dependent kinases 2 and 5 (Cdk) (reviewed in Ref. 13). Likewise, several protein phosphatases (PP) of the 1, 2A, and 2B families can dephosphorylate tau proteins in vitro, and PP2A directly binds to tau and microtubules (14). To determine the relevance of the yeast two-hybrid interaction between G12 and the A subunit of PP2A, we demonstrate direct and specific G12-dependent stimulation of PP2A activity in vitro. Furthermore, we show that G12-mediated signaling in COS cells and primary cultured neurons stimulates PP2A-mediated dephosphorylation of the microtubule-binding protein, tau.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Subunits, and Antibodies—cDNAs for G12, G13, QL12, (Q229L), and QL (Q229) were obtained from the Guthrie cDNA Resource Center (www.cDNA.org). GST- and pcDNA3-G12, GST, G13, and Gs were described previously (11). Myc-tagged PP2A A subunit was provided by Dr. David Virshup (University of Utah), and Cdk5 and p25 plasmids (15) were provided by Li-Huei Tsai (Harvard Medical School). 4R GFP-tau has been described previously (16). Baculovirus purified Gi1 was provided by Stephen Graber (West Virginia University), and G12 and Gs were generously provided by Patrick Casey (Duke University). Polyclonal rabbit anti-G12 and G13 and goat-anti A antibodies were from Santa Cruz Biotechnology, monoclonal anti-Myc mouse antibodies from Invitrogen. Antibodies to tau were from Upstate Biotechnology (Lake Placid, NY), and GFP was from Abcam Inc. (Cambridge, MA). Secondary antibodies were all obtained from Molecular Probes. Polyclonal rabbit anti-A was provided by D. Virshup, and tau phosphospecific antibody CP9 was generously provided by Peter Davies (Albert Einstein College of Medicine). Lysophosphatitic acid (LPA) was from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL).
Yeast Two-hybrid Screening—The MatchmakerTM (Clontech, Palo Alto, CA) was utilized as described previously (17). Mouse G12 cDNA was cloned into the bait vector pAS-2 and a human fetal kidney library, in pACT-2, was screened using standard selection methodology as described previously (17) except that co-transformed yeast were incubated at room temperature for 6–10 days. Clones growing on selective plates at room temperature were regrown and screened for -galactosidase activity.
Immunohistochemistry, Immunoprecipitation, Immunoblot Analysis, and GST Pull-downs—Caco-2 cells and primary cortical neurons were cultured on sterile glass cover slips and costained with rabbit anti-G12 and goat anti-A both at 1:50 as described previously (10). Donkey anti-rabbit FITC (1:1000) and donkey anti-goat Texas Red (1:1600) were used for costaining and images were obtained on a Nikon Labophot-2 microscope and Spot Digital camera and software (www.diginc.com/SpotSoftware). Images were processed in Adobe Photoshop. For immunoprecipitations and Western blots, cells were washed in phosphate-buffered saline, and lysates prepared in modified RIPA buffer (20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% Na-deoxycholate, 0.02% azide, 1 mM Na3VO4, 25 mM NaF + protease inhibitor mixture). Immunoprecipitation with various antibodies (rabbit A, rabbit G12, mouse myc, or control antibodies) was described previously (11). SDS-PAGE and Western blot were done as described previously (11). GST pull-downs were performed as described previously (11) from cell lysates prepared from primary cortical neurons.
PP2A Phosphatase Activity—Purified PP2A core enzyme (A and catalytic unit) was obtained from Upstate Biotechnology and kinetic analysis of phosphatase activity determined by Malachite Green phosphatase assay (Upstate Biotechnology). The phospho-peptide (K-R-pT-I-R-R) was used as substrate to determine PP2A activity alone and in combination with GST, and GST-G12, GST-G13, and GST-Gs at 1:5 molar ratio). A similar comparison was done with recombinant G12, Gs, and Gi1 at 1:1 molar ratio. G proteins were incubated with GDP or GTPS (50 µM) or 
(3 mm NaF + 50 µM AlCl3) for 15 min at 30 °C followed by incubation with PP2A for 30 min at 23 °C. The reaction was initiated by addition of substrate at t = 0, and after 30 min the reaction was terminated, absorbance measured at 624 nm, and enzyme activity determined (nmol of phosphate/min/units).
