Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB

Cell, Vol. 70, 105-113, July 10, 1992, Copyright 0 1992 by Cell Press

Transcriptional Attenuation Following CAMP Induction Requires PP-1 -Mediated Dephoschotylation of CREB Masatoshi Hagiwara, l Arthur Alberts,t Paul Brindle,* Judy Meinkoth,t James Feramisco,t Tiliang Beng,* Michael Karin,* Shirish Shenolikar,§ and Marc Montmlny” “The Clayton Foundation Laboratories for Peptide Biology The Salk Institute La Jolla, California 92037 tDepartment of Medicine *Department of Pharmacology University of California at San Diego La Jolla, California 920936636 §Department of Pharmacology Duke University Durham, North Carolina 27710

We have examined the mechanism by which the transcriptional activity of the CAMP-responsive factor CREB is attenuated following Induction with forskolin. Metabolic labeling studies reveal that, after an Initial burst of phosphorylation in response to CAMP, CREB is dephosphorylated and transcription of the CAMP- responsive somatostatln gene is correspondingly reduced. The phosphatase inhibitor 1 protein and okadaic acid both prevented the dephosphorylation of CREB at Ser-133 in PC12 cells and also augmented the transcriptional response to CAMP. of the four Ser/Thr phosphatases described to date, only PP-1 appears to be similarly inhibited by these agents. As PP-1 specifically dephosphorylates CREB at Ser-133 and inhibits CAMP-dependent transcription, we propose that this phosphatase is the major regulator of CREB activity in CAMP-responsive cells.

A number of studies describing signal transduction mechanisms that stimulate gene activity have recently emerged. However, the manner by which such transcriptional signals are then down-regulated remains relatively uncharac- terized. The transcriptional response to CAMP stimulation, for example, follows’burst-attenuation” kinetics with maximal rates of transcription usually occurring 30 to 60 min following stimulation (Lewis et al., 1967; Sasaki et al., 1964). Transcription of CAMP-responsive genes declines steadily thereafter, typically reaching a nadir at 6-l 0 hr. Such attenuation does not appear to simply reflect loss of CAMP or PK-A activity in stimulated ceils, as both remain elevated throughout the treatment. Indeed, CAMP-stimulated cells are often refractory to subsequent induction (by CAMP), suggesting that the attenuation reflects true down-regulation of the transcriptional apparatus (Sasaki et al., 1964).

CAMP mediates the hormonal stimulation of a number of eukaryotic genes through a conserved CAMP-responsive

element (CRE) that is recognized by the transcription factor CREB (Gonzalez and Montminy, 1969; Gonzalez et al., 1969; Montminy and Bilezikjian, 1967). CREB activity, in turn, is regulated by the CAMP-dependent protein kinase PKA, which phosphorylates CREB at a single phosphoacceptor site Ser-133. As Ser-133 phosphorylation specifically stimulates the transcriptional activity of CREB without affecting its DNA-binding properties, we have pro- posed that such phosphorylation induces a structural change in the molecule that subsequently alters its interac- tions with other proteins in the RNA polymerase II complex (Gonzalez et al., 1991).

Attenuation of CREB activity could occurthrough a number of mechanisms. For example, CAMP may induce an inhibitor protein that would associate with and inhibit CREB activity. Indeed, such a candidate inhibitor, termed CREM, has recently been characterized and shown to repress CREB activity in transient cotransfection experiments (Foulkes et al., 1991). Expression of the CREM protein has not been demonstrated in any cell type to date, however, and we have been unable to detect CREM protein expression in a number of CAMP-responsive cell lines by Western blot analysis (Gonzalez and Montminy, 1969).

Alternatively, CAMP may shut down the transcriptional response by stimulating specific proteases that degrade the CREB protein. CREB protein levels appear to remain unchanged up to 12 hr after CAMP stimulation (Gonzalez and Montminy, 1969), however, prompting us to examine whether dephosphorylation is involved. To date, the Ser/ Thr protein phosphatases have been classified into four groups termed PP-1, PP-2A, PP9B, and PP-2C (Cohen, 1969). The biochemical properties of these enzymes have been well-characterized in vitro, and representative phosphatases from each group have recently been purified and cloned.

