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J. Biol. Chem., Vol. 280, Issue 52, 42644-42654, December 30, 2005
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1
¶2
From the
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, Kinexus Bioinformatics Corporation, Vancouver, British Columbia V6T 1Z3, Canada, ¶The Brain Research Centre, University of British Columbia, Vancouver, British Columbia V6T 2B5, Canada, and ||Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242
Received for publication, March 4, 2005 , and in revised form, October 12, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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regulatory subunits by 
3-fold or knock-down of B by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-B/ subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in B knock-down cells but elevated in B- and B-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing B or B. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes ABC and ABC act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A ABC and ABC holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Numerous protein kinases have been shown to form stable complexes with PP2A including p70 S6 kinase and p21-activated kinases (22, 23), I
B kinases (24, 25), calcium-calmodulin-dependent protein kinase IV (26, 27), protein kinase B/Akt (28), Janus-activated kinase (29), casein kinase II (30), and mitogen-activated protein kinase (MAPK) family members (31–33). The identification of multiple signaling modules containing both kinases and phosphatases suggests that the control of the respective signal transduction pathway is mediated via the interplay of these opposing enzymes. Because dephosphorylation of the associated protein kinase by PP2A has been linked to both positive and negative regulatory effects on kinase activation (24, 26, 27, 32, 33), the regulatory subunit likely plays an important role in controlling the function of these protein kinase-PP2A signaling complexes.
The MAPKs represent a large family of protein kinases that regulate diverse intracellular signal transduction cascades controlling cellular proliferation, differentiation, and apoptosis (34). Thus far, four well characterized families of mammalian MAPKs have been identified: the extracellular signal-regulated kinases (ERK1 and ERK2), c-Jun NH2-terminal/stress-activated protein kinases, p38 MAPKs, and ERK5 (for a review, see Ref. 34). Immunoprecipitation studies have revealed that PP2A associates with ERK2 and the MAPK kinase MEK1 (31) as well as with the kinase suppressor of Ras (33) and Raf1 (also known as c-Raf) (32), two upstream kinases in the prototypical MAPK signal transduction pathway (Raf1-MEK1/2-ERK1/2). Although PP2A has been reported to negatively regulate ERK1/2 activity (16, 35–38), recent studies have demonstrated that PP2A can also function as a positive regulator of Raf1 (32, 39–41) and kinase suppressor of Ras (33). The positive regulation of these kinases by PP2A is thought to be mediated via dephosphorylation of phosphorylated serine or threonine residues that inhibit kinase activity. Cell type- or species-specific regulatory effects on MAPK signaling have also been documented for PP2A regulatory subunits. For example, knock-down of the single Drosophila B family subunit results in ERK1/2 activation (16), whereas overexpression of a member of the B family of regulatory subunits, B
, in a mammalian neuronal cell line leads to ERK1/2 activation (41). Furthermore the Caenorhabditis elegans B homolog SUR-6 has been identified as a positive regulator of Raf1 and MPK-1 (the C. elegans ERK ortholog) (42).
Although the PP2A catalytic subunit has been identified in multiple protein kinase macromolecular complexes, the regulatory subunits in these complexes remain largely unknown. Additional studies are needed to determine the oligomeric composition of these complexes as well as the precise function of individual regulatory subunits. To elucidate the role of specific PP2A holoenzymes in MAPK signaling, we altered the cellular levels of B or B regulatory subunits in target cells by overexpression and RNAi approaches. Western analyses of the stable cell lines using phosphospecific antibodies revealed opposing effects on the phosphorylation state of ERK1/2 that were dependent upon overexpression or repression of the targeted B subunit protein. Our findings indicate that PP2A B-orB-containing holoenzymes control ERK1/2 activation via their interaction with and dephosphorylation of the upstream kinase Raf1, leading to sequential phosphorylation/activation of MEK1/2 and ERK1/2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/B- and B-specific antibodies were as reported previously (13). PD98059 was obtained from Calbiochem, and microcystin-LR was from Alexis Biochemicals (San Diego, CA).
FLAG-tagged B Subunit Constructs—The FLAG epitope was added to the carboxyl terminus of B and B using a two-step PCR protocol. In the first reaction, PCR amplification of a 300-bp fragment was accomplished using the sense primer 5'-GTC ATG ACT GGA TCC TAC AAT-3' (which overlaps the BamHI site 300 bp from the stop codon), the antisense primer 5'-GTC GTC CTT GTA GTC ATT AAT TTT GTC CTG-3', and rat B/pcDNA5/TO as a template. The resulting PCR product served as a template for the second reaction using the same sense primer and the antisense primer 5'-GGC GGC CGC CTA CTT GTC ATC GTC GTC CTT-3'. Underlined nucleotides indicate BamHI and NotI restriction endonuclease sites in the sense and antisense primers, respectively. The second PCR product was subcloned into pGEM-T (Promega, Madison, WI), and the 300-bp B-FLAG fragment was excised with BamHI and NotI and ligated into B/pcDNA5/TO digested with BamHI and NotI. A similar strategy was utilized to epitope tag the B subunit. Using rat B/pcDNA5/TO as template, the first PCR was performed with the sense primer 5'-ACT GCC GCG GAG TTC CAC CCA-3' and the antisense primer 5'-GTC GTC CTT GTA GTC ATT AAT TTT GTC CTG-3'; primers for the subsequent PCR were the same sense primer and the antisense primer 5'-GGC GGC CGC TTA CTT GTC ATC GTC GTC CTT-3'. Underlined nucleotides indicate SacII and NotI restriction endonuclease sites in the sense and antisense primers, respectively. The second PCR product was cloned into pGEM-T, and the B-FLAG fragment was excised with SacII and NotI and ligated into B/pcDNA5/TO digested with the same enzymes. Generation of the FLAG-tagged B'
construct was as described previously (43). Proper construction of the plasmids encoding the FLAG-tagged B subunits was verified by automated DNA sequencing (Vanderbilt University DNA Core Facility).
