Similarly, transient expression of the PP2A catalytic subunit with c-Jun resulted in ... signal transduction pathways that regulate AP-1 activity and c-Jun ...
MOLECULAR AND CELLULAR BIOLOGY, Apr. 1993, p. 2104-2112 0270-7306/93/042104-09$02.00/0 Copyright X 1993, American Society for Microbiology
Vol. 13, No. 4
Protein Phosphatase 2A Potentiates Activity of Promoters Containing AP-1-Binding Elements ARTHUR S. ALBERTS,' TILIANG DENG,' ANNING LIN,' JUDY L. MEINKOTH,2 AXEL SCHONTHAL,1"2 MARC C. MUMBY,3 MICHAEL KARIN,' AND JAMES R. FERAMISCOl,2* Departments of Pharinacology' and Medicine,2 Cancer Center, University of California at San Diego, La Jolla, California 92093-0636, and Department of Pharmacology, University of Texas,
Southwestern Medical Center at Dallas, Dallas, Te-xas 75235-9041 Received 2 September 1992/Returned for modification 20 October 1992/Accepted 21 December 1992
The involvement of serine/threonine protein phosphatases in signaling pathways which modulate the activity of the transcription factor AP-1 was examined. Purified protein phosphatase types 1 (PP1) and 2A (PP2A) were microinjected into cell lines containing stably transfected lacZ marker genes under the control of an enhancer recognized by AP-1. Microinjection of PP2A potentiated serum-stimulated 13-galactosidase expression from the AP-1-regulated promoter. Similarly, transient expression of the PP2A catalytic subunit with c-Jun resulted in a synergistic transactivation of an AP-1-regulated reporter gene. PP2A, but not PP1, potentiated seruminduced c-Jun expression, which has been previously shown to be autoregulated by AP-1 itself. Consistent with these results, PP2A dephosphorylated c-Jun on negative regulatory sites in vitro, suggesting one possible direct mechanism for the effects of PP2A on AP-1 activity. Microinjection of PP2A had no effect on cyclic AMP (cAMP)-induced expression of a reporter gene containing a cAMP-regulated promoter, while PP1 injection abolished cAMP-induced gene expression. Taken together, these results suggest a specific role for PP2A in signal transduction pathways that regulate AP-1 activity and c-Jun expression.
Specific phosphorylation events mediated by protein kiand phosphatases have been shown to modulate the activity of a variety of transcription factors within the cell (32). For example, the phosphorylation state of the Jun and Fos proteins, components of the transcription factor AP-1, is altered upon stimulation by growth factors and phorbol esters which activate protein kinase C (4, 6-9, 11, 15, 43). In some cases, these changes in phosphorylation have correlated with alterations in AP-1 activity. For c-Jun, expression of activated Ha-Ras stimulates phosphorylation on its activation domain, while both Ha-Ras and treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) lead to rapid dephosphorylation of sites adjacent to the DNA binding region, resulting in increased DNA binding activity (11, 15, 53). Conversely, c-Jun is phosphorylated by casein kinase II (CKII) on sites that inhibit DNA binding, and microinjection of peptides that inhibit CKII activates AP-1 activity in living cells (37). In one study, however, similar changes in phosphorylation of c-Jun resulted in only minor changes in AP-1 binding or in vitro transcription activity, suggesting that this regulation may be complex (8). The DNA binding activity of transcription factor c-Myb is also inhibited by phosphorylation by CKII, and this inhibition is relieved by incubation with protein phosphatase type 2A (PP2A) (38). c-Jun can be also phosphorylated on inhibitory sites by cytosolic kinases, termed extracellular signal-regulated kinases (ERKs) or mitogen-activated protein kinases (MAP), in vitro, which may also inhibit c-Jun function (3), although recent experiments indicate that overexpression of ERK1 participates in activation of AP-1 (25). In the case of certain growth factor receptors which contain or are associated with tyrosine kinase activities (31, 58), ligand-receptor interaction results in activation of ERKs nases
*
Corresponding author.