In the present study we compare the phosphorylation of CREB with transcription of the cAMP-responsive somatostatin gene. The CAMP-stimulated phosphorylation of CREB at Ser-133 is indeed followed by dephosphorylation of that site. Moreover, the rate of Ser-133 dephosphorylation following CAMP treatment mirrors a steady decline in somatostatin transcription. Based on this evidence, we propose that a CREB phosphatase shuts down the transcriptional response to CAMP. We proceed by characteriz- ing the putative phosphatase in vivo, in nuclear extracts, and finally with purified phosphatase preparations. Our results identify one particular Ser/Thr phosphatase, PP-1, as the major down-regulator of CREB activity following CAMP stimulation.

Results

To examine the kinetics of CAMP induction, we performed run-on transcription assays using PC12 and 3T3 cell clones stably transfected with a somatostatin reporter gene. In these cells, somatostatin mRNA is correctly initi- ated and is induced 5 to 1 O-fold within 4 hr of CAMP treat-

. 1

ment (Montminy et al., i 988). Run-on transcription assays in NIH 3T3 Cells (Figures 1 C and 1 D) showed a 4-fold Surge in somatostatin transcription peaking 30 min after forskolin stimulation and then declining to near baseline levels by 4 to 8 hr. Similar results were obtained for PC12 cells (data not shown). ,By contrast, transcription of the non-CAMP- responsive a-tubulin gene remained stable over the entire treatment period (Figure 10).

Having previously determined that phosphorylation of CREB at Ser-133 is critical for the transcriptional response to CAMP (Gonzalez and Montminy, 1989), we examined whether reductions in CREB phosphorylation at this site could account for the transcriptional attenuation of the somatostatin gene. To this end, we prepared immunoprecipitates of CREB from PC12 cells labeled with IZP]ortho- phosphate and harvested at various times after forskolin addition (Figure 1A). Two-dimensional ttyptic analysis of each precipitate showed two phosphopeptides: the major peptide (labeled A) containing the previously characterized PK-A phosphoacceptor Ser-133 (Gonzalez and Mont-

miny, 1989) and a minor phosphdpeptide (labeled B) corresponding to the principal casein kinase II (CKII) phosphorylation site in the CREB protein (M. M., unpublished data). Phosphorylation of the PK-A-phosphorylated peptide A was rapidly induced by CAMP, rising from near unde- tectable levels in unstimulated cells to about 30-fold over baseline within 30 min (Figure 1B). Phosphotylation of peptide A declined steadily thereafter reaching 25% of peak levels by 8 hr. Only minor changes in peptide B phosphorylation could be observed during forskolin treatment, suggesting that dephosphorylation of CREB was restricted to Ser-133. Moreover, the levels of Ser-133 phosphotylation for each time point showed positive corre- lation with the relative rates of somatostatin transcription, as determined by the run-on assay (compare Figures 16 and 1C).

Previous results showing that CREB’ protein levels remain invariant up to 12 hr after forskolin induction (Gonza- lez and Montminy, 1989) prompted us to hypothesize that a Ser/Thr phosphatase may be present that would selec-