B RNAi Plasmid—For tetracycline-inducible RNAi, we used the pcDNA3.1/H1-TO plasmid described previously (18) in which two tetracycline operator (TO) sites flank the TATA box of the H1 promoter. The short hairpin RNA-encoding sequence targeting B was synthesized as two complementary primers (forward, GAT CCC CGT GGC AAG CGA AAG AAA GAT TCA AGA GAT CTT TCT TTC GCT TGC CAC TTT TTG GAA A; reverse, AGC TTT TCC AAA AAG TGG CAA GCG AAA GAA AGA TCT CTT GAA TCT TTC TTT CGC TTG CCA CGG G; B complementary sequence underlined), phosphorylated, and ligated into pcDNA3.1/H1-TO digested with BglII and HindIII.
Inducible Cell Lines for B Subunit Overexpression or Knock-down—Human embryonic kidney (HEK) T-Rex cells (Invitrogen) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 5 µg/ml blasticidin. Monoclonal cell lines stably expressing tetracycline-inducible expression plasmids encoding B-FLAG, B-FLAG, or FLAG-B' were generated by transfecting (FuGENE 6, Roche Applied Science) HEK T-Rex cells with B-FLAG/pcDNA5/TO, B-FLAG/pcDNA5/TO, or FLAG-B'/pcDNA5/TO followed by selection of individual clones in medium containing 175 µg/ml hygromycin B. Monoclonal cell lines for tetracycline-inducible repression of B were generated by transfecting HEK T-Rex cells with the B RNAi vector followed by selection in medium containing 1 mg/ml G418. Control cell lines stably expressing pcDNA5/TO or pcDNA3.1/H1-TP were selected in medium containing 175 mg/ml hygromycin or 1 mg/ml G418, respectively. Expression of B-FLAG and B-FLAG and suppression of endogenous B was accomplished by treating the respective cells with 1 µg/ml tetracycline for 48 h at 37 °C.
Reverse Transcription-PCR—Total RNA was obtained from tetracycline-treated HEK T-Rex cells using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA concentrations were determined by spectrometric analysis and adjusted to equal concentrations with Milli-Q H2O prior to cDNA synthesis. Synthesis of cDNA was accomplished using Superscript reverse transcriptase (Invitrogen) and 1 µg of total RNA. The resulting cDNA products were subjected to PCR amplification using primers specific for B, B, and GAPDH. PCR primers for the reactions were as follows: B forward, 5'-GGA GGG AAT GAT ATT-3'; B reverse, 5'-TAG TGT AGT AAC TGT AGT-3'; B forward, 5'-GCG GGC GGC AAC GAC TTC-3', B reverse, 5'-TAG CGC CGT GAT CCT AAA-3'; GAPDH forward, 5'-GGT CAT CAT CTC TGC CCC-3'; and GAPDH reverse, 5'-CAC CAC CCT GTT GCT GTA G-3'. PCR amplification (30 cycles) was performed in a 50-µl reaction containing 2.5 units of Amplitaq, 0.2 mM dNTPs, and 0.5 µM primers; each cycle consisted of denaturation at 95 °C for 30 s, annealing at 50 °C (B/B) or 55 °C (GAPDH) for 30 s, and extension at 72 °C for 30 s.
Purification of ABC, ABC, and AB'C Holoenzymes—Stable HEK T-Rex cell lines harboring B-FLAG/pcDNA5/TO, B-FLAG/pcDNA5/TO, or FLAG-B'/pcDNA5/TO were treated with 1 µg/ml tetracycline for 48 h to induce protein expression. Stable HEK T-Rex cell lines harboring the empty vector (pcDNA5/TO; EV) were used as controls. Cells were lysed in buffer containing 20 mM Tris-HCl, pH 7.6, 0.1% Igepal CA-630, 150 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 17 µg/ml aprotinin, and 5 µg/ml leupeptin. Following centrifugation for 15 min at 15,000 x g, the clarified lysates (1 mg of protein) were incubated with 20 µl of a 50% slurry of anti-FLAG-agarose for 4 h. Bound proteins were washed twice with PAN buffer (10 mM PIPES, pH 7.0, 100 mM NaCl, and 17 µg/ml aprotinin) containing 0.5% Igepal CA-630, once with PAN buffer, and once in phosphatase assay buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 0.25 mg/ml bovine serum albumin). Bound proteins were eluted by incubation for 30 min at 4 °C in phosphatase assay buffer (150 µl) containing 100 µg/ml FLAG peptide. The amount of PP2A catalytic subunit in the purified PP2A holoenzymes was determined by SDS-PAGE and silver staining using serial dilutions of bovine serum albumin as standards. These values were used to calculate the protein concentration of purified PP2A holoenzymes based upon stoichiometric levels of the A, B, and C subunits in each preparation (see Fig. 1A). Aliquots of the purified holoenzymes were either assayed for phosphatase activity or subjected to SDS-PAGE followed by silver stain or immunoblot analysis.