Cell Culture and COS Cell Transfections—COS cells were cultured and transfected as described previously (18) using LipofectamineTM (Invitrogen) according to the manufacturer's protocol. Total cDNA was constant and combinations of GFP-4R, Cdk5, p25, and wild type or QL12 were transfected using equivalent amounts. Cells were lysed 48 h after transfection in 0.5 ml modified RIPA or HEDL buffer (50 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10 mM MgCl2, 1 mM Na3VO4, 25 mM NaF + protease inhibitor mixture).
Primary Neuronal Cultures and Agonist Stimulation—The cortex of E18 Sprague-Dawley rats was separated in 2.5% trypsin (Invitrogen) and 0.5–1 ml 0.16% (w/v) DNase I (Sigma) dissolved in HBSS. Cells were plated at 1 x 105 cells/cm2 on poly-L-lysine-coated plates or coverslips. HBSS was replaced with Neurobasal media (Invitrogen) supplemented with glutamine and B-27 and penicillin/streptomycin antibiotic solution. Experiments were done after 10–14 days in culture by adding vehicle, isoproterenol (1 µM), or LPA (10 µM) for 30–60 min followed by washing, scraping in modified RIPA buffer, and Western analysis on identical amounts of total protein using CP-9 antibody. Blots were stripped and reprobed with tau antibodies.
Statistics and Quantization—Western blots with exposures in the linear range were scanned using a desktop scanner and the images analyzed in NIH Image 1.63 (Wayne Rasband). Statistics were compiled with GraphPad Prism (GraphPad Software, San Diego, CA). Results are expressed as the mean ± standard deviation. Statistical significance was determined using two-tailed t test.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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12, we identified an interaction with the A subunit of PP2A. This interaction was confirmed in several assays and we found direct G12-dependent stimulation of PP2A activity in vitro. Furthermore, studies in COS cells and primary neurons reveal G12-dependent stimulation of PP2A activity resulting in reduced phosphorylation of a target protein, tau.
Yeast two-hybrid screening of a human embryonic kidney library with mouse wild type G12 subunit resulted in five confirmed positive clones. Two of these were identical and encoded a 1.1-kb fragment of the regulatory subunit A (PR 65) of PP2A (GenBankTM accession number NM_014225
[GenBank]
). The coding sequence in this fragment included amino acids 225 to the C terminus (590) encompassing repeats 7–15. PP2A is one of eight classes of serine/threonine phosphatases, is ubiquitously expressed, and is a major regulator of many fundamental cellular processes (reviewed in Ref. 19). PP2A is composed of a catalytic subunit (C), a scaffolding subunit (A), and a regulatory subunit (B). The catalytic and scaffolding subunits bind tightly to form a core dimer (AC) that is a functional unit. The regulatory subunits are numerous and diverse and provide specificity and localization for the many functions of PP2A. Many proteins interact with PP2A including signaling proteins and transcription factors, membrane receptors and transporters, protein kinases, cytoskeletal proteins, and others (reviewed in Ref. 20).