D. Som. e II: Ilr

Tub. - .‘*I_

0 30 lh 4h 8h

15’30’ 60 2h 4h 6h 8h Tii

Figure 1. Kinetic Profiles of CREB Phosphorylation and Somatostatin Transcription in Response to CAMP (A)Twodimensional tryptic maps of =P-labeled CREB immunoprecipitates from PC12 cells following forskolin treatment (IO pM). 0, origin; A and B, CREB tryptic phosphopeptides corresponding to PK-A- phosphorylated Ser-133 (A) and CKII site (B). Time points at which cells were harvested are shown at the top left of each chromatogram. (B) Graph showing phosphorylation of peptides A (PK-A site) and B (CKII site) over time in forskolin-stimulated cells as determined by densitomeby. Peptide A phosphorylation, open circles; peptide B, closed circles. (C and D) Run-on transcription of the rat somatostalin gene in response to CAMP. Graph (C) shows the rate of somatostatin transcription relative to a-tubulin control following forskolin (10 PM) stimulation as determined by densitometry of slot-blot autoradiogram below. Time points indicated on abscissa. In (D) is an autoradiogram of slot-blotted somatostatin (Som.) and a-tubulin (Tub.) cONAs probed with “P-RNA from CAMP-stimulated NIH 3T3 cells stably transfected with the rat somatostatin gene. Time points at which cells were harvested following forskolin addition (10 MM) are shown below each lane.

PP-1 Mediates Attenuation of CREB Activity 107

tively dephosphorylate CREB at Ser-133. To characterize the putative phosphatase, we employed a potent phosphatase inhibitor, okadaic acid (OA), and examined the effects of this agent on CREB phosphorylation in situ (Figure 2A) in PC12 cells. At 1 uM concentrations, OA completely re- versed the dephosphorylation of CREB phosphopeptide A, maintaining maximal levels of phosphorylation there for at least 4 hr after forskolin addition. No effect on CREB dephosphorylation was obtained by 1 nM OA. Peptide B phosphorylation was unaffected by OA, however, suggesting that OA could specifically inhibit the phosphatase that normally down-regulates CREB activity in response to CAMP.

The ability of OA to prevent CREB dephosphorylation at Ser-133 (peptide A) prompted us to examine whether this inhibitor would correspondingly potentiate the transcriptional response to CAMP (Figure 28). Using transient transfection analysis, we observed that forskolin treatment of PC1 2 cells induced a 5-fold response in CRE-CAT activity, and OA further enhanced CAT activity 2-to 3-fold in cells harvested after only 4 hr of treatment. A mutant CRE-CAT reporter that cannot bind CREB (and is unresponsive to CAMP) was unaffected by OA, suggesting that the stimulation we observed was indeed dependent on CRE. OA also had no effect on the activity of a cotransfected RSV+gal reporter gene (data not shown), suggesting that OA may not generally increase RNA polymerase II-dependent transcription.

To determine whether the effect of OA on somatostatin promoter activity was indeed CREB dependent, we employed a eukaryotic expression plasmid encoding a Gal4- CREB fusion protein (Figure 28). This protein contains the Gal4 DNA-binding domain fused to the N-terminus of the entire CREB molecule. When cotransfected with the GaH- CREB expression plasmid into PC12 cells, the activity of

A. (Z, 0 1

a Gal4-CAT reporter vector in PC12 cells was induced 5- to 1 O-fold by forskolin. Supplementing forskolin-treated PC1 2 cells with OA further augmented the CAMP response 3-to Cfold. However, a mutant Gal4-CREBMl expression plasmid containing a Ser-133 to Ala-133 substitution at the PK-A site showed greatly reduced GaH-CAT activity in response to forskolin, and OA was similarly ineffective. As OA appears to block dephosphorylation of CREB at Ser-133 specifically, the ability of this inhibitor to augment CREB activity in a Ser-133dependent manner suggested that aCREB phosphatase might indeed attenuate the transcriptional response to CAMP.

To characterize this phosphatase, we prepared nuclear extracts of PC12 cells and tested their ability to dephosphorylate CREB at Ser-133 (Figure 3). Indeed, recombinant CREB protein that had been phosphorylated in vitro with PK-A was rapidly dephosphorylated within 10 min of extract addition. OA blocked CREB phosphatase activity with an I& (50% inhibitory dose) of 20 nM, which is close to the lC& of PP-1. In addition, treatment with 1 uM OA abolished over 95% of CREB phosphatase activity in these extracts. Of the four characterized SerfThr phosphatase groups, PP9B and PP-PC appear to be effectively OA resistant (Cohen, 1989) enabling us to exclude these as candidate CREB phosphatases. PP-1 and PPQA activities, however, are readily inhibited by OA with characteris- tic ICsosof 20 nM and 0.2 nM, respectively. As dephosphorylation of CREB was inhibited by concentrations of OA above 10 nM, we speculated that PP-1 might indeed con- stitute the CREB phosphatase.