Preparation of Phosphorylated Substrates and Phosphatase Assays— Radiolabeled substrates (e.g. phosphorylase a and cAMP-dependent protein kinase-phosphorylated histone H1) were prepared as described previously (14, 44). Purified FLAG-B/ PP2A holoenzymes (9 ng) were assayed for phosphatase activity in a 50-µl reaction containing phosphatase assay buffer and 32P-labeled substrate. Following a 15-min incubation at 30 °C, the reactions were terminated by the addition of trichloroacetic acid (20% final concentration). The samples were incubated on ice for at least 30 min, and proteins were pelleted by centrifugation at 13,000 x g for 10 min. Supernatants were collected and quantified for 32Pi release by liquid scintillation counting. Less than 20% of total phosphorylated substrate was dephosphorylated during the assays, thus assuring that substrate was not limiting. In some experiments, purified PP2A holoenzymes were pretreated for 15 min with 2 nM okadaic acid, a concentration of inhibitor that selectively inhibits PP2A (45), prior to assay of phosphatase activity.
KinetworksTM Phosphoprotein Analysis—Detergent-solubilized extracts (350 µg of protein) from stably transfected HEK T-Rex cells were subjected to Kinetworks Phospho-Site Screening (KPSS) 1.3 and 4.1 as described on the Kinexus Bioinformatics Corp. website (www.kinexus.ca) and in Ref. 46. These screens use panels of 31–34 highly validated commercial phosphosite-specific antibodies and 20-lane multichannel blotters. The intensity of the ECL signals for the target protein bands on the Kinetworks immunoblots were quantified with a FluorS Max Imager and Quantity One software (Bio-Rad).
Gel Electrophoresis and Immunoblot Analysis—HEK T-Rex cells were cultured for 24 h in the presence of tetracycline and for an additional 16 h in serum-free medium containing tetracycline. As indicated, some cells were incubated with 50 ng/ml epidermal growth factor (EGF) for 10 min or 10% fetal bovine serum for 15 min prior to cell lysis. Cells (from one confluent 10-cm dish) were quickly washed two times with ice-cold phosphate-buffered saline and lysed in buffer A (20 mM Tris-HCl, pH 7.2, 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 40 mM -glycerophosphate, 20 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine, 5 µM pepstatin, 10 µM leupeptin, and 0.5% Igepal CA-630). Clarified cell lysates were obtained by centrifugation at 12,000 x g for 10 min. Cellular extracts (15 µg of protein) were subjected to 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the indicated primary antibodies and corresponding secondary antibodies. Visualization and quantification of the immunolabeled proteins was accomplished using the OdysseyTM infrared imaging system (Li-Cor, Lincoln, NE) and Odyssey software, which measures integrated pixel intensity. Measurements for p-ERK1/2 or p-MEK1/2 were scaled to reflect the value observed for total ERK1/2 or MEK1/2 in each lane. After scaling, a two-sample t test (two-sided) was performed to determine the significance of the differences in the mean p-ERK1/2 and p-MEK1/2 signals in B/B-overexpressing or B knock-down cell lines relative to control cells (EV).
Luciferase Reporter Assays—The PathDetect Elk1 trans-reporting system (Stratagene) was modified for the dual luciferase assay (Promega) to quantify ERK activation according to the manufacturer's instructions. B/-inducible HEK T-Rex cells were plated at 100,000 cells/well in 24-well plates and transfected 24 h later with up to 50 ng/well activator plasmid (Ha-Ras V12, MEK1 S218D,S222D; adjusted to 50 ng with pcDNA3.1) and 450 ng/well reporter plasmid mixture (85% pFR-Luc, 5% pFA2-Elk1, 10% pRL-SV40) using LipofectamineTM 2000 (Invitrogen). Doxycycline (1 µg/ml) or 0.1% ethanol vehicle was added at the same time. After 2 days, cells were lysed and subjected to dual luciferase assays on a Berthold Sirius luminometer. Photinus and Renilla luciferase activity ratios were expressed relative to basal conditions without ERK activator plasmids.
Raf1 and FLAG-B Immunoprecipitations—Cells were grown in the presence of tetracycline for 24 h, serum-starved for 16 h, and then treated with or without EGF for 10 min immediately prior to harvest. Cells were lysed in buffer B (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Igepal CA-630, 17 µg/ml aprotinin, 10 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4) or buffer A. Raf1 was immunoprecipitated from the clarified cell lysates (500 µg of protein) by incubation with Raf1 antibody (1 µg; sc-133) for 1 h followed by subsequent incubation with protein G-Sepharose (20 µl of a 50% slurry) for 90 min at 4 °C. FLAG-B complexes were immunoprecipitated from the clarified cell lysates (500 µg of protein) by incubation with anti-FLAG M2-agarose beads (20 µl of 50% slurry) for 4 h at 4°C. Immune complexes were washed three times with the respective buffer, eluted in SDS sample buffer, resolved by 10% SDS-PAGE, and immunoblotted with the indicated antibodies.