To confirm this interaction, we cotransfected myc-tagged PP2A A and wild type G12 or constitutively active Q229L12 in COS cells. Endogenous G12 protein is not detectable nor can any be immunoprecipitated (last lane in Fig. 1A). Immunoprecipitation of myc-tagged A subunits precipitated the co-expressed G12 subunits (wild type and QL, Fig. 1A). Control immunoprecipitations with G12 antibody and myc antibody in vector-transfected cells did not detect any G12. Stripping and reprobing the blot with A rabbit polyclonal antibody revealed the myc-tagged A subunit migrating at 80 kDa. Fig. 1B shows that immunoprecipitating the endogenous A subunit coprecipitated a fraction of the transfected wild type and QL12 subunits. Controls with serum and beads alone were negative. Parallel experiments with G13, Gi2, and Gs failed to detect any interaction with A (results not shown). To document this interaction in non-transfected cells, GST pull-downs from primary cortical neurons were performed. Fig. 1C shows interaction of endogenous A from neurons with GST-G12 but not GST, GST-G13, or GST-Gs. These findings suggested that the interaction of G12 with A subunits did not depend upon G12 conformation. To confirm this, wild type G12 and myc-A were cotransfected into COS cells and divided into three equal fractions. Lysates were incubated at 23 °C for 20 min in the presence of GDP, GTPS, or . Following immunoprecipitation of myc-A subunits and analysis by G12 Western blot, there were no significant differences in the amount precipitated (Fig. 1D). It was previously shown in separate studies that G12 and PP2A are localized in epithelial cell tight junctions (21, 22). Endogenous G12 and A were colocalized by immunofluorescent microscopy using antibodies to G12 and A in Caco-2 cells and primary cultured neurons (Fig. 1E). The two proteins colocalize in the lateral membrane of Caco-2 cells (Fig. 1E) and in the cell body and processes of primary neurons (Fig. 1E).
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12 and A interact, there is no known regulation of PP2A by G proteins. To determine whether G12 affects PP2A activity, phosphatase activity of purified core enzyme (A and catalytic subunit) was measured in the presence and absence of several G subunits. Fig. 2A shows that GST-G12 significantly stimulates PP2A phosphatase activity by nearly 300% above the activity of the enzyme alone. Parallel assays done with GST alone or GST-G13 failed to demonstrate any significant effect on PP2A activity. Consistent with binding results in Fig. 1, preincubation of GST- subunits with GDP or GTPS did not significantly affect the G12-mediated stimulation of PP2A activity. modestly decreased base-line PP2A activity (by 25%), but there was still nearly 3-fold stimulation of PP2A activity (data not shown). These results suggest that the effect on PP2A activity was specific for G12 and independent of bound nucleotide. However, GST-G proteins may not appropriately exchange guanine nucleotides. To address this, PP2A phosphatase activity was measured in the presence and absence of purified G subunits. Fig. 2B shows that recombinant G12 stimulates PP2A phosphatase activity over 300%, whereas recombinant Gs and Gi1 had no demonstrable effect. Similar to the results with GST-G12, and consistent with the binding data (Fig. 1D), the G12-stimulated phosphatase activity was similar for GDP- and GTPS-liganded G12.
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12-stimulated PP2A activity would result in less phosphorylation of a target substrate. To test this in cells, we utilized the microtubule associated protein, tau (GFP-tagged) and determined the effect of G12 expression on its phosphorylation in COS cells. We have previously demonstrated that the function of GFP-tau is indistinguishable from wild type tau (16). Fig. 3A shows a Western blot of COS cells transfected with GFP-tau (4R), Cdk5+p25 (the active kinase), and either wild type or QL12. As expected for COS cells, expression of tau (4R, GFP-tagged) alone reveals no detectable Thr-231 phosphorylation (as detected with CP9 antibody; Fig. 3A, lane 1). When GFP-tau is coexpressed with the active kinase (4R+kinase) a robust signal is apparent. Cotransfection of either wild type or QL12 along with tau and the active kinase results in 60% reduction in Thr-231 phosphorylation. The total tau in each lane was similar, and the bar graph (Fig. 3A) summarizes the relative effect of G12 expression on Thr-231 phosphorylation. Consistent with the effects of G12 occurring through regulation of PP2A, the PP2A phosphatase inhibitor, okadaic acid (50 nM), nearly completely inhibited the G12 mediated stimulation of PP2A activity (+O.A., Fig. 3). As expected from the lack of PP2A stimulation by other G subunits (Fig. 2), parallel experiments with cotransfected wild type and QL13 in COS cells showed no change in tau phosphorylation (results not shown). Finally, we determined the extent of tau phosphorylation in primary cortical neurons stimulated with the G12/13 agonist, LPA. Fig. 3B shows a 50% reduction in endogenous tau phosphorylation when the cells were stimulated with LPA for 60 min. There was no effect of isoproterenol on tau phosphorylation, and the effect of LPA was inhibited by preincubation with okadaic acid (Fig. 3B).