by phosphorylation on serine and threonine residues via a protein kinase cascade (1, 14, 19). The ERKs can be inactivated in vitro by incubation with the serine/threonine-specific protein phosphatases, particularly PP2A. ERK- or MAP-dependent phosphorylation is increased in vivo by treatment of cells with okadaic acid (OA), a specific inhibitor of PP1 and PP2A (10, 26, 29, 30, 56). Several recent studies using OA have implicated an important role for protein phosphatases in growth control and gene expression (20, 21, 46). OA stimulates the expression of a subset of growth-related immediate-early genes (17), including c-fos and c-jun, through TPA response elements (TRE or AP-1-binding sites) and serum response elements in the promoters of these genes (35, 47, 50, 54). OA has no effect on cyclic AMP (cAMP) response element (CRE)modulated gene expression in NIH 3T3 fibroblasts, although it has been shown to increase forskolin-induced phosphorylation of the CRE-binding transcription factor CREB in PC12 cells (28, 47). CREB has been shown to be a substrate of PP1 and PP2A in vitro, although cytosolic microinjection of PP1, but not PP2A, blocks CRE-regulated gene expression (28). OA can also act as repressor of cell growth or transformation in tissue culture assays (13, 45, 49, 57). Though the ability of OA to inhibit two major classes of protein phosphatases has been useful, the reliance on this agent alone to study the role of specific phosphatases in growth control and gene expression has proven to be confusing, probably because OA inhibits multiple phosphatases (46). Microinjection studies have been useful in comparing the biological activities of PP1 and PP2A, which share multiple substrates in vitro yet appear to have distinct specificities in vivo (2, 24, 51). In this study, we sought to examine the role of PP2A in signal transduction pathways that regulate gene expression. Toward this end, preparations of purified PP2A catalytic subunit (PP2Ac) or catalytic subunit complexed to the A regulatory subunit (PP2AAC) (41) were introduced into 2104
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living fibroblasts and assayed for their effects on gene expression by two different microinjection methods. In one method, cell lines harboring stably transfected reporter constructs were used as recipients for the microinjected phosphatase preparations (39, 40); the second approach employed the coinjection of reporter constructs along with phosphatase preparations or expression constructs for the phosphatase (28, 55). We also tested the effects of expression of PP2A subunits on c-jun transactivation by transient transfection techniques (5, 18). The results of these experiments suggest a role for PP2A in the positive regulation of genes under the control of AP-1-binding site (TRE)-containing promoters. Overexpression of PP2A by either microinjection or transfection increased gene expression from TREcontaining promoters. Moreover, microinjection of PP2A stimulated endogenous c-jun expression. In vitro, incubation of c-Jun prephosphorylated by CKII with purified PP2AC resulted in significant dephosphorylation of c-Jun on sites previously shown to inhibit DNA binding, which suggests a possible direct mechanism underlying the increase in AP-1 activity caused by PP2A (15, 37). These results suggest that the signaling pathways which control AP-1-regulated promoter activity include the enzyme PP2A. MATERIALS AND METHODS Cell culture and microinjection. F9 cells and REF-52 fibroblasts were grown at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 100 U of penicillin per ml, 100 jig of streptomycin sulfate per ml, and 10% (vol/vol) fetal calf serum (FCS) (GIBCO). Rat2 cell lines stably transfected with lacZ expression vectors under the control of five copies of a TRE derived from the human collagenase gene (5'-TCGAGAT GAGTCAGCTGATGAGTCAGC-3') or five copies of a CRE from the human vasoactive intestinal peptide gene (5'TCGAGCTGCGTCATACTGTGACGTCC-3') (39, 