Raf1 and ERK1/2 Dephosphorylation Assays—HEK T-Rex cells stably expressing empty vector were serum-starved for 16 h, treated with or without EGF for 10 min, and then lysed in buffer A. Raf immune complexes were isolated from the clarified cell extracts as described above. After washing with buffer A, the immune complexes were washed three times with phosphatase assay buffer lacking or containing 500 nM microcystin-LR and then incubated for 30 min at 37 °C with the purified PP2A holoenzyme (90 ng) that had been pretreated with (+) or without (–) 500 nM microcystin-LR. For experiments examining the dephosphorylation of phospho-Raf1 and phospho-ERK1/2 by purified ABC, ABC, or AB'C holoenzymes, Raf1 immune complexes were isolated from extracts of HEK cells grown under normal conditions and lysed in buffer A. ERK1/2 immune complexes were also isolated from these extracts by incubation with ERK1/2 antibody (1 µg) and 20 µl of a 50% slurry of protein G-Sepharose for 90 min at 4 °C. After washing with buffer A and phosphatase assay buffer, the immune complexes were incubated with purified ABC, ABC, or AB'C holoenzymes (90 ng) for 30 min at 37 °C. The phosphatase reactions were terminated by the addition of SDS sample buffer and subjected to immunoblot analyses using antibodies recognizing phospho-Raf1(Ser-259), total Raf1, phospho-ERK1/2(Thr-202 + Tyr-204/Thr-185 + Tyr-187), and total ERK1/2. The blots were also immunoblotted with a PP2A catalytic subunit (PP2Ac) antibody to ensure equivalent amounts of phosphatase catalytic subunit were added to the dephosphorylation reactions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ Subunits—A tetracycline-inducible system was utilized for controlled expression of FLAG epitope-tagged PP2A regulatory subunits in HEK T-Rex cells. B-FLAG or B-FLAG complexes were isolated from the respective extracts using anti-FLAG-agarose affinity resin, eluted from the resin with FLAG peptide (FLAG eluates), resolved by SDS-PAGE, and examined by immunoblot and silver stain analysis. The cellular levels of B-FLAG and B-FLAG proteins were 2.6- and 3.5-fold higher, respectively, than the total levels of endogenous B and B subunits (Fig. 1A). Similar results were obtained with multiple independent clones (data not shown). Consistent with previous reports (41, 47), inducible overexpression of B or B did not alter expression of the A and C subunits (Fig. 1A). To determine whether the ectopic B subunits bind endogenous A and C subunits, the FLAG eluates were probed with specific antibodies recognizing the PP2A subunits or the FLAG epitope (Fig. 1A). Both A and C subunits were detected in the FLAG eluates from tetracycline-treated cells, whereas no PP2A subunits were observed in FLAG immune complexes isolated from lysates of control cells.
The relative purity and subunit stoichiometry of the isolated PP2A holoenzymes were addressed by SDS-PAGE and silver stain analysis. Three major proteins migrating at 65, 55, and 36 kDa (the predicted molecular masses of the A, B, and C subunits, respectively) were detected in the FLAG eluates (Fig. 1B). Analysis of these proteins by mass spectrometry verified their identity as the A, B or B, and C subunits of PP2A (data not shown). The relative staining intensity of the A, B, and C subunits also indicated that these proteins were present in roughly stoichiometric levels in the FLAG eluates.
To test whether the isolated PP2A holoenzymes exhibited phosphatase activity, aliquots of the FLAG eluates were assayed for catalytic activity toward several phosphorylated substrates. ABC and ABC holoenzymes efficiently dephosphorylated all substrates and exhibited specific activities of 0.062 and 0.074 mmol/min/mg, respectively, when [32P]histone H1 was used as a substrate. The catalytic activity of the purified holoenzymes was completely inhibited by 2 nM okadaic acid (data not shown), a concentration that selectively inhibits PP2A activity (45). Together with the immunoblot and protein staining analyses, these results demonstrate the utility of this system for the successful isolation of functional PP2A holoenzymes from cells expressing a FLAG-tagged PP2A regulatory subunit.
Knock-down of PP2A-B by Inducible RNAi—As a complementary strategy to study the function of select PP2A holoenzymes, we exploited an RNAi strategy for knock-down of the B regulatory subunit. HEK T-Rex cell lines inducibly expressing short hairpin RNA for RNAi-mediated degradation of B mRNA were generated and analyzed for B mRNA and protein levels following tetracycline induction. Reverse transcription-PCR was used to verify B mRNA knock-down and to ensure experimental effects were specific for the B subunit. Tetracycline-inducible knock-down of B mRNA was observed in multiple clones harboring the B RNAi plasmid but not in control cell lines expressing the empty vector (Fig. 2A). Immunoblot analyses of cells expressing B-targeted RNAi revealed a greater than 75% reduction in B subunit protein compared with control cells (Fig. 2B). The inducible knock-down of B, like the inducible overexpression of B or B, did not alter the expression of the A and C subunits (see Figs. 1A and 7B). In comparison with the immunoblot analysis of cell lines expressing B/-FLAG (Figs. 1A and 2B), reverse transcription-PCR analysis of these cells revealed similar increases in the corresponding regulatory subunit transcript (Fig. 2A).