To determine whether PP-1 can catalyze the dephosphorylation of CREB, we incubated the purified enzyme with PK-A-phosphorylated CREB protein (Figure 4A). PP-1 completely dephosphorylated the CREB substrate over a 3 hr period, and the dephosphorylation reaction could be

1000

A

Figure 2. Effect of the Phosphatase Inhibitor OA on CREB Phosphorylation and CAMP- Dependent Transcription in PC12 Cells (A) Two-dimensional tryptic maps of “P-labeled CREB immunoprecipitates prepared after 4 hr treatment with forskolin plus either 0, 1, or loo0 nM OA as indicated over each chromatogram. 0. origin; A and 8, CREB phosphopeptides A (PK-A site) and B (CKII site) as described in Figure 1. (B) Representative (CAT) assay of PC12 cells transfected with CRE-CAT or Gal4-CAT reporter plasmids as indicated. For Gaul-CAT reporter assays, cotransfected Gal4-CREB and Gal4-CREBMI effector plasmids are shown over each lane. Percent conversion (% CONV.) of “Cchloramphenicol is shown in the box below each lane. Forskolin (FORSK.) (10 uM) or OA (0.1 uM) treatment is indicated by a plus sign under each lane. A minus sign indi- cates no treatment. All assays were normalized to b-galactosidase activity derived from the cotransfected RSV-bgal expression plasmid. The CRE-CAT experiments were repeated four times, and the average induction by OA on forskolin-treated cells was 2.1 f 0.47.

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CREB +

Time 0 1 3 10 (min)

OA (nM) 0 1

inhibited by OA with an lCso of 20 nM. A purified PPPA catalytic subunit also dephosphorylated CREB, albeit less efficiently and with afar lower I& (0.2 nM) of OA inhibition than PP-1 (data not shown). Thus, the effects of OA on the CREB phosphatase in PC12 cells and in nuclear extracts appeared to be identical with purified PP-1 protein.

To assess further whether PP-1 is indeed a CREB phosphatase in vivo, we examined the ability of this enzyme to dephosphorylate CREB at the principal CKII phosphorylation site corresponding to peptide B (Figure 48). As peptide B was minimally dephosphotylated following CAMP treat-

A. CREBIPK-A + PP-1

Time (min) 0 M 60 12s 180 0 1 10 100 1OW OA(nM)

B. CREB Mi/CKtt PP-1 PP-2A

Time(min) 0 ‘30 M) 1P 180’ ‘30 60 1P ld

+W- , ^yLI “a*/ c

Figure 4. Activity of PP-1 on CREB Phosphorylated at Peptide A (PK-A Site) or Peptide B (CKII Site) In Vitro

(A) Left, autoradiogram showing SDSgel of recombinant CREB protein phosphorylated with purified PK-A in vitro. Arrow points to 43 kd CREB band. Free %P generated by hydrolysis of phospho-Ser-133 appears at bottom of gel. Time (in minutes) following incubation with purified PP-1 is shown above each lane. Right, effect of OA on PP-1 -mediated dephosphorylation of CREB. All samples contain 32P-labeled CREB incubated for 190 min with purified PP-1 plus OA at concentrations (nM) shown over each lane. (B) Effect of purified phosphatases PP-1 and PP-2A on dephosphorylation of CREB phosphorylated by CKII. CREBMl/CKII refers to purified recombinant CREBMI protein containing a Ser-133 to Ala-133 substitution phosphorytated with CKII in vitro. SDS-polyaclylamide gel electrophoresis is shown with arrows pointing to YP-labeled CREB. PP-1 or PP-2a enzymeswere incubated with CKII-phosphorylated CREBMI for times indicated (in minutes) over each lane.