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12 but not the related G13 subunit. In addition, G12-stimulated PP2A activity may be an important regulatory pathway in cells that could affect the phosphorylation state of proteins such as tau. Strategies to decrease tau phosphorylation have received intense interest, since some features of Alzheimer and other neurodegenerative diseases are associated with hyperphosphorylated tau in neurofibrillary tangles. Furthermore, there is complex regulation of tau phosphorylation by multiple kinases and phosphatases (reviewed in Ref. 13). Several lines of evidence support the notion that G12 may have an important role in regulating tau phosphorylation in the central nervous system. The expression pattern of G12 in the central nervous system overlaps with the locations in which neurofibrillary tangles are known to develop including the cortex and hippocampus (23). Ligands for many G12/13-coupled receptors have been implicated in Alzheimer and other neuropathologic processes. For example, endothelin and thrombin can couple to G12/13, and activation of these receptors may contribute to neuronal cell death and amyloid plaque development in Alzheimer disease (24–26). In addition, GSK-3 is one of several kinases that phosphorylates tau, and recent studies reveal regulation of GSK-3 activity by G12/13 (27, 28).
These results plus recent studies suggest that the G12/13 family of G proteins may regulate serine/threonine phosphatases through multiple mechanisms. An interaction of QL12 with the Ser/Thr protein phosphatase type 5 (PP-5) was recently identified. Both QL12 and QL13 interacted with the N-terminal tetratricopeptide repeat unique to the PP-5 family of Ser/Thr phosphatases and stimulated phosphatase activity (29, 30). In contrast, our studies reveal similar binding of both wild type and QL12 to PP2A, and we do not detect any interactions with G13. Taken together, this suggests that protein phosphatase activities of several families may be regulated by G12/13 and raises the possibility that G protein regulation of serine/threonine phosphatases is a more general phenomenon.
The stimulation of PP2A phosphatase activity by G12 independent of nucleotide bound suggests a novel mechanism of regulation. The G12-stimulated PP2A phosphatase activity in vitro reveals that core enzyme (A and catalytic subunit) and G12 are sufficient for the stimulation. As a result, G and PP2A regulatory B subunits are not necessary for this effect, but future studies are needed to determine any potential regulatory role of G and/or B subunits on this activity. Our findings are consistent with a direct interaction of A and G12, although we cannot exclude the possibility that G12 also interacts with the catalytic subunit of PP2A. Additional studies are needed to address this question and whether the mechanism of stimulation by G12 is direct or through a secondary conformational change in A. The observation that G12 binds to the scaffolding A subunit of PP2A and regulates phosphatase activity is another example of signaling modulation through multiprotein complexes. This novel pathway of G12-stimulated PP2A phosphatase activity was not seen with other related G subunits and raises the possibility for specific modulation of some PP2A target proteins in vivo. This may have major implications for treating specific diseases such as neurodegenerative processes mediated by tau hyperphosphorylation.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Renal Division, Brigham and Women's Hospital, Harvard Institutes of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5809; Fax: 617-525-5830; E-mail: bdenker{at}rics.bwh.harvard.edu.
1 The abbreviations used are: G protein, guanine nucleotide-binding protein; Cdk, cyclin-dependent kinase; PP, protein phosphatase; FITC, fluorescein isothiocyanate; LPA, lysophosphatidic acid; GSK, glycogen synthase; GST, glutathione S-transferase; GFP, green fluorescent protein; GTPS, guanosine 5'-O-(thiotriphosphate); HBSS, Hanks' balanced salt solution.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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