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/ Subunit Levels on Cellular Phosphoproteins—To monitor signaling pathways targeted by ABC holoenzymes, we utilized two Kinetworks phosphoprotein analyses to examine the phosphorylation status of at least 59 phosphosites in over 50 known proteins (not including alternative splice variants) in control, B-overexpressing, and B knock-down cells treated with or without EGF (Fig. 3). These screens apply phosphosite-specific antibodies to monitor the phosphorylation state of select proteins in cellular lysates. With 10-min EGF treatment, control cells exhibited dramatic increases in the phosphorylation status of numerous proteins including Raf1, MEK1/2, and ERK1/2 (Fig. 3, compare A and B). Several prominent changes in the phosphorylation status of these members of the MAPK pathway were observed in B knock-down cells and B-overexpressing cells relative to control cells. In the absence of EGF, ERK2 showed a loss of phosphorylation (–70%) in the B knock-down cells and an increase of phosphorylation (+181%) in cells overexpressing B (Fig. 3A). In the presence of EGF, overexpression of B caused a 214% increase in ERK2 phosphorylation, whereas knock-down of B resulted in a 43% decrease in ERK2 phosphorylation (Fig. 3B). A modest increase (+20%) and decrease (–36%) in EGF-induced MEK1/2 phosphorylation was observed in B-overexpressing cells and B knock-down cells, respectively. In contrast to MEK1/2 and ERK1/2, EGF-induced Raf1 (p70) Ser-259 phosphorylation was decreased (–32%) in cells overexpressing B and increased (+50%) in cells lacking B. The opposing effects on Raf1, MEK1/2, and ERK1/2 phosphorylation observed in cells with elevated B levels versus cells with reduced B levels suggest that these members of the MAPK pathway may be physiologic targets for B-containing PP2A holoenzymes.
Activation of MEK1/2 and ERK1/2 by ABC and ABC Holoenzymes—PP2A exhibits diverse regulatory roles in the control of various MAPK signal transduction pathways (16, 41, 48–50). Based upon the Kinetworks analysis of our cell lines, we chose to further address the role of PP2A ABC and ABC holoenzymes in the regulation of Raf1-MEK1/2-ERK1/2 signaling. Stable cell lines with increased or decreased expression of B family subunits were serum-starved (basal phosphorylation) or serum-starved and then stimulated with fetal bovine serum. The phosphorylation states of MEK1/2 and ERK1/2 were assessed by immunoblot analysis of the resulting cell lysates using phosphosite-specific antibodies recognizing the active forms of these MAPK kinase and MAPK members (supplemental Fig. 1). In comparison to control cells, a significant increase in ERK1/2 (Thr-202 + Tyr-204/Thr-185 + Tyr-187) phosphorylation was observed in the cell lines overexpressing B or B under both basal and serum-stimulated conditions. In contrast, the phosphorylation of ERK1/2 was significantly decreased in B knockdown cells, relative to control cells, under both conditions. Similar changes in the phosphorylation of MEK1/2 (Ser-217 + Ser-221) were observed in these cell lines (data not shown).
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or B exhibited increased phosphorylation of MEK1/2 and ERK1/2 as compared with control cells at the respective time points. This effect was most pronounced at 10 and 15 min of EGF treatment (Fig. 4, compare lane 10 with lanes 11 and 12 or lane 13 with lanes 14 and 15) at which times control cells showed near basal levels of MEK1/2 and ERK1/2 phosphorylation, whereas B- and B-overexpressing cell lines still exhibited increased phosphorylation of these kinases. Quantitation of the 10-min EGF time points revealed a significant increase in ERK1/2 phosphorylation in the cell lines overexpressing B or B (2.97- or 2.83-fold, respectively; p < 0.05; n = 13) compared with control cells. Likewise MEK1/2 phosphorylation was significantly increased in the cell lines overexpressing B or B (4.12- or 3.67-fold respectively; p < 0.05; n = 4) relative to control cells. In contrast, ERK1/2 phosphorylation was reduced by 30% in B knock-down cells relative to control cells following a 10-min EGF treatment (Fig. 5, compare lane 6 with lane 5). Together these findings demonstrate increased phosphorylation of MEK1/2 and ERK1/2 in cells with elevated levels of B or B and decreased phosphorylation of these kinases in cells lacking B. Thus, PP2A ABC and ABC holoenzymes appear to positively regulate the activation of these MAPK kinases and MAPKs.