Figure 3. Dephosphorylation of PK-A-Phos- phorylated CREB in Nuclear Extracts of PC12 Cells and Reversal by OA (A) Autoradiogram of SDS gel showing dephosphorylation of CREB labeled with “P-ATP plus PK-A in vitro and then incubated with PC12 nuclear extracts (25 pg of protein) at 39% for time points (in minutes) indicated below each lane. (B) Effect of OA on dephosphorylation of PK-A- phosphorylated CREE protein incubated with PC12 nuclear extracts as indicated above. SDS-polyactylamide gel electrophoresis shows “P-labeled CREB and free =P generated from hydrolysis of Ser-133 following incubation with extract. Concentration (nM) of OA used is indicated below each lane.

ment and was unaffected by OA treatment (see Figure 1 A), we hypothesized that the CREB phosphatase should be unable to dephosphorylate that site. We phosphorylated recombinant CREBM 1 protein (which lacks the PK-A phosphoacceptor site) with CKII in vitro and tested the ability of PP-1 or PPQA to dephosphorylate this site. Surprisingly, the CKII-phosphorylated CREB protein was readily dephosphorylated by PP-PA but was completely resistant to PP-1 treatment. As minimal changes in peptide B phosphorylation were obsenred with OA treatment of forskolin- stimulated PC12 cells, these results further strengthened the hypothesis that P,P-1 indeed constitutes the major CREB phosphatase.

To test this model further, we performed a series of transient assays in F9 cells using eukaryotic expression plasmids encoding the catalytic subunits of PP-1 and PP-2A (Figure 5). As previously noted (Gonzalez and Montminy,

CREB - + - + + +

PK-A - - + + + + pp-, _ _ - - + - PP-PA - - - - - +

Figure 5. Effect of Phosphatases PP-1 or PP-2A on CREB Activity in F9 Teratocarcinoma Cells Transient (CAT) assay of F9 cells transfected with somatostatin CRE- CAT reporter. Bar graph shows reporter activity as percent of [“CCjchloramphenicol conversion. Cells were transfected with CRE- CAT reporter plus effector plasmids indicated by plus signs under each lane. Assays were normalized to 5galactosidase activity derived from cotransfected RSV+gal plasmid.


1989), cotransfection of PK-A and CREB expression vector stimulated CRE-CAT reporter activity lO-fold. Cotrans- fecting an expression vector for the PP-1 phosphatase, however, severely abrogated the PKA response. An expression plasmid encoding the catalytic subunit of PP-2A had no inhibitory effect on CRE-CAT activity and in fact stimulated the CRE-CAT reporter modestly in these cells.

To verify further the observed differences between PP-1 and PP-2A activities in transfection assays, we used purified enzyme preparations to perform microinjection experiments with living cells (Figures 8,7, and 8). Two independent cell lines, each expressing a stably transfected CRE-IacZ reporter plasmid, were used for the microinjection studies: WRT CRE-Z, a rat thyroid follicular cell line, and Rat2 CRE-Z, a fibroblast cell line. Upon stimulation with forskolin or IBMX/8BrcAMP, these cells induce the production of 8galactosidase protein, which can be readily detected after staining with the chromogenic substrate Xgal (Riabowol et al., 1988). Moreover, microinjection of CREB antiserum significantly reduces the CAMP response in these cells, suggesting that such expression is indeed CREB dependent (Meinkoth et al., 1991).

When introduced into the cytoplasm of either cell type (WRT or Rat2) by microinjection, the purified PPPA catalytic subunit had no effect on expression of the CRE+gal reporter gene (Figure 7). By contrast, PP-1 markedly inhibited 6gal induction in both cell lines (Figures 6 and 8). Interestingly, PP-1 had no effect on the activity of a TPA- responsive reporter gene (TRE+gal), suggesting that PP-1 acts differentially in signal transduction pathways that control gene transcription. As the TRE element is rec-

ognized by AP-1 (Angel et al., 1988) but not CREB, these results demonstrate that PP-1 may act specifically on proteins that mediate the CAMP response.