40) were maintained in the same medium but supplemented with 300 p,g of G418 (GIBCO) per ml. For injection into quiescent cells, cells were incubated in DMEM containing 0.05% FCS for 24 to 36 h. For direct injection of reporter constructs and assay of c-Jun expression, REF-52 cells lacking such stably transfected reporters were used. Proteins. Purified PP2AC and PP2AAC were purified from bovine cardiac tissue and PP1 (kindly provided by S. Shenolikar) was purified from rabbit skeletal muscle as described previously (22, 41). All preparations were at stored at -70°C in 50% (vol/vol) glycerol-25 mM Tris (pH 7.4)-l mM dithiothreitol-1 mM EDTA. Immediately prior to microinjection, the proteins were exchanged into injection buffer (50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 7.2], 40 mM NaCl or 100 mM KCl, 5 mM NaPO4 [pH 7.3]). The final concentration of purified PP2AC was -1.0 mglml (700 nmol/min/mg as assayed with 2 ,uM myosin light chain as a substrate), and that of PP2AC was between 1.5 and 2.0 mg/ml (240 nmollmin/mg). The specific activity of PP1 with 10 ,M phosphorylase a as a substrate was 2,000 nmol/min/mg. PP1 was injected at between 0.2 and 1.0 mg/ml. Protein concentrations were determined by the method of Bradford (16). The proteins were 80 to 90% pure, as estimated by analysis of Coomassiestained gels iFig. 1). On the basis of an injected volume of -50 x 10-1 liter (23), 105 to 106 phosphatase molecules were injected into each cell. Since PP2AC subunit represents approximately 0.1% of total cellular protein, injections gave rise to at least a doubling of the amount of endogenous
ROLE OF PP2A IN REGULATION OF AP-1 ACITIVITY C (0.5
97
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AC
l.g)
(1.0 pKg)
-
66
45
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31
-
1 -
1
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FIG. 1. SDS-12.5% polyacrylamide gel of purified PP2A. Lanes: 1, 0.5 pLg of purified catalytic subunit of PP2A; 2, 1.5 ±Lg of purified PP2AAC.
PP2AC (44). Nonspecific mouse immunoglobulin G (mIgG) or sheep immunoglobulin G (sIgG) (Sigma, St. Louis, Mo.) at concentrations of 1 to 4 mg/ml were coinjected with the phosphatase preparations for positive identification of the injected cells by indirect immunofluorescence. Transient transfections. Transfections into F9 cells by calcium phosphate coprecipitation were performed as previously described (18). In each experiment, 2 ,ug of a TREcontaining reporter plasmid derived from the collagenase promoter (-73-coll-CAT) (7) was cotransfected with 0.1 jig of Rous sarcoma virus (RSV)-controlled plasmid RSV-c-jun and 1 ,ug of a pRSV-PP2A expression plasmid bearing either the C or A subunit of mammalian PP2A (27, 52). Chloramphenicol acetyltransferase (CAT) activity was assayed 16 h later as previously described (5). Immunofluorescence and histochemistry. Following injection, the cells were stimulated for 6 h with DMEM containing 20% FCS or 200 ng of TPA per ml and then fixed in 3.7% (wt/vol) formaldehyde in phosphate-buffered saline (PBS) for 5 min at ambient room temperature. The cells were washed with PBS and incubated for up to 12 h at 37°C with 1 mg of 5-bromo-4-chloro-3-indolyl-13-D-galactopyranoside (X-Gal; IBI) per ml-5 mM potassium ferrocyanide-5 mM potassium ferricyanide-1 mM MgCl2 in PBS. Cells expressing ,B-galactosidase (C-Gal) contained a dark blue precipitate from the reaction product. To identify injected cells, the coverslips were washed with PBS and the cells were permeabilized with 0.3% (vol/vol) Triton X-100-PBS. Following several washes with 0.1% Tween 20-PBS, the coverslips were incubated with either a biotinylated goat anti-mIgG (1:100; Vector Laboratories, Burlingame, Calif.) followed by Texas red-streptavidin (1:100; Amersham, Arlington Heights, Ill.) or Texas red-conjugated goat anti-mIgG (1:100; Jackson Laboratories, West Grove, Pa.). Each antiserum was diluted in 0.5% (vol/vol) Nonidet P-40-PBS. Coverslips were then washed extensively with Tween 20-PBS and mounted in PBS containing 15% (wt/vol) Gelvatol (polyvinyl alcohol), 33% (vol/vol) glycerol, and 0.1% (wt/vol) NaN3 onto glass slides. In some experiments, ,3-Gal expression was assessed by indirect immunofluorescence. With labeling for the presence of injected marker antibody, expression of 1-Gal and endogenous c-Jun expression was performed after fixation and permeabilization as described above. Injected cells were
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identified by staining with a donkey anti-sIgG conjugated to the fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA; Jackson Laboratories). 1-Gal was detected with a monoclonal mouse anti-C-Gal antibody (Promega) followed by a fluorescein isothiocyanate-conjugated donkey antimouse antibody (Jackson Laboratories). c-Jun expression was monitored with a rabbit polyclonal antibody (Oncogene Science) followed by a biotinylated goat anti-rabbit antibody (Vector Laboratories) and Texas red-streptavidin (Amersham). Primary and secondary antibodies were diluted 1:100 in 0.5% (vol/vol) Nonidet P-40-PBS. Fluorescence microscopy. All cells were observed and photographed with a Zeiss Axiophot fluorescence microscope. Fields of cells stained with X-Gal (phase-contrast) were photographed under x20 or x40 magnification (1.4 numerical aperture) with Kodak Technical Pan film (ASA 100). The fluorescent fields were photographed with Kodak T-Max film (ASA 800). Phosphorylation and dephosphorylation assays. Purified recombinant c-Jun protein (100 ng) was incubated at 30°C for 30 min in a reaction mixture (35 ,ul) containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 10 mM MgCl2, 20 ,uM [_y-32P]ATP, and 60 ng of CKII. The reaction was terminated by addition of 0.1% (wtlvol) sodium dodecyl sulfate (SDS)-1% (vol/vol) Nonidet P-40. c-Jun protein was immunoprecipitated with polyclonal antibody 601 (18). The immunocomplexes were washed twice with radioimmunoprecipitation assay buffer (33) and subjected to dephosphorylation. For PP2AC and alkaline phosphatase treatment, the immunoprecipitate was washed twice with 20 mM HEPES (pH 7.0)-i mM dithiothreitol-1 ,ug of leupeptin per ml and resuspended in 30 Ru of the same buffer containing 0.5 U of either PP2A or alkaline phosphatase (Boehringer). For acid phosphatase treatment, the immunoprecipitate was washed twice with 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES)-HCl (pH 6.0)-i jig of leupeptin per ml and resuspended in 30 jil of the same buffer containing 1 U of acidic phosphatase (Boehringer). Dephosphorylation reactions were carried out at 30°C for 30 min. Samples were boiled for 4 min, resolved on an SDS-10% polyacrylamide gel, and then subjected to autoradiography. RESULTS Microinjection of PP2A potentiates serum stimulation of TRE-regulated promoters. The activation of c-Jun can be accompanied by the specific dephosphorylation of sites adjacent to its DNA binding domain (15). To assess whether PP2A might participate in c-Jun activation, purified PP2A was injected into quiescent Rat2 fibroblasts stably transfected with a TRE-regulated 3-Gal expression vector (TRElacZ). The purity of injected enzymes was estimated by Coomassie-stained SDS-polyacrylamide gels to be >90% pure (Fig. 1). Injected cells were subsequently stimulated with medium containing 20% FCS or 0.05% FCS as a control for refeeding; 100 to 200 cells were injected in each experiment, and the experiments were repeated three to four times. Stimulated cells were incubated for 4 h and fixed, and 13-Gal expression was monitored by staining with the chromogenic substrate X-Gal. Blue cells were scored as 13-Gal positive, and colorless cells were scored as 1-Gal negative. Representative fields of cells stimulated with medium containing 20% FCS that were injected with PP2AC, PP2AAC, or marker antibody alone were photographed by both phasecontrast and fluorescence microscopy. The fluorescent photomicrographs of the cells showed the presence of the
MOL. CELL. BIOL.
coinjected marker antibody, and the phase-contrast photomicrographs of the same fields showed cells expressing 13-Gal. Serum stimulation of TRE-lacZ cells typically results in 40% of the cells expressing 1-Gal. Injection of various control proteins generally lowered this response slightly to approximately 30% overall. Examples of control injections are shown in Fig. 2E and F. Injection of PP2AC (Fig. 2A and B) or PP2AAC (Fig. 2C and D) resulted in 55 and 75% of the injected cells expressing 1-Gal, respectively. The arrows are provided only for orientation between fluorescence micrographs and phase-contrast micrographs. The results of the injections into the TRE-lacZ cell lines are summarized in Fig. 3. In quiescent cells injected with PP2AC or PP2AAC, 1-Gal expression was also increased severalfold in comparison with those injected with marker antibody alone (Fig. 3). However, in the absence of serum, the level of 1-Gal expression was low, making quantitation of the data difficult. These results were confirmed by using an alternative assay for the effects of PP2A on AP-1 activity, based on the comicroinjection of an expression plasmid bearing PP2AC directly into the cell nucleus and the TRE-lacZ plasmid (27, 28, 55). Similar to the results observed following injection of purified proteins, serum-stimulated expression from coinjected TRE-lacZ reporter plasmid was also potentiated by coinjection of the PP2A expression plasmid (data not shown). PP2A catalytic subunit potentiates the ability of c-Jun to transactivate a TRE-responsive promoter. AP-1, composed of c-Jun-c-Jun homodimers, is able to transactivate TREcontaining promoters (4, 7, 18). To independently assess the effect of PP2A on TRE-regulated gene expression, cotransfection studies were conducted with F9 embryonal carcinoma cells, which have low endogenous AP-1 levels, thus allowing a system wherein levels of AP-1 constituents can be manipulated (18, 36). Exponentially growing F9 cells were cotransfected with a TRE-containing reporter plasmid (-73coll-CAT) containing a consensus TRE sequence (4, 18), a c-Jun expression vector (RSV-c-jun), and expression vectors encoding PP2A C subunit, A subunit, or both subunits. As shown in Fig. 4, both c-Jun and C-subunit expression vectors stimulated collagenase promoter-CAT activity approximately fivefold. Cotransfection of c-Jun and PP2AC expression vectors resulted in greater than 40-fold stimulation of CAT activity. Interestingly, while the A-subunit vector alone had no effect on collagenase promoter activity, cotransfection of both the A- and C-subunit expression vectors abrogated the stimulation of AP-1 activity seen with use of the C-subunit vector alone. These latter results suggest that the A subunit may regulate the C subunit in some cases, perhaps by either modifying enzyme activity or redistributing the enzyme within the cell (discussed below) (42). PP2A potentiates c-Jun expression. Expression of c-Jun has been shown to be positively autoregulated via the binding of AP-1 to a TRE contained in the c-jun promoter (5). As PP2AC increased the apparent AP-1 activity in cells stably transfected with the TRE-lacZ reporter (Fig. 2), we wished to examine the effects of PP2AC and PP1 on endogenous c-Jun expression, which was done by immunofluorescence. In this set of experiments, PP2AC was coinjected with the TRE-lacZ reporter plasmid into quiescent REF-52 cells, which lacked any reporter, in order to assess both c-Jun expression and AP-1 activity by reporter expression. As nuclear injection is required for expression of injected DNA, the mixtures were injected directly into the nucleus (55).
VOL. 13, 1993
ROLE OF PP2A IN REGULATION OF AP-1 ACTIVITY
* IF
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FIG. 2. Microinjection of PP2A into Rat2 TRE-lacZ cells. Representative fields of injected cells are shown. Each enzyme preparation was coinjected with 2 mg of mIgG per ml to identify injected cells. Coinjected mIgG was detected by indirect immunofluorescence as described in Materials and Methods. Arrows are provided for orientation between panels illustrating indirect immunofluorescence of injected cells (left) and cells expressing 3-Gal (right). PP2AC (-1 mg/ml) was injected into serum-deprived cells, which were immediately stimulated with DMEM containing 20% FCS (A and B). Microinjection of PP2AAC (-2 mg/ml) increased ,8-Gal expression (C and D) similarly to injection of PP2AC. Microinjection of mIgG alone (E and F) had no effect on serum-stimulated 1-Gal expression. These results are summarized in Fig. 3. (Photographed under x20 magnification [1.4 numerical aperture]; bar represents 60 ,um.)
Because X-Gal staining for lacZ-encoded a-Gal activity can quench fluorescence emission in stained cells, we used indirect immunofluorescence to detect 1-Gal expression instead. This approach allows for the simultaneous imaging, in single injected cells, of TRE-lacZ expression (fluorescein isothiocyanate immunostain), endogenous c-Jun expression
(Texas red immunostain), and coinjected marker sIgG (AMCA immunostain). These experiments were performed several times, with approximately 50 cells injected in each experiment. Again, representative fields of injected cells were photographed (Fig. 5). Microinjection of PP2AC was found to potentiate both
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MOL. CELL. BIOL.
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CAT activity 50 Starved; Refed 0.05% FCS/DME Serum Stim.; 20% FCS/DME .
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FIG. 3. Summary of the effect of PP2A on TRE promoter activity. Rat2 fibroblasts containing the TRE-lacZ reporter gene were injected with either PP2A catalytic subunit (C) or holoenzyme (AC) and refed with medium containing 0.05% or 20% FCS, as indicated. Cells were scored for 1-Gal expression (blue versus white), and the presence of injected marker antibody was detected by immunofluorescence. Bars represent the mean standard deviation of two to three separate experiments (100 to 200 cells were injected in each). ±
serum-stimulated TRE-lacZ expression and c-Jun expression (Fig. 5, row C). In contrast, injection of PP1 (row B) or marker antibody alone (row A) had no effect on TRE-lacZ or c-Jun expression. As previously reported in studies using cells lines stably transfected with CRE-lacZ plasmids (28), PP1 was able to inhibit expression from the coinjected CRE-lacZ plasmid when stimulated with 0.5 mM isobutylmethylxanthine (IBMX)-8-bromo-cAMP (8Br-cAMP) following injection (row E). Injection of either PP2AC (row F) or sIgG (row D) had no detectable effect on the CRE promoter, as expected. Basal c-Jun expression in quiescent cells was unaffected by PP1 or PP2A injection (data not shown). These results lend further support to the idea that PP2A participates in the activation of AP-1. Moreover, these experiments demonstrate that PP2A does not increase 1-Gal expression in injected cells by a nonspecific mechanism. PP2A dephosphorylates c-Jun on negative regulatory sites. The effect of PP2A on AP-1-regulated gene expression could be due to a direct effect on AP-1 or some indirect effect on cellular pathways regulating AP-1 activity. One possible direct effect would be dephosphorylation of c-Jun on negative regulatory sites. It has been demonstrated that specific sites at the C terminus (or in the C-terminal half) of c-Jun are dephosphorylated upon treatment of cells with TPA (15). It has further been shown that these sites can be phosphorylated by CKII in vitro (37). To determine whether PP2A could dephosphorylate these inhibitory sites in vitro, bacterially expressed recombinant c-Jun was phosphorylated with radiolabeled ATP in vitro by CKII and purified by immunoprecipitation from the kinase reaction. 32P-labeled c-Jun was then incubated with either PP2AC, potato acid phosphatase, or calf intestinal alkaline phosphatase. PP2A treatment resulted in the complete dephosphorylation of c-Jun within 30 min (Fig. 6, lane 2). In contrast, c-Jun was only partially dephosphorylated by alkaline phosphatase (lane 3) and almost completely resistant to dephosphorylation by acid
+A +A and C coll-CAT +C FIG. 4. Evidence that PP2AC potentiates c-Jun transactivation of a TRE-containing promoter. Logarithmically growing F9 cells were transfected with 2 ,ug of reporter plasmid -73-coll-CAT (containing the AP-1 consensus sequence) alone or in the presence of 1 pg of an RSV-controlled vector expressing bovine PP2A catalytic subunit (C), regulatory subunit (A), or both subunits. Parallel transfections that also included 0.1 pg of RSV-c-jun were performed. Bars indicate the mean ± standard error of the mean of three to four experiments (except for the assay for c-Jun plus A subunit, which was performed twice) standardized to the amount of CAT activity in cells transfected with reporter plasmid alone.
phosphatase (lane 4). These results demonstrate that inhibitory phosphorylation sites in c-Jun are substrates for PP2AC in vitro. DISCUSSION We examined the effect of purified PP2A on gene expression from promoters containing AP-1-binding sites (TREs). Our results demonstrate that overexpression of PP2A positively affects genes containing TREs in their promoters. In microinjection assays, both PP2AC and PP2AAC potentiated serum-stimulated 3-Gal expression from AP-1-regulated reporters and expression of the endogenous c-jun gene. Similarly, in transient transfection experiments, expression of PP2AC induced expression from a TRE-containing reporter gene and resulted in a synergistic enhancement of the ability of c-Jun to activate an AP-1-responsive promoter. However, unlike the results obtained in the microinjection experiments, cotransfection of an A-subunit expression vector inhibited the ability of the PP2AC to synergize with c-Jun in transactivation. Since the cotransfections were performed in F9 cells, which have low endogenous levels of c-Fos and AP-1 (36), most of the TRE-CAT activity presumably results from binding of c-Jun-c-Jun homodimers to this promoter (5). It is possible that the effects of the cotransfected A and C subunits on the c-Jun-c-Jun homodimers differ from the effects of injected PP2AAC on the AP-1 heterodimer consisting of c-Fos and c-Jun, which presumably accounts for the majority of the AP-1 activity in Rat2 fibroblasts after serum stimulation (34). It is also possible that the differences observed between microinjection and transfection experiments are due to higher efficiency of expression of the transfected A subunit relative to the C subunit (27), whereas both subunits of microinjected PP2AAC were present in equimolar amounts. The differential effects of cotransfected A and C subunits versus the C subunit alone suggest that the presence of the A subunit within the enzyme complex may
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FIG. 5. Evidence that PP2A potentiates serum-stimulated endogenous c-Jun. Serum-starved REF-52 fibroblasts were coinjected with a TRE-lacZ (rows A to C) or CRE-lacZ (rows D to F) expression vector (0.5 mg/ml) along with either sIgG (3 mg/ml) alone, PP1 (0.2 mg/ml), or PP2AC (1.0 mg/ml), as indicated. The sIgG was included in the injections of phosphatases to provide a marker for injected cells. Immediately following injection, the cells were refed with 20% FCS-DMEM (TRE; A to C) or 0.5 mM IBMX-8Br-cAMP (CRE; D to F) and incubated for an additional 2 h. Following fixation in 3.7% formaldehyde-PBS, cells were immunostained for the presence of injected sIgG (left column), 1-Gal (middle column), and c-Jun (right column) as described in Materials and Methods. Each row of panels shows the same field of injected cells. Arrows indicate the injected cells in panels showing c-Jun expression. Note that c-Jun expression in cells injected with marker antibody alone (A) or PP1 (B) was similar to that in uninjected cells. c-Jun expression in cells injected with PP2AC (C), however, was more intense than that in uninjected cells or cells injected with either PP1 or sIgG. Also, note that PP1 abolished 1-Gal expression in IBMX-8Br-cAMP-stimulated cells coinjected with the CRE-lacZ (E), while sIgG (D) and PP2AC (F) had no effect. (Bar represents 30 p.m.) 2109
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ALBERTS ET AL.
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3 2 4 1 FIG. 6. Evidence that PP2A dephosphorylates c-Jun phosphorylated by CKII on a negative regulatory domain (15, 37). Recombinant c-Jun (100 ng) was incubated with purified CKII (60 ng) in the presence of 20 p,M [y-32P]ATP as described in Materials and Methods. c-Jun protein was immunoprecipitated with polyclonal anti-c-Jun antibody 601. The immunoprecipitates were mock treated (lane 1) or treated with PP2A (lane 2), alkaline phosphatase (AP; lane 3), or acid phosphatase (AcP; lane 4) for 30 min at 30°C. Proteins were resolved by SDS-10% polyacrylamide gel electrophoresis and autoradiography.
regulate the activity or specificity of PP2AC under some circumstances in vivo. This possibility is further supported by other experiments in which we have shown that PP2AC but not the PP2AAC complex appears to regulate the function of the retinoblastoma gene product (2). Similarly, microinjection of the catalytic subunit of PP2A, but not PP2AAC, can inhibit serum-induced reporter gene expression controlled by the serum response element of the c-fos gene (48). Whether the different forms of PP2A have altered specificity or altered subcellular distribution remains to be elucidated. Both microinjection of PP2A (this study) and treatment with OA (35, 47, 50, 54) induce AP-1 activity. This apparently contradictory effect may be due to the inhibition of multiple classes of phosphatases, including PP1 and PP2A, by OA (20). In addition to inhibiting PP2A, OA can lead to activation of ERKs and possibly other protein kinases that are negatively regulated by PP2A (19). Since c-Jun is phosphorylated on both activating and inhibitory sites in response to various stimuli (11, 15), regulation of AP-1 activity may reflect the action of multiple phosphorylation-dephosphorylation pathways, several of which may be affected by OA (54). While positive-acting sites of phosphorylation in the N-terminal half of c-Jun may be dephosphorylated by PP2A and/or other phosphatases (12), our results suggest that a PP2A-dependent dephosphorylation event may also participate in the activation of AP-1 in vivo. We have shown that in vitro, PP2A can completely dephosphorylate c-Jun phosphorylated by CKII (Fig. 6). CKII has been shown to phosphorylate c-Jun on sites which inhibit DNA binding (carboxyl-terminal sites) (37). Completely dephosphorylated c-Jun is capable of DNA binding (8, 37) and is partially active
in transactivation (8, 53). Dephosphorylation of fully phosphorylated c-Jun by PP2A could therefore result in increased AP-1 activity in the cell. Activation of protein kinase C by TPA treatment also results in the rapid dephosphorylation of the sites next to the DNA binding domain of c-Jun that are recognized by CKII and results in increased DNA binding activity (15). Whether these sites are dephosphorylated by PP2A in vivo remains to be determined. Microinjection of PP2AC had no effect on TPA-induced ,3-Gal expression from the TRE-regulated promoter (data not shown). It is likely that TPA-induced 1-Gal expression is already maximal, since TPA is a potent stimulator in this cell line and was previously shown to be a better inducer of c-Jun than is serum (5). Microinjection of PP2A, on the other hand, had no effect on CRE-regulated 1-Gal expression, while microinjection of PP1 inhibited CRE-regulated 1-Gal expression stimulated by IBMX-8Br-cAMP treatment (Fig. 5) (28). Together, these results suggest that PP1 and PP2A act upon distinct substrates which may participate in discrete signaling pathways and may thereby regulate specific transcription factors. PP2A further augmented the expression of the AP-1dependent reporter and the endogenous c-jun gene by serum. This result may suggest that PP2A participates in the regulation of other serum-stimulated reactions, in addition to a possible direct dephosphorylation of c-Jun. While the activation of ERKs by serum may be a positive signal for increased AP-1 activity, there may be members of the ERK family that inhibit AP-1 activity. One member of the ERK family, ERT, or epidermal growth factor threonine 669 kinase, has been shown to phosphorylate c-Jun in vitro on one of the inhibitory sites (3). It is conceivable that injection of PP2A causes an inhibition of the serum-dependent activation of the various ERK family members, thereby inhibiting both potential positive and negative phosphorylation signals on c-Jun. This effect, combined with the effect of PP2A in relieving the inhibition of AP-1 activity due to phosphorylation of c-Jun by CKII, would potentially yield active AP-1. Direct tests of this possibility are currently under way. The results presented above demonstrate that PP2A acts differentially in the cell toward specific synthetic promoter elements which control gene expression. Microinjection of PP2AC and PP2AAC resulted in a potentiation of serum induction from TRE-containing promoters but had no effect on cAMP induction of CRE-containing promoters. To elucidate the mechanism of action of PP2A, it will be necessary to identify the substrates for this enzyme in vivo which are involved in signaling pathways. Candidates include c-Jun and c-Fos, as both are regulated by phosphorylation-dephosphorylation. It is of interest that maximal effects of microinjected PP2A required prior serum stimulation. Serum is known to result in the rapid activation of several protein kinases (e.g., ERK) (19) which may be involved in the regulation of gene expression, including AP-1 activity (3). Further studies into the molecular details of the relationships among ERKs, PP2A, and transcription factors will be required for understanding the interaction of these kinases and phosphatases and the results on gene expression. ACKNOWLEDGMENTS We are grateful to S. Shenolikar for the purified PP1. We also thank A. M. Thorburn, J. A. Frost, and S. Shenolikar for helpful discussions. We thank C. J. Buckmaster for excellent technical assistance.
VOL. 13, 1993
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