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and B subunits exert their effects on the MAPK activation cascade, we first tested whether pretreatment of our cell lines with a MEK1/2 inhibitor (PD98059) influenced EGF-induced phosphorylation of MEK1/2 and ERK1/2. PD98059 reduced EGF-induced ERK1/2 phosphorylation in all cell lines (Fig. 5, lanes 9–12). Consistent with a previous report (51), we found that PD98059 did not completely abolish ERK phosphorylation in response to 50 ng/ml EGF. However, somewhat surprisingly, increased MEK1/2 phosphorylation was observed in all cells treated with the MEK inhibitor and EGF (Fig. 6, lanes 9–12). The EGF/PD98059-induced hyperphosphorylation of MEK is most likely due to blockade of an inhibitory feedback loop, which requires ERK-dependent phosphorylation and inactivation of Raf1 (Ref. 52; also see "Discussion"). The presence of elevated, yet comparable, levels of MEK1/2 phosphorylation in EGF/PD98059-treated cell lines overexpressing either the B or B subunit and the lack of any increases in MEK1/2 and ERK1/2 phosphorylation in the EGF/PD98059-treated cell lines lacking B suggest that these MAPK family members are not themselves direct substrates for ABC and ABC holoenzymes. Instead these findings support the idea that the PP2A holoenzymes act upstream of MEK1/2 and ERK1/2 to activate this signal transduction pathway.
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C and ABC Holoenzymes Activate the MAPK Cascade Downstream of Ras—To further explore the target(s) of ABC or ABC holoenzymes within the MAPK cascade, we utilized a reporter assay to monitor MAPK-dependent transcriptional activation of Elk1 in our cell lines after transfection of oncogenic Ras V12 or constitutively active MEK1 (CA-MEK1). Both Ras V12 and CA-MEK1 stimulated MAPK reporter activity severalfold in all cell lines (Fig. 6). However, in comparison with the uninduced cell lines (–Dox), the inducible expression of B or B (+Dox) significantly augmented the Ras V12-induced MAPK activation at three different doses of Ras V12. In contrast, the inducible expression of B or B did not have any significant effect on CA-MEK1-induced MAPK activation. Furthermore the addition of doxycycline did not influence Ras V12- or CA-MEK1-induced reporter activity in the control cells, thus demonstrating that the augmentation of Ras V12-induced MAPK activation was specific to cells with elevated levels of B regulatory subunits. Together with results obtained with the MEK inhibitor (Fig. 5), these findings indicate that the ABC and ABC holoenzymes act downstream of Ras and upstream of MEK1/2 to promote MAPK activation.
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or B levels relative to control cells (–35 or –50%, respectively; p < 0.05; n = 5); in contrast, increased phosphorylation of this site (+17%) was observed in the B knockdown cell line relative to control cells (Fig. 7A). A similar trend in Raf1 Ser-259 phosphorylation status was observed in the unstimulated cell lines. To confirm these findings, Raf1 was immunoprecipitated from the corresponding cell lysates and analyzed for phospho-Raf1 Ser-259. Similar to the analyses of the cell lysates, Ser-259 phosphorylation was decreased 38 and 78% in the Raf1 immune complexes from EGF-stimulated cells with elevated B and B levels, respectively (Fig. 7B). These results are consistent with our Kinetworks phosphoprotein analyses of EGF-stimulated cells (Fig. 3B) that revealed decreased (–32%) phosphorylation of Ser-259 in the B-overexpressing cell line and increased (+50%) phosphorylation of this residue in the B knockdown cell line relative to control cells. Dephosphorylation of the Raf1 inhibitory site (phospho-Ser-259) and subsequent activation of this kinase would account for the phosphorylation/activation of the downstream kinases MEK1/2 and ERK1/2 in cell lines overexpressing B or B. Likewise increased phosphorylation of Ser-259 in B knock-down cell lines would explain the inactivation of MEK1/2 and ERK1/2 in these cell lines. Together these findings support the idea that PP2A holoenzymes dephosphorylate Raf1 Ser-259, leading to the phosphorylation and activation of the downstream kinases MEK1/2 and ERK1/2.
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C and ABC Holoenzymes Associate with Raf1—To determine whether PP2A regulatory subunits modulate Raf1-MEK1/2-ERK1/2 signaling by targeting PP2A holoenzymes to Raf1, we performed a series of co-immunoprecipitation experiments. Western analysis of FLAG immune complexes from cells expressing B-FLAG or B-FLAG revealed that endogenous Raf1 together with the endogenous PP2Ac specifically co-precipitated with FLAG-tagged B subunits (Fig. 8A). In reciprocal immunoprecipitation experiments, FLAG-B and FLAG-B were found to co-precipitate with Raf1 in both the presence and absence of EGF stimulation; however, the associations of B and B with Raf1 were enhanced by 47 and 38%, respectively, following EGF treatment (Fig. 8B). PP2Ac also co-immunopurified with Raf1, and this interaction was markedly increased as well with EGF treatment. Furthermore in the presence of EGF, the amount of phosphatase catalytic subunit co-immunoprecipitating with Raf1 from lysates of B knockdown cells was 40% less than the corresponding immunoprecipitation from control cell lysates (Fig. 8B, top panel, compare lane 8 with lane 2). In contrast, the amount of PP2Ac co-immunoprecipitating with Raf1 from lysates of cells overexpressing B/B was increased 35% as compared with the immunoprecipitation from control cell lysates (Fig. 8B, top panel, compare lanes 4 and 6 with lane 2). Interestingly EGF stimulation of cells expressing empty vector or FLAG-B/ subunits produced a subtle upward shift in the migration of Raf1 on SDS-polyacrylamide gels (Fig. 8B, top panel). Because PP2A ABC and ABC appear to be recruited to Raf1 following EGF treatment, the slower migrating form of Raf1 is likely due to PP2A-mediated dephosphorylation of phospho-Ser-259 and subsequent phosphorylation of several activating residues in the kinase (e.g. Ser-338 and Ser-621). Consistent with this idea, no EGF-induced shift in the migration of Raf1 was observed in the B knock-down clones, indicating that Raf1 Ser-259 dephosphorylation (and subsequent activating site phosphorylations) is prevented by the reduced levels of B subunit. Together these data provide compelling evidence that B and B subunits target the PP2A holoenzyme to Raf1.
PP2A-mediated Dephosphorylation of Raf1 Ser-259—Because previous reports have established that phosphatase inhibitors block dephosphorylation of Raf1 Ser-259 and prevent maximal activation of the kinase (32, 33, 39, 40), we tested whether phospho-Ser-259 is a direct target of PP2A. Raf1 immune complexes were isolated from lysates of target cells prepared in the presence of phosphatase inhibitors (i.e. to retain the phosphorylation state of Raf1 prior to dephosphorylation assays). After washing, the immune complexes were incubated with purified ABCorABC holoenzymes that had been pretreated with or without the PP2A inhibitor microcystin-LR. Immunoblot analysis of the reaction mixtures revealed that both holoenzymes completely dephosphorylated phospho-Ser-259 in a microcystin-sensitive fashion (Fig. 9A). Prior cell treatment with EGF did not influence the ability of the purified holoenzymes to dephosphorylate phospho-Ser-259 in these in vitro dephosphorylation assays.
To determine the specificity of Raf1 Ser-259 dephosphorylation, a B'-containing PP2A holoenzyme was also tested for activity toward phospho-Raf1. In contrast to ABC and ABC, AB'C failed to dephosphorylate Ser-259 (Fig. 9B). Furthermore we tested whether these purified PP2A holoenzymes could dephosphorylate activating phosphosites in ERK1/2 (Thr-202 + Tyr-204/Thr-185 + Tyr-187); no detectable dephosphorylation of ERK1/2 was observed for ABC, ABC, or AB'C (Fig. 9C). Presumably the dephosphorylation of ERK1/2-activating sites in cells is catalyzed by one of the ERK-specific dual specificity phosphatases (35). The results of our phosphatase assays reinforce our conclusion that Raf1 is the preferred ABC and ABC substrate within the Raf1-MEK1/2-ERK1/2 pathway and also indicate that dephosphorylation of the Raf1 inhibitory site (phospho-Ser-259) is specifically mediated by these two holoenzyme forms of PP2A.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C and ABC holoenzymes, we initially exploited the Kinetworks screen for analysis of a wide range of phosphoproteins in our cell lines treated with or without EGF. Among the few proteins that exhibited opposing changes in their phosphorylation state in cells overexpressing versus cells lacking the B subunit were the MAPKs (Fig. 3).
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or B levels and reduced phosphorylation of these kinases in B knock-down cells relative to control cells (Figs. 3, 4, 5). In contrast to MEK1/2 and ERK1/2, the phosphorylation of Raf1 Ser-259 was decreased in cells with elevated B family regulatory subunit levels and increased in B knock-down cells relative to control cells (Figs. 3 and 7). Because phosphorylation of Ser-259 is inhibitory with respect to Raf1 activity, our data lend additional support to the idea that PP2A functions as a positive regulator of Raf1-MEK1/2-ERK1/2 signaling by dephosphorylating phospho-Ser-259 resulting in Raf1 activation and consequential phosphorylation and activation of MEK1/2 and, in turn, ERK1/2 (33, 41, 52). Consistent with this hypothesis, our immunoprecipitation experiments indicate that the B and B regulatory subunits play an important role in targeting PP2A to Raf1 (Fig. 8). Although the B subunit has been shown to associate with Raf1 in a growth factor-dependent manner (33), our results provide evidence that both B and B subunits interact with Raf1 even in serum-starved cells and that these interactions increase following growth factor treatment. Of particular importance, we now demonstrate that the novel B family subunit member B also functions as a positive regulator of Raf1-MEK1/2-ERK1/2 signaling.
Multiple mechanisms control Raf1 activity including intra- and intermolecular protein interactions as well as inhibitory and activating phosphorylations (for a review, see Ref. 55). In support of the idea that Raf1 is subject to feedback inhibitory phosphorylations, recent studies have revealed that Raf1 activation is prolonged following pharmacological inhibition of MEK1/2 (51, 52). Consistent with this hypothesis, we show hyperphosphorylation of MEK1/2 in our cell lines treated with EGF and the MEK1/2 inhibitor PD98059 (Fig. 5). Importantly the presence of elevated levels of MEK1/2 phosphorylations in EGF/PD98059-treated cell lines with increased B family regulatory subunit expression and the lack of any increases in MEK1/2 and ERK1/2 phosphorylations in EGF/PD98059-treated B knock-down cell lines indicate that these MAPK family members are not themselves direct substrates for ABC and ABC holoenzymes. If MEK1/2 and ERK1/2 were direct substrates for the PP2A holoenzymes, we would have expected to see decreased phosphorylation of these proteins in the cell lines with elevated B subunit levels and increased phosphorylation of these proteins in cells lacking B; however, this was not observed in our model system. Furthermore our studies of RasV12- and constitutively active MEK1-dependent MAPK activation indicate that ABC and ABC holoenzymes act downstream of Ras and upstream of MEK1 (Fig. 6). These observations together with our findings showing that both PP2A holoenzymes form stable interactions with Raf1 and directly dephosphorylate Raf1 Ser-259 (Figs. 8 and 9) demonstrate that the major target of ABC and ABC holoenzymes in the Raf1-MEK1/2-ERK1/2 pathway is in fact Raf1.
Dougherty et al. (52) recently described a feedback mechanism for Raf1 inactivation following PDGF stimulation that involves ERK1/2-dependent phosphorylation of six additional inhibitory sites in Raf1. Results from cellular experiments performed with the phosphatase inhibitor okadaic acid implicated PP2A in the dephosphorylation of these inhibitory residues and suggested that the hyperphosphorylated/desensitized Raf1 is returned to a signaling-competent state via a PP2A-dependent process. In support of a role for PP2A in the direct dephosphorylation of these newly identified inhibitory phosphorylation sites, we noticed a pronounced shift in the migration of Raf1 on SDS-polyacrylamide gels to a faster migrating species following incubation of phospho-Ser-259 Raf1 (–EGF) or hyperphosphorylated Raf1 (+EGF) with purified PP2A holoenzymes (Fig. 9A). Thus, in addition to phospho-Ser-259, PP2A appears to target other inhibitory phosphorylation sites and thereby plays a key role in the activation of Raf1.
The regulation of MAPK signaling by individual PP2A holoenzymes may be cell type-specific. For example, overexpression of the neuron-specific B family regulatory subunit B, but not B, leads to ERK1/2 activation in PC6-3 cells (a neuronal PC12 subline) (41). Furthermore we have recently reported a role for B/B and B' family members in the modulation of ERK and Akt activation, respectively, in PC6-3 cells (43). Of note, PC12 cells predominantly express the neuronal B-Raf isoform, which may be regulated by phosphorylation/dephosphorylation in a manner distinct from Raf1 (56, 57). Our present studies exploit the powerful combination of complementary overexpression and knockdown strategies to delineate the precise role of ABC and ABC holoenzymes in the control of Raf1-MEK1/2-ERK1/2 signaling in HEK cells. Our findings provide compelling evidence that B- and B-containing holoenzymes control activation of this pathway via their interaction with Raf1 and dephosphorylation of the inhibitory phospho-Ser-259 site, resulting in consequential activation of MEK1/2 and ERK1/2. Taken together with other recent reports (33, 41, 43), our studies indicate that specific PP2A holoenzymes target multiple steps in this important signal transduction cascade and appear to do so in a cell type-specific manner.
Additional physiologic targets for ABC and ABC holoenzymes likely exist in vivo. In addition to ERK1 and ERK2, the phosphorylations of numerous other proteins (e.g. CDK1, GSK3, GSK3, MKK3/6, protein kinase B, Rb, and cAMP-response element-binding protein) were altered by B overexpression or knock-down (Fig. 3). However, in most cases, the phosphorylation of these proteins did not display dramatic opposing changes in cells with increased versus decreased PP2A regulatory subunit levels. Whether or not opposing changes in the phosphorylation of any protein are observed in these cells likely depends on the relative contributions of PP2A and the counteracting kinase on the steady state phosphorylation levels under a given stimulus condition. A more comprehensive analysis of the phosphoprotein levels in these cell lines over an extended EGF time course will allow us to test whether any of these proteins are true physiologic targets for ABC and/or ABC holoenzymes.
The tetracycline-inducible systems for overexpression or knockdown of select PP2A regulatory subunits in mammalian cells provide a means to identify physiologic substrates of closely related PP2A holoenzymes. Although B and B display 90% amino sequence identity, these two subunits may differentially target the PP2A heterotrimer to specific substrates within the cell. Consistent with this idea, we previously demonstrated that B and B exhibit differences in their subcellular localization (13). However, our current findings indicate that these two PP2A regulatory subunits likely exhibit redundant functions in the control of Raf1-MEK1/2-ERK1/2 signaling because no major differences in the phosphorylation of these proteins were observed in B-versus B-overexpressing cells. Nonetheless specific cellular functions for these B subunit family members may exist and might be revealed as we examine additional signal transduction pathways in cell lines with altered B or B levels.
The large number of protein kinases regulated by PP2A and the molecular mechanisms controlling protein kinase-PP2A signaling complexes are subjects of intense investigation. Given the presence of 14 genes coding for regulatory subunits that bind to the AC core dimer, more than 70 different PP2A heterotrimers are predicted to exist in mammalian cells. Our present studies define the role ABC and ABC holoenzymes play in activation of the Raf1-MEK1/2-ERK1/2 signal transduction cascade. Furthermore as reported herein, the development of cell lines that can be induced to overexpress or knock down specific regulatory subunits provides an elegant strategy to elucidate the functional role of specific PP2A holoenzymes in control of various biological processes.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.
1 Supported in part by National Institutes of Health Training Grant 5T32DK07563.
2 To whom correspondence should be addressed: Dept. of Pharmacology,