To obtain independent confirmation that PP-1 indeed mediates down-regulation of CREB activity following CAMP induction, we examined whether the PP-l-specific inhibitor 1 (l-l) and inhibitor 2 (l-2) proteins could also augment the CAMP response by modulating CREB phosphorylation (Figures 9, 10, and 11). Both l-l and l-2 bind with high affinity (Cohen, 1989) and inhibit PP-1 but not PP-2 activity. When added to crude PC12 nuclear extracts, pori- fied l-2 protein inhibited dephosphorytation of CREB at Ser-133 (Figure 9).

To test whether the inhibitor proteins could enhance CREB activity by inhibiting cellular PP-1 in vivo, we performed microinjection experiments (Figures 10 and .1 1) with an expression plasmid encoding I-1 (Elbrecht et al., 1990). As I-1 binds to and inhibits PP-1 only when phosphorylated at Thr-35 by PK-A, we employed a constitu- tively active mutant form of I-1 containing a Thr-35 to Asp- 35 substitution (S. S., unpublished data). In the absence of CAMP induction, the I-1 expression plasmid stimulated CRE-IacZ activity 5-fold in rat embryo fibroblasts (Figure 10). I-1 also further augmented the CAMP response when subthreshold levels of BBr-cAMP were employed, suggesting that endogenous PP-1 does indeed inhibit CREB activity.

Discussion

Various intracellular events such as contractility, mem-

Figure 6. Microinjection of Phosphatase PP-1 Inhibits Transcription of the CRE+gal Re- porter Gene in Response to CAMP

Quiescent Rat3 fibroblasts stably transfected with a CRE+gal indicator plasmid were microinjected with 0.1 mg/ml purified PP-I plus IgG (3 mglml) to identify microinjected cells. Immediately following injection, cells were stimulated for 6 hr with IBMX (0.5 mM) plus BBr-CAMP (0.5 mM). Following fixation, cells were stained with X-gal to visualize 6galactosi- dase expression and treated with secondary antibody (Jackson Labs) to detect coinjected marker antibody. Left, dark-field photomicrographs of cells showing immunofluorescence from cells coinjected with marker IgG antibody. Right, light-field photomicrographs (corresponding to dark-field photomicrographs on left), showing 6-galactosidase activity derived from stably transfected CRE+gal plasmid. (A-D) Two representative fields of cells injected with PP-1 plus IgG are shown. (E and F) Cells injected with marker IgG alone.

Cdl 110

brane transport, glycogen metabolism, and cell division are regulated through interconversion processes cata- lyzed by protein kinases and phosphatases. The impor- tance of phosphorylation in stimulating a number of transcription factors suggests that,‘by analogy with CREB, phosphatases will be correspondingly important in shut- ting off the transcriptional response.

Although CREB has been shown to be phosphotylated by PKA following CAMP induction, no information on po- tential phosphatases that catalyze CREB dephosphorylation has previously been available. In this report, we ob-

served that CREB is in&d dephosphorylated following an initial peak of phosphorylation in response to CAMP. Although a 75% reduction in Ser-133 phosphorylation appears sufficient to down-regulate somatostatin transcription following CAMP stimulation, we could not determine which fraction of the CREB protein in these immunoprecipitates was bound to chromatin and actively regulating transcription. Thus, the fraction of CREB in transcriptionally active chromatin may be more extensively dephosphorylated than these assays suggest.

OA did not increase the stoichiometry of CREB phos-

40 BLUE CELLS

‘80

70

60

50

40

Q PPPA catalytic sub.

Figure 8. Bar Graph Summarizing Effects of PP-1 and PPPA on Expression of a Stably Transfected CRE-ggal Indicator Plasmid in WRT (WRT CRE-Z) Thyroid Follicular Cells or Ra12 Fibroblasts (Rat2 CRE-2) Effect of PP-1 and PP-2A on activity of serum- stimulated Rat2 cells stably transfected with a TRE-IacZ (TRE-Zj reporter also shown. Bars represent the mean f standard deviation of three experiments where 100-150 cells were injected in each.

0 WRT CRE-Z Rat2 cm-z TRE-Z

:;;I Mediates Attenuation of CREB Activity

Mr

27-

Extract - + + I-2 - - +

Figure 9. Effect of l-2 Protein on CREB Phosphorylation Effect of l-2 protein on dephosphorylation of PK-A-phosphorylated CREB protein following incubation with PC12 nuclear extracts. SDS- polyacrylamide gel electrophoresis shows “P-labeled CREB (arrow)

phorylation, but rather extended the time during which CREB was phosphorylated. Thus, the transcriptional effects of OA should become more pronounced with longer treatment periods. Indeed, OA caused a 3- to 4-fold en- hancement in CREB activity after 4 hr, but toxic effects related to OA treatment prevented us from examining later time points. Nevertheless, the ability of OA to inhibit CREB dephosphotylation at concentrations above 10 nM strongly implicated PP-1 as the predominant CREB phosphatase.

Remarkably, PP-1 appears to be quite specific in de- phosphorylating CREB and in down-regulating CREB-acti- vated transcription. Transcriptional responses to TPA and serum, for example, are unaffected by PP-1 (figure 8). In contrast, phosphatase PPPA, which has no effect on CREB activity, appears to be important in regulating SRE- and AP-l-containing promoters (A. A., J. F., and M. K., unpublished data). These results suggest that the bio- chemically defined Ser/Thr phosphatases may also have

before (lane 1) and following (lanes 2 and 3) incubation with extract. Lane 3 shows the effect of l-2 protein (20 ng) on CREB dephosphorylation upon addition to crude extract. Mr. molecular size (in kilodaltons).

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46 BLUE CELLS LOO

80

‘0

40

20

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m CUE-IacZ 0 CRE-IacZ + INH-1

0 0.1 0.5

[IBMWSBrcAMP] mM

Figure 11. Summary of Injections Shown in Figure 10 I-1 activates and potentiates CREB activity in REF-52 cells. Bars represent the mean ( f standard error of the mean) of three to four experiments where 50-100 cells were injected in each.

distinct roles in regulating transcription. The expression vectors we have developed should provide a useful tool to analyze the functional role of these phosphatases.

The ability to repress the CAMP response pathway appears to be an important mechanism in negative control of gene expression. The TSE-1 extinguisher locus, for example, mediates repression of liver-specific genes in hep atoma x fibroblast cell hybrids (Boshart et al., 1991; Jones et al., 1991). Indeed, this locus has recently been shown to encode the Rla subunit of PK-A, indicating that repression is maintained by reducing levels of free catalytic subunit that can phosphorylate CREB. The ability of PP-1 to inhibit transcriptional induction by CAMP selectively suggests that, in some cell types, this phosphatase may func- tion as an extinguisher.

In this respect, it is interesting to speculate that the phosphatases themselves may be regulated by cellular signals. The activity of PP-1, for example, can be inhibited by intrin- sic small proteins, l-l and l-2 (Cohen, 1989). As I-1 can inhibit PP-1 activity only when phosphorylated in response to PK-A or calmodulin kinase II, our results suggest that, in l-l -expressing cells, the CAMP and CaZ+ responses may be amplified by preventing the dephosphotylation of CREB and other targets of PP-1 that are important for CREBdependent transcription. Further studies into the mechanism of phosphatase regulation will allow us to un- derstand better the role of these important modulators in cellular signaling. Other cellular signals like phorbol es- ters, retinoic acid, glucocorticoids, and thyroid hormone may also be attenuated by posttranslational modifications of their corresponding transcription factors.

Experimental Procedures

Cell Lines, Transtectlons, and Microinjectlon PC12 and F9 teratocarcinoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum plus 5% horse serum. WRT CRE-2 cells were maintained in a six hormone (BH)-containing medium (Ambesi-lmpiombato et al., 1980) supplemented with 150 &ml G418. Rat2 CRE-Z and TRE-Z fibroblasts were maintained in DMEM containing 10% fetal calf serum

.

and 300 &ml 6418. Transfections were performed as described previously (Gonzalez and Montminy, 1989) using 10 pg of reporter ((-71)CAT and Gal4-CAT). 4 ug of RSV-Bgal plasmid, and, when indicated, 5 ug each of expression plasmids of Mt C (C subunit of PK-A), RSV-PP-1 (PP-1), CMV-PTC (PPgA), GaH-CREB, and 7.5 pg of RSV-CREB plasmid. Cells were assayed for CAT activity after normalizing to f3galactosidase. For OA experiments, PC1 2 cells were transfected and treated for 4 hr with 0.1 pM OA f 10 pM forskolin. Longer incubations with OA were toxic to PC12 cells. NIH 3T3 D5 cells expressing a stably transfected somatostatin reporter plasmid were maintained in DMEM plus 10% calf serum supplemented with 400 ug/ ml 6418 (Montminyet al., 1986). For microinjection experiments, WRT CRE-Z, Rat2 CRE-Z. and Rat2 TRE-Z cells were plated on scored glass coverslips and rendered quiescent for at least 24 hr prior to injection. Purified phosphatases were coinjected with affinity-purified IgG in or- der to identify injected cells unambiguously. Cytoplasmic injections were performed.

For I-1 microinjection experiments, I-1 expression vector (1 mg/ml) and CRE-IacZ reporter (0.1 mg/ml) were injected directly into the nu- cleus of logarithmically growing REF-52 fibroblasts almg with sheep IgG (4 mg/ml) marker antibody. Three hours after injections, cells were stimulated with IBMX/8Br-CAMP where indicated. After 3 hr of further incubations, cells were fixed and stained for Pgalactosidase expression and presence of marker antibody.

Immunopreclpltatlons and Phosphopeptlde Mapplng All immunoprecipitations were performed using CREB antiserum 244 as described previously (Gonzalez and Montminy, 1989). PC12 and 3T3 cells were labeled with inorganic “P for 4 hr and treated with 10 uM forskolin for indicated times. =P-labeled cell extract was immuno- precipitated with antiserum 244 and electrophoresed. =P-labeled bands, visualized by autoradiography. were cut out, digested by tryp sin, and analyzed by two-dimensional mapping as described previously (Gonzalez and Montminy, 1989).

Nuclear Run-On Transcr?ptlon Assey PC12 and NIH 3T3 cells (2.5 x lo5 cells per point) stably transfected with the somatostatin gene were treated with 10 PM forskolin for the indicated time. After harvesting nuclei of treated cells, YP-labeled RNA probe was prepared and hybridized to immobilized somatostatin and a-tubulin plasmids on nitrocellulose filters as described by Greenberg and Ziff (1984). Autoradiographic densities were quantitated with a scanning densitometer. Each somatostatin signal was normalized to the a-tubulin signal on the same strip.

Plasmlds The RSV-PP-la expression vector was constructed by inserting an EcoRI-BamHI fragment of the PP-la cDNA from plasmid N ill/21.9 (Cohen, 1988) into Bluescript SK-II. A Hindlll-Notl fragment containing the RSV promoter from the RSV &fun plasmid was then cloned into the PP-la cDNA-Bluescript plasmid. The CMV-PP-2A catalytic subunit expression vector was a generous gift of M. Mumby (Green et al., 1987). The I-1 expression vector was constructed by inserting the I-1 cDNA, encoding a Thr to Asp substitution at Thr-35, into the pUC plasmid containing the metallothionine promoter.

Acknowledgments

We thank P. Cohen and M. Mumby for gifts of PP-la and PPOA plasmids and Dave Rose for helpful advice with reporter plasmid injections. We also thank T. Hunter for critical review of this manuscript and members of the Montminy laboratory for their helpful insights. This work was supported by National Institutes of Health grants GM37828, CA50528, CA141 95, and CA3981 1 and by the Foundation for Medical Research, Inc. M. M. is a Foundation for Medical Research Investiga- tor. J. F. was supported by the Council for Tobacco Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact.

Received February 13. 1992; revised April 21, 1992.


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Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB