A Secondary Phosphorylation of CREB341 at Serf2' Is Required for ...

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tion at the PKA and GSK-3 sites of CREB are essential for CAMP control of CmB. A functional role for protein phosphorylation in regulating nuclear processes ...
Vol. 269, No. 51, Issue of December 23, pp, 3218732193, 1994 Printed in U.S.A.

TKEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

A Secondary Phosphorylation of CREB341at Serf2'Is Required for the CAMP-mediated Control of Gene Expression A ROLE FOR GLYCOGEN SYNTHASE KINASE-3 IN THE CONTROL OF GENE EXPRESSION* (Received for publication,June 20, 1994, and in revisedform, Octaber 11, 1994)

Carol J. FiolS, John S . Williams§, Chin-Hua ChouOn, Q. May Wang$,Peter J. Roach$, and Ourania M. AndrisaniOll From the Wepartment of Biochemistry and Molecular Biology,Indiana University School of Medicine, Indiana~ol~s, Indiana 46202 and the $Department of Physiology and Pharmacology, School of Veter~naryMedicine, Purdue University~West Lafayette, I n d ~ n a47907

The CAMP-dependentprotein kinase (PKA)phospho- been reported (reviewed in Ref. 2). The nuclear factor CREB interacts specifically with the cyclicAMP response element rylates CREBS27/541 at a single serine residue, Ser""", respectively. Phosphorylationat this sitecreates the se- (CRE)l in genes controlled by the cyclic AMP-mediated pathquence motifsxxxS(P), a consensussite of the glycogen ways of signal t r ~ s d u c t i o n(3,4). Phosphorylation of CREB by synthase kinase-3(GSK-3) enzyme (Fiol, C. J., Mahren- cyclic AMP-dependent protein kinase was initially thought to holz, A. M., Wang,Y., Roeske,R. W., and Roach, P. J. (1987) be necessary and sufficient for the activation of transcription by J. Biol. Chem. 262,14042-14048). We examined the phos- CAMP (5). In either of twoforms of CREB(CREB327 and phorylation of CREB at the sxxxS(P) consensus site CREB341)that result from alternative splicing, a mutation of and its role inCREB transactivationto C A M P induction. the PKA phosphorylation site Ser119/'33to an alanine or an asNeither isoform of the GSK-3 enzyme (GSK-3 a or p) partic residue prevented activation by PKA in vitro and abolutilizes CREB as its substrate unless CREB is already ished the CAMPcontrol of transcription in vivo (6).Aphosphate A 13-amino acid peptide conphospho~lated Ser1*wK'89. at group in thetransactivating region is therefore essential for a taining the sequence surrounding Ser11s/189 was phosphorylated byGSK-3, at Ser'1N12s, onlyafter the primary transcriptionally active protein. Later, measurements in vivo of phosphorylation of the peptide by PKA (at Serll"'sa),s u e the activities of deletion and point-mutated chimeric fusion that Serllgwas necessary but not gesting that Ser"''2v is a GSK-3 phosphoacceptor site. proteins of CREB327 showed sufficient for the full CAMP-inducible transactivation functions +Ala substi. Mutant CREBSnM1 proteins containing Ser tutions confirmed Ser11"29as the only GSK-3 phospho- of CREB (7). It was suggested that phosphorylation within a rylation site. Transfection assays of wild type and mu- domain termed the P-box (a 46-amino acid sequence, residues tant Gal4-CREB fusion proteins in PC12 cells 92-137) was required for the full response to CAMP.This dodemonstrated that Ser 4Ala substitution of residue 129 main includes consensus phosphorylation sites for protein kiofCREB341 impairs the transcriptional response to nase C, casein kinase 11, and the well established PKA site CAMPinduction. Analogous mutation in CREB"' results (Ser119/1331. in 70%decrease in itstransactivationresponse to CAMP. It is now recognized from the study of protein phosphorylaIn undifferentiated F9 cells, which are refractory to tion in general that multiple phosphorylation of proteins is the C A M P induction, transfected GSK-3p kinase induces a norm rather than the exception (8). Furthermore, in several AMP response element-depend- instances the multiple phosphorylations do not occur independ60-fold increase in cyclic ent transcription, mediated via the endogenous CREB ently (1, 9) but follow an obligate order in what has been protein. We propose that the hierarchical phosphoryla- termed hierarchical phosphorylation (8)and involves "primary" tion at the PKA and GSK-3 sites of CREB are essential and "secondary" kinases. The mechanistic basis for the phefor C A M P control of CmB. nomenon is that thesecondary kinases appear to require prior

A functional role for protein phosphorylation in regulating nuclear processes has been predicted for many years, and recently, phosphorylation of several transcription factors, including CREB, AP-l/c-Jun and C-Fos,c-Myb, c-Myc,and ADR-1, has

phospho~lationof the substrateby primary kinases. The best studied example of this synergism is thephosphorylation of the metabolic enzymeglycogen synthase (1)by GSK-3. GSK-3preferentially phosphorylates substrates where primary kinases have produced the primary sequence motif SXXXS(P). This motif is found in other GSK-3 substrates such as ATP citrate lyase (10)and the G subunit of type I protein phosphatases (11). The role of GSK-3 as theactivator of the A ~ - ~ ~ - d e p e n d e n t phosphatase is also well studied and involves a synergistic phosphorylation of the inhibitor 2 protein, However, in this instance the secondary site is 13 residues N-terminal to the primary CK-I1 site (12). The cDNAs encoding two isoforms of GSK-3 termed GSK-3a (51 kDa) and GSK-3p (47 kDa) have been isolated from a rat brain library by Woodgett (13).These two proteins share 85% homology at the amino acid level. It is becoming clear that

* This work was supported in part by Juvenile Diabetes Foundation Research Grant 18922 (to C . J. F.), by the Diabetes Research Training Center, and Indiana University Medical School Public Health Service Grants NTP1246 and DK44533 (to 0. M. A,) and DK27221(to P. J. R.). Peptide synthesis and protein sequencing was performed by the Biochemistry Biotechnology Facility of the Biochemistry and Molecular Biology Department of Indiana University School of Medicine. 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 with18 U.S.C. Section 1734 solely to indicate this fact. nRecipient of PredactoralTrainingGrant94040130fromthe American Heart Association,IndiandOhio AffYiate. The abbreviations used are: CRE, cyclic AMP response element; /I To whom correspondence should be addressed. Tel.: 317-494-8131; PKA,cAMP-dependentproteinkinase;GSK-3,glycogensynthase Fax: 317-494-9193;E-mail [email protected]. kinase-3; CAT, chloramphenicol acetyltransferase.

32187

32188

The Role of a Secondary P ~ o ~ p h o ~ofl CRE€3341 ~ ~ ~ o nat Ser129

there isa family of related GSK-3-like enzymes.A third mammalian form, DM,-1, has been identified by polymerase chain reaction.' Two yeast enzymes, MCK-I and MDS-1, appear to recognize the SXXXS(P) sequence motif. MCK-I has 45% sequence identity with GSK-3 within the catalytic domain, and MDS-1 has 57%sequence identity over a 296-amino acid overlap with GSK-3a from rats (14). Zeste white/Shaggy has been shown to be the Drosophila melanogaster homologue of GSK3p, with 88%homology within the catalytic domains and similar specificity for the known GSK-3 protein substrates. It is therefore reasonable to expect that Shaggy will participate in hierarchical phosphorylations, although it has not beendirectly demonstrated. In transgenic flies, it has been shownthat GSK-3p can substitutefor Shaggy (15).Shaggy plays a crucial role in embryogenesis, and it is speculated that it may be involved in theregulation of transcription factors involved in the expression of genes leading to embryonic segmentation. It was apparent from inspection of the CREB amino acid sequence that phosphorylation by PKA at Ser119/133 generates a GSK-3 site at Ser1155/'29 with the potential for hierarchical phosphorylation of the CREB protein by PKA and GSK-3. A synthetic peptide, based on the transactivationdomain of CREB, is a substrate for GSK-3 (16). In this study, recombinant CREB proteins are shown to be substrates for either isoform of GSK-3 only after primary phosphorylation byPKA. The functional significance of this secondary phosphorylation site isexamined in vivo using mutated forms of CREB bytransient transfection assays in PC12 cells, a well d o c u m e n ~ d c ~ P - r e s p o n scell ive system (17). The direct requirement of GSK-3 kinase activity in CREB transactivation is assessed by transfection assays of GSK-3p encodingmammalian expression vectors in the undifferentiated F9 embryonal carcinoma cell line. The undifferentiated F9 cell line, UF9, has been used by others (6, 18) as a model system for assessing the transcriptional involvement of the CREB protein in the CAMPresponse. It has been demonstrated (6) that thecAMP-unresponsiveness of CRE-dependent promoters in F9 cells is overcome by overexpression of exogenous CREB and the catalytic subunit of PKA. The studies by Masson et al. (19) demonstrated that theCAMP refractory nature of the UF9 cells is not due to the absence or reduced levels of known positive-acting factors, such as CREB or PKA. In the present study, we employed the undiff~rentiated F9 cell line as a model system to examine if the GSK-38 kinase is an additional positive factor in the CAMP transduction pathway. EXPERIMENTALPROCEDURES Peptides-CREB peptidewas synthesized using the solid phase method with an Applied Biosystems Model431A machine run with the small scale (0.1 mmol) t-Boc program. Purification of the crude peptide was by reverse phase chromatography using Aquapore C8 semi-preparative (1 x 10 cm) cartridge columns(Brownlee Laboratories). Sequences wereconfirmed using a Porton Instruments model2090 microsequencer. Peptide Phosphorylation-The CREB peptide (100 p ~was ) incubated with PKA (3.6 pg/ml), GSK-3a (3 pg/ml) or GSK-3P (8 pg/ml), or the sequential combination of PKA and either isoform of GSK-3 in a 50-pl reaction volume containing C-Y-~~PJATP (500 c p ~ p m o l )Other . reaction conditions were as described in Ref. 1. Peptide phosphorylation was analyzed by isoelectric focusing with a modified, more basic gradient (pH 3.5-9.5) as described in Ref. 1. The peptides werelocalized by autoradiography; for quantitation, the phosphopeptides were excised from the gel, and the radioactivity was measured. The locations of phosphorylated residues were identified according to the method described in Ref. 20. phosphorylated peptide was purified with SEPAK C18 cartridges and reacted (1-nmol samples) with ethanethiol for 1h at 50 "C. This reaction converts the phosphoserines to S-ethylcysteines, which could then be identified by sequencing using a Porton Instruments microsequencer model 2090.

Y. Wang, D. Brahmi, and F? J. Roach, unpublished results.

In Vitro P ~ o s p h o r y ~ t i oofn CREB Proteins-CREB proteins were phosphorylated by PKA (3.6 pg/mlf, GSK-30 (3 pg/ml), or GSK-38 (8 pglmlf, i n ~ v i d u a l ~oryin combination. The reaction mixture contained 42 m~ Tris-HC1, pH 7.5, 1.4 mM [y32PlATP(1000 cpndpmol), 8.6 mM Mg(CH,COOf,. The labeled proteins were locatedby autoradiography of SDS-polyacrylamide gel separations. Quantitation was carried out by excising the bands and dissolving the gel in 30% hydrogen peroxideat 60 "C followed by liquid scintillation counting. Enzymes-Rabbit skeletal muscle GSK-3a was purifiedas described in Ref. 21. Using antibodies raised to a synthetic peptide common to both isoforms (a gift from Dr. John C. Lawrence, Washington University), we identified a single immunoreactive species of 57 kDa. We conclude that the enzyme purified from rabbit skeletal muscle was predominantly GSK-3a Rabbit skeletal muscle GSK-3P was expressed and purified from Escherichia coli (221. Homogenous bovine cyclic Ah4Pdependent protein kinase catalytic subunit was the gift of Dr. Edwin G. Krebs (U~versityof Washington). Expression and Ptlrification of R e c o ~ b i n a ~ CREB-Wild t type or mutant forms of CREB127'3*1 proteins were expressed in bacteria via the T , 4 1 2 1 Lys S(DE3) system (23, 241. Purification of the recombinant protein was carried out as described in Colbran et al. (251. Construction of Ser -3 Ala CREB Mutants-Site-directed Ser --f Ala substitutions were constructed a t the GSK-3 consensus phosphorylation sites, Ser"' and SerlZ9of CREBSZ7 and CREB341, respectively, employing the site-directed mutagenesis protocol of Olsen and Eckstein (26). The introduced mutation was verified by dideoxy sequencing. CREB-dependent Assay System-The indicator plasmid contains the somatostatin promoter(-750 to +50) fused to the chloramphenicol acetyltransferase (CAT) reporter gene (27). The CRE site atnucleotide position -43 has been replaced by the insertion of five Gal4 DNA binding sites. Specifically, the PstI site of the pBxSSTb4 construct, described in Andrisani et al. (27), was used to insert the synthetic Gal4 DNA binding site. The resulting indicator plasmid is pBxSS~Gal4)CAT.The CREB expressor vector used is described by Berkowitz and Gilman (28) and was kindly provided by Dr. M. Gilman (Cold Spring Harbor Laboratory). In thisvector, the Ga141"47 DNAbinding domain is fused at the NH,-terminal end of CREB. The resulting Gal4-CREB fusion proteins are functional, as demonstrated in earlier studies (281. Dansfection of PC12 Cells-Expressor plasmid (5 pg) was transfected with indicator plasmid (10 pg) in PC12 cells by the Ca(PO,), coprecipitation method employing the Life Technologies, Inc. transfection kit. PC12 cells were grownin Dulbecco's modified Eagle's medium containing heat-inactivated horse serum t 10%) and heat-inactivated fetal bovine serum (5%) on 100-mmtissue culture dishes coated with rat collagen. PC12 cells were transfected at passage 18. 48 h following introduction of the DNA, the cells were harvested, and extracts were prepared in the lysis buffer described in Ref. 29, which yields higher CAT extract activity. Cellular extract (25 pg) was assayed for CAT enzyme activity for 30min a t 37 "C as previously described(301. In uiuo metabolic labeling of transfected PC12 cells, with 135Slmethionine, was carried out by employing the protocol described by Lee et al. (7). Immunoprecipitation reactions with Gal4 antibodies were carried out as described (7). TheGal4 antibody was kindly provided byDr.M. Ptashne. Zkansfection of UndifferentiatedF9 Cells-Undifferentiated F9 cells were maintained as monolayer cultures on 100-mmdishes, coated with 0.1%gelatin, in Dulbecco's modifiedEagle's mediumsupplemented with 15%heat-inactivated fetal bovine serum. Subconfluent (90%)monolayers were passaged 1:30 the day before the transfection. Transfections were carried out via the CaPO, coprecipitation method, employing the Life Technologies, Inc. transfection kit. The Gal4 programmed transfection assay system utilized 8 pg of indicator plasmid pBxSSTfGal4)CAT, 5 pg of expressor Ga14-CREB32"~1,and 10 pg of CMV4-GSK-38 plasmid DNA. The amount of total DNA transfected was kept constant by addition of CMV4 vector DNA. The cells were harvested 48 h posttransfection following 20 p~ forskolin stimulation. Cell extracts were prepared in the lysis buffer described in Ref. 29 and assayed for CAT enzyme activity for 2 h at 37 "C as described earlier (30). The pBxSST transfections employing the endogenous CREBwere carried out with 5-8 pg of pBxSST indicator plasmid, 1 pg of CbfY4PKA (catalytic subunit), and10 pg of CMV4-GSK-3P expressor plasmid DNA. Cells were harvested 24 h post-infection. Cellular extracts were prepared and assayed as described above. RESULTS

In Vitro P ~ ~ ~ p h o ~of~ CREB-To u t ~ o n examine if the transcription factor CREB is a substrate for the GSK-3 protein

The Role of a Secondary Phosphorylation of CREB341at SerI2'

MW

A

CREB

Transactivation domain a peptide P-box

N

P

88-102

/

c- 116 c- 94

/ /

/ /

c .67 +- 53

2

3

4

5

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c-

/ /

" \

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/ /

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30

6

MW C

170

116 94 t 76 c 67 C

c 53

CREB

"c

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\\

GSK-3

B

C

284-341

\ \

/

c 43

1

B

\

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C. 76

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DNA-bindIng domain leucinezi r

P

106160

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-

32189

1 2 3 4

FIG.2. Mapping of the GSK-3phosphorylation site in CREB. Schematic diagram ofthe GSK-3 phosphorylation site within the CREB transactivation domain. The GSK-3 consensus sequence sxxrIS(P)is formeduponprimaryphosphorylationbyPKA.Theautoradiogram shows the isoelectric focusing analysis ofCREBpeptide (amino acid sequence shown) phosphorylated by GSK-3a (3 pg/pl) in lune 2, by PKA (3.6 pg/pI) in lune 3, and by PKA and GSK-3a synergistically in lune 4, using [Y-~~PIATPdescribed as under "Experimental Procedures."Lune 1, phosphorylation of the kinases without added CREB peptide.

+ 43

phosphorylated by PKA alone or in combination with GSK-3 was reacted with ethanethiol and subjected to sequence analysis. The sequencing pattern shown in Fig. 3 is consistent with a phosphate being present only on Ser119/133 after phosphoryla1 2 3 4 5 6 tion with PKA (panel B ) and with phosphate also present at FIG.1. Synergistic phosphorylationof CREB. Purified recombi- SerllS/129 after phosphorylation with GSK-3 (panel C).Thus, we nant CREB proteins phosphorylated with PKA (3.6 pg/pl), GSK-3a (3 were confident that we had mapped the GSK-3 site toSer"5/'29 pg/pl), or GSK-3P and their combination. A, in vitro phosphorylation of CREB. with GSK-3u; B, in vitro phosphorylationwith GSK-3P. Lanes 2-4 show To confirm the identification of the phosphorylation site in phosphorylation of wild type CREB"j7 by GSK-3 alone, PKA alone, and a combination of PKA and GSK-3. Lanes 5 and 6 show phosphorylation the intact native CREB protein, we carried out site-directed of mutant CREB(Ala'33)by PKA or the PKA-GSK-3 combination, re- mutagenesis of Ser""'29 to Ala, as shown in Table I. Ser"'/'29 is spectively.Lane 1 is the autophosphorylationof PKAand GSK-3 with no the putativephosphorylation site for GSK-3, based on the pepCREB protein added. tide phosphorylation studies. The mutant proteins were exkinase, recombinant CREB327protein was used in in vitro en- pressed in bacteria,purified, and utilized for in vitro phosphozymatic reactions employing PKA and eitherof the two GSK-3 rylation reactions(Fig. 4). Controls for theseexperiments isoforms, GSK-3a or GSK-3P. The CREB327proteins were phos- include the wild type CREB327protein (lanes 1 4 ) and the phorylated in vitro by PKA to a stoichiometry of 0.6 (Fig. 1, A CREB proteins that contain Ser"' Ala substitutions at the and B, lanes 3 ) .A substitution of Ser1Igby an alanine inCREB PKA phosphorylation site (lanes 5 and 6 ) . Lane 7 in panel A abolished PKA phosphorylation, confirming that Ser1Igis the shows that the mutantCREB327with the Ala"' substitution is only phosphorylation site for CAMP-dependent protein kinase a substrate for PKA with a stoichiometry essentially equal to (Fig. 1, A and B,lanes 5).GSK-3a (Fig. lA, lane 2 ) or GSK-3P that of wild type CREB327shown in lane 3. Unlike the native (Fig. lB, lane 2 ) alone did not phosphorylate CREB. However, CREB327(lane 4 ) , mutant CREB did not become a substrate for if CREB was previously phosphorylated by PKA at Ser1Ig, either GSK-3a (panel A, lune 8)or GSK-3P (data not shown) GSK-3a or GSK-3P could stoichiometrically introduce another after phosphorylation by PKA. This resultconfirms that Ser"' phosphate (Fig. 1, A and B, respectively, lanes 4 ) . The S e P g was the site for GSK-3 phosphorylation in vitro. The same mutant was unable to act as a substrate for either isoform of results were obtained withmutant CREB341protein containing GSK-3 (lane 6 ) . Thus, the in vitro phosphorylation reactions SerIz94Ala substitution, employing GSK-3P (panel B, lane 8) shown in Fig. 1demonstrate thatCREB can be phosphorylated or GSK-3a (data not shown). by either isoform of GSK-3 but only after primary phosphorylI)-ansactivation Propertiesof GSK-3 Site MutantCREB327'34' ation by PKA. Proteins-"he role of this secondary phosphorylation reaction Mapping of Phosphorylation Sites in CREB-To map the of CREB in its transactivation response to CAMPwas examined GSK-3 phosphorylation sites in CREB, a peptide was synthe- by in vivo functional assays in the well documented CAMPsized with sequence as shown in Fig. 2. The CREB peptide was responsive PC12 cell line (17). The transcriptional activity of stoichiometrically phosphorylated bycyclic AMP-dependent the wild type CREB327/341 protein was compared with the activphosphorylated by ity of the CREB protein mutants containing Ser -+ Ala substiprotein kinase (lane 3 ) , but it was not GSK-3 (lane 2). Once the CREB peptide had been phosphoryl- tutions at the GSK-3 phosphorylation site. ated by PKA, it became a substrate for both GSK-3a and Theassay systemused has been previously reported by Gsk-3preproducing the synergisticphosphorylation of the Berkowitz and Gilman (281, Lee et al. (7) and Shenget al. (31). CREB protein consistent with previous results (16). Stoichio- The DNAbinding domainof CREB is reprogrammed, via fusion metric phosphorylation by GSK-3a of the monophosphopeptide to theheterologous Ga141"47 DNAbinding domain. A schematic produced by PKA is shown in lane 4 of Fig. 2. CREB peptide diagram of the vector is shown in Fig. 5A. A diagram of the I-

30

--f

The Role of a Secondary Phosphorylation of CREB341at Ser12'

32190

-

A

ww

6 170

116 94 76 c 67 C

-

CREE

C

C

C

1

2

3

4

5

6

7

53

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30

8

Mw + 170

B

c 116

c 94 c 76 c 67

CREB

c 53 c 43

-b

30 5

10

15

20

TIME (mln)

FIG.3. Determination of phosphorylationsites in CREB peptide. Phosphorylated residues identified by amino acid sequence analysis, aftermodification of the phosphoserines with ethanethiol, is shown. A, elution profile of the phenylthiohydantoin-serine of the unphosphorylated peptide.B, disappearance of phenylthiohydantoin-serine and appearance of S-ethylcysteine (S-ET-Cys) a t cycle 11,which corresponds to phosphorylation of Ser1Ig. The sequenced peptide is phosphorylated by the CAMP-dependent protein kinase.C , appearanceof S-ethylcysteine a t cycle 7, which corresponds toSer115.The sequenced peptideis phosphorylated by GSK-3 and CAMP-dependent protein kinases. Automated Edman degradation was performed ainPorton instrument1090 microsequencer. DTT, dithiothreitol; DPTU, diphenylthiourea.

1

2

3

4

5

6

7

8

FIG.4. Mapping of the GSK-3site in the CREB proteins. Purified recombinant wild type CREB327phosphorylated using GSK-3 (3 &PI), PKA (3.6 pg/pl),or their combination (lanes 2 4 ,respectively) is shown. A, lanes 5 and 6 show the phosphorylation of mutant CREB32'(Ala119) by PKA or the PKA-GSK-3a combination, respectively. Lanes 7 and 8 show the phosphorylation of mutant CREB3"(Ala1") by PKA and the PKA-GSKa combination, respectively. B , lanes 5 and 6 show the phosphorylation of mutant CREB"41(Ala133) by GSK-3P. Lanes 6 and 7 show the phosphorylation of mutant CREB341(Ala129) by PKA and the PKA-GSK-3P combination, respectively.

CREB. Lanes 1 and 2 show the level of transactivation obtained with the wild type Ga14-CREB327and Ga14-CREB341,respectively, in response to forskolin. As expected, the Ser + Ala substitutions within thePKA phosphorylation site inCREB341 TABLE I Mutagenesis of CREB327'34'Proteins (lane 3 ) and CREB327(lane 4 ) are transcriptionally inactive in The constructed site-directed Ser+ Ala mutants are listed below. response to CAMP, in agreement with earlier studies (6, 27). The Ser+ Ala substitution within the GSK-3 phosphorylation GSK-3site mutants PKA site mutants site in CREB341(lane 5) also renders the CREB protein transcriptionally inactive. However, in CREB327,the Ser"' -+ Ala mutation at theGSK-3 site reduces its transcriptional response to CAMPinduction, on average, to 30% of its wild type activity. The histogram in Fig. 5C shows the quantitation of three inindicator plasmid used in the above CREB-dependent assay dependent transfection assays in PC12 cells. system isalso shown in Fig. 5A. The indicatorplasmid contains The in vivo synthesis and stabilityof the transfected Ga14the rat somatostatin promoter, spanning the sequences be- CREB proteins was confirmed by in vivo metabolic labeling tween nucleotideposition -750 to +50.The CRE site at position studies, employing [35S]methionineas shown in Fig. 6, A and B . -43 of the promoter is replaced by the Gal4DNA binding site. The radiolabeled proteins were immunoprecipitatedwith Ga14Thus, this Gal4-CAT indicator plasmid maintains the native specific antibodies. Because we have encountered difficulties in arrangement of the somatostatin promoter as opposed to a transfecting the PC12 cell line (Fig. 6A), we carried out additional in vivo labeling assays in HeLa cells (Fig. 6B). The reminimal GalCdriven promoter. The wild type CREB327'341 and the Ser+. Ala mutants shown sults of the immunoprecipitation reactions confirm the expresin Table I were also cloned into the vector shown in Fig. 5A. sion of the CREB341proteins as shown in lanes 1 and 2. The Following transfection inPC12 cells and forskolin stimulation, control immunoprecipitation reaction shown in Fig. 6B, lane 3, thetranscriptional activity of the GSK-3 sitemutants of employing extracts of 3sS-labeled cells transfected with vector CREB327/341 was compared with the activity of the wild type lacking the Gal4-CREB cDNA insert, confirms the specificity of CREB327'341 proteins. In addition, the inactive PKAsite mutants the Gal4 antibody and supports that theimmunoprecipitated of CREB327/341 were also transfected in parallel and usedas a proteins shown in Fig. 6 correspond to the Gal4-CREB fusion. negative control. An additional negative control in the transDansfection of GSK-3P Encoding Plasmids in UF9 Cellsfection assays included an expression vector encoding only the The undifferentiated F9 cell line is refractory to CAMPinducGa141-147 DNAbinding domain and lacking an activation do- tion of the CRE-dependent promoters of somatostatin and vamain. The resultsof the transient transfection CAT assays are soactive intestinal peptide (19), although the known positive functional (19). Overshown in Fig. 5B.The percentconversion of chloramphenicol to effectors CREB and PKA are present and its acetylated form is indicative of the transcriptionalactivity of expression of exogenous CREB and PKA compensates for the

The Role of a Secondary Phosphorylation of CREB341at Ser129 A

Gal4

kd

Gel4 Bindingsites I4

CAAT

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98+I TATAA I

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1

2

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,

CAT

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kd 1 98 -

2

3

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4hp deletion of CRE sile

Somatostatin promoter

B

A

pBySST(Ga14)CATIndicator Plasmid

32191

Expressor Plasmid " . I

B %conversion 99 1

27 2

5.6 3

1.7 4

1.6 5

8.5 6

1.6 7

FIG.6. Immunoprecipitations of in vivo S6SS-labeled Gal4-CREB proteins. A, immunoprecipitations of 36SS-labeledextracts prepared from PC12 cells transfected with wild type Gal4-CREBY4'expressor (lane I ) , GSK-3 mutant Gal4-CREB"' (lane 2), and GSK-3 mutant Ga14-CREB327(lune 3 ) . B, immunoprecipitation of 35S-labeledextracts prepared from HeLa cells transfected with wild type Gal4-CREB3'I expressor (lune 1), GSK-3 mutant Ga14-CREB341(lune 2), and vector DNA lacking the Ga14-CREB"41insert. Analysis of the immunoprecipitates in A and B is by SDS-polyacrylamide gel electrophoresis and autoradiography. Autoradiography was carried out at -80 "C for 3 days. Sizes of molecular mass markers are shown; urrow denotes thespecific Gal4-CREB band.

endogenous positive-acting factors of the cAMP transduction pathway. We have employed the UF9 cell line to examine if GSK-3 kinase activity is an additional positive factor required for the CAMP transduction pathway. We have carried outtransfection 100 assays of the mammalian expression vector CMV4, encoding the GSK-3P kinase, to assess the effect on transcription directed from the CREKREB-dependent promoter of the rat so80 matostatin gene. Initially, we employed the Gal4-CREB-dependent assay system described earlier inFig. 5. The pBxSST(Gal4)CATindicator plasmid (Fig. 5A) and the Gal4-CREB expressor (Fig. 5 4 ) were cotransfected in thepresence of a CMV4 expressor vector Iencoding the GSK-3P isoform. The transfection assays were < carried out in the presence of 20 PM forskolin. The histogram 0 40 activity shown in Fig. 7 demonstrates that the transcriptional of CREB341is induced between 5-25-fold in thepresence of the cotransfected GSK-3P kinase. Similarly, CREB327activity is 20 induced approximately 10-foldby the cotransfected GSK-3P --.f Ala substitution at the kinase. In contrast, the Ser*15/129 GSK-3 site of CREB327R41 displayed an approximately 2-fold 0 activation in the presence of cotransfected GSK-3P plasmid, 1 2 3 4 5 6 7 suggesting the importance of the GSK-3 phosphorylation on FIG.5. Gal4-CREB-dependentassays in PC12 cells.A, schematic CREB-dependent transcriptionandthe CAMP transduction diagrams of the pBxSST(Gal4)CAT indicator plasmid and the CREB expressor, described by Berkowitz and Gilman (28), used in the CREB- pathway. The histograms of the independent transfection asdependent assay system.B , functional assaysof CREB and its mutants says depicted in Fig. 7 show some variability in the level of in PC12 cells. Transient transfections cells ofPCl2 were carried out withtranscriptional induction of CREB341by the cotransfected 10 pg of indicator and 5 pg of expressor DNA in thepresence of 20 p~ GSK-3P kinase; this variation is most likely due to differences forskolin. Cells were harvested 48 h later,and extracts were prepared. 25 pgof cellular extract was assayed at 37 "C for 30 min. Expressor DNA in thetransfection efficiency of the UF9 cell line. However the overall inducible is as follows: lune 1, wild type Ga14-CREB341;lune 2, wild type Ga14- results of these experiments demonstrate the CREB327;lune 3, PKAmutant Gal4-CREB3'I; lane 4, PKA mutant Ga14- effect of the GSK-3P kinase on CREB transactivation. CREB"'; lane 5,GSK-3 mutant Ga14-CREB341;lune 6,GSK-3 mutant To further demonstrate thatGSK-3P is a positive effector in Ga14-CREB3"; and lune 7, Ga14l-I4' DNAbinding domain. Percentchloramphenicol conversion to the acetylatedform is shown above each lane. the CAMP transduction pathway, we examined in UF9 cells C , histograms show the quantitation of three independent transfection whether exogenously transfected GSK-3P kinase could comassays in PC12 cells. Percent CAT activity is plotted against each ex- pensate for the overexpression of exogenous CREB and PKA pressor plasmid tested. Eachbur is one independentassay. Groups 1-7 required to obtain transcription from the CRE-dependent socorrespond tothe expressor plasmid testeda s follows: 1, wild type Ga14matostatin promoter. We carried out transient expression asCREB?"; 2, wild type Ga14-CREB327;3, PKA mutant Gal4-CREB3'I; 4, PKA mutant Gal4-CREB"'; 5, GSK-3 mutant Ga14-CREB3"; 6, GSK-3 says employing the pBxSST reporter plasmid (271, which conmutant Ga14-CREB"'; and 7, Gall-"' DNA binding domain. tains the rat somatostatin promoter (+50 to -750) in frontof the CAT gene. This CRE/CREB-dependent reporter plasmid was with the expression plasmid encoding the lack of CRE-dependent induction by the endogenous cAMP ef- cotransfected fector molecules (6). This observation has been interpreted to GSK-3P kinase. In the resultsshown in Fig. 8, the CREB promean (19) that negative regulators block the activity of the tein mediating the transcriptionalresponse is the endogenous

c

The Role of a Secondary Phosphorylation of CREB34’at SerI2’

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FIG.7. Gall-CREB-dependent assays in undifferentiated F9 cells. Histograms show the quantitationof independent transfections in UF9 cells, employing the Gal4-CREB assay systema s a function of GSK-3P kinase co-expression. Schematic diagramsof the indicator and expressor plasmids are shown in Fig. 5A. Transfections contained 8 pg of pBxSST(Gal4)CAT indicator, 5 pg of Ga14-CREB”27’341 expressor, and where indicated,10 pg of CMV4-GSK-3P plasmid DNA or CMV4 vector DNA. The transfected cells were treated with20 UM forskolin for 48h.

CREB; the catalyticPKA activity was obtained either by transfection of an expression vector encoding the catalytic subunit of the PKA enzyme (Fig. 8 A ) or via forskolin stimulation of UF9 cells (Fig. 8B).An approximately 60-fold induction in the transcriptional activity of the somatostatinpromoter mediated via the endogenous CREB is observed in the presence of cotransfected GSK-3P kinase. The cotransfected GSK-3 kinase requires the presence of the catalytic subunitof PKA for CREB transactivation in agreement with thewell documented mode of action of this class of enzymes. In agreement with earlier studies (6, 191, overexpression of the catalytic subunitof PKA without overexpression of exogenous CREB is notsufficient for CRE-dependent transcriptional induction in UF9cells. The results shown in Fig. 8 support thepositive effector role of GSK-3 type kinases in theCAMPtransduction pathway.

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FIG.8. cAIW induction of the somatostatin promoter in UF9 cells by overexpression of GSK-3/3 kinase. A, transfections in UF9 cells contained 8 pg of the somatostatin gene reporter plasmid pBxSST (27), 1 pg of CMV4-PKA (catalytic subunit) expressor, and, where indicated, 10 pg of CMV4-GSK-3P or CMV4 vector DNA. Cells were harvested 24 h post-transfection, and CAT assays were carried out as described in Ref. 30. The percent conversion to the acetylated chloramphenicol is indicated above each lune. B , histograms show the quantitation oftwo independent transfection assays incells, UF9as a function of GSK-3P co-expression, in thepresence of cotransfected catalytic subunit of PKA or 20 PM forskolin addition.

for the activation of CREB327’341.However, the PKA phosphorylation is not sufficient sincewe demonstrated thatablation of DISCUSSION the secondary phosphorylationsite of CREB327”41, Ser115’129, imThis study provides evidence in vitro and in vivo to support paired the transactivating function of the protein. Furthera role for hierarchical phosphorylation in the regulationof the more, analysis of the UF9 cell system directly demonstrates transactivation properties of CREB. The amino acid residues that GSK-3P is a positive effector of the CAMPtransduction 92-137 are critical for the transcriptional activation of CREB pathway. Transfection of the GSK-3P expression vector results in response to CAMP induction (7). This domain of CREB is in a 60-fold induction in transcription of the CRE-dependent serine-rich and hasbeen shown to be multiply phosphorylated promoter, employing the available endogenous CREB. This in addition to thephosphorylation by PKA. We therefore tested transcriptional induction requires thepresence of the catalytic the possibility of hierarchical phosphorylation reactions occur- PKA subunit. The results of these two types of in vivo experiring within CREB327’341 in the regulationof its transcriptional ments in PC12 cells (Fig. 5) and UF9 cells (Fig. 8) are interresponse to CAMP. A synthetic CREB peptide and site-directed preted to mean thattwo phosphorylations must occur to genCREB mutants were usedto map thesecondary CAMP-depend- erate a fully activated form of CREB. This is consistent with ent phosphorylation to Ser*15‘129 within theactivation domainof the proposal that PKA phosphorylation is notsufficient for full CREB327’341. The in vitro data strongly suggested that the pri- CREB activation. Overexpression of exogenous CREB and PKA mary kinase is PKA and the secondary kinase is one or both is required for detectable CRE-dependent transcription inUF9 isoforms of GSK-3, though it is not certain which kinase(s) cells, which compensate for thepartial activation state of carry out the sequentialphosphorylations in vivo. Our results CREB. The in vivo data support theconcept that a secondary phosfrom the in vivotransfection assays PC12 in cells are consistent with the conclusion that phosphorylation by PKA is essential phorylation at Ser12’ is an importantcomponent of the trans-

The Role of a Secondary Phosphorylation of CREB341at Ser129 activation response of CREB and suggest a mechanism for integration of signals from different signal transduction pathways. Evidence for CREB beinginvolved in thecross-talk between different signaling pathways has appeared (31). There are reports of CREB341functioning as a Ca2+-regulated transcription factor through thephosphorylation of Ser'33 (31).It is also possible that a secondary phosphorylation, dependent on primary phosphorylation of Ser'33, mediates the response to Ca2+ signal since mutation of Ser133would destroy thephosphorylation at the secondary site aswell. activity At the moment,it is not clear what may regulate the of GSK-3 in the cell. One possibility is that GSK-3 activity is on the level of substrate maintainedby constant and dependent primary kinases responding to several different signal transduction pathways. There is evidence that suggests thatGSK-3 must be phosphorylated on a tyrosine residuet o be in a n active state (32). Another report suggests that one form of GSK-3, GSK-3P, may be a target for protein kinase C phosphorylation resulting in down-regulation (33). It has been proposed that a down-regulation of GSK-3 activity by protein kinase C in response to phorbol esters results in a reduced level of c-Jun phosphorylation and subsequent stimulation of c-Jun binding to DNA. Though there is no evidence for inactivation of CREB upon 12-0-tetradecanoylphorbol-13-acetatetreatment,such down-regulation of GSK-3P activity would also implya possible role for protein kinaseC inactivation of CREB341via a decrease in phosphorylation of SerlZ9by GSK-3. Based on these models, GSK-3 would appear to have opposing roles in theexpression of CRE or TRE containing genes. It is important to note that presently it is not known which cellular isoform of GSK-3 carries out thephosphorylation of Ser'29, and GSK-3a appears not to bedown-regulated by protein kinaseC phosphorylation.This isconsistentwiththe observation that12-0-tetradecanoylphorbol-13-acetate treatment does not influence CRE-mediated stimulation of transcription (7). More recent reports (34, 35) have shown that phosphorylation of GSK-3a or GSK-3P by mitogen-activated proteinkinase-activatedproteinkinase-1 (34) and p70 S6 kinase (35) results in almostcomplete inactivation. Thus, GSK-3 has been implied as a target for inactivation through the insulin response pathway. This inactivationby insulin would provide a mechanism to antagonize CAMP-dependentgene expression. A secondary phosphorylation of CREB341at SerlZ9 potentiatedby the primary phosphorylation of Ser133by PKA appears to be necessary to fully evoke the changeintranscriptional function of CREB341.Another example in which GSK-3 has been shown to act synergistically with CAMP-dependent protein kinase is in the phosphorylation of two sites in the glycogen binding subunit of type 1 protein phosphatase (11).Judging from these two examples, one can postulate that the sequence SRR(G/P)S is a consensus for a coupled pair of P W G S K - 3 sites. At present, the only enzymes known to have the appropriate specificity, for the sequence

32 193

motif sxxXS(P) areenzymes designated GSK-3 as previously discussed. This work strongly suggests a role for GSK-3 or a related family of acidotropic kinases in the control of transcriptional events through hierarchical protein phosphorylations, in particular the expression of genes responding to changes in CAMPlevels. REFERENCES 1. Fiol, C. J.,Mahrenholz, A. M., Wang, Y., Roeske, R. W., and Roach, P. J. (1987) J. Biol. Chem. 262, 14042-14048 2. Meek, D. W., and Street, A. J. (1992) Biochem. J. 287, 1-15 3. Montminy, M. R., and Bilezikjian, L. M. (1987) Nature 328, 175-178 4. Andrisani, 0.M., Zhu, Z., Pot, D. A,, and Dixon, J. E. (1989) Proc. Natl. Acad. Sci. U. S. A . 86, 2181-2185 5. Yamamoto, K. K., Gonzalez, G.A,, Biggs, W. H., 111, and Montminy, M. R. (1988) Nature 334,494-498 6. Gonzalez, G. A., and Montminy, M. R. (1989) Cell 69,675-680 7. Lee, C. Q., Yun, Y., Hoeffler, J. P., and Habener, J. F. (1990) EMBO J. 9, 4455-4465 8. Roach, P. J. (1991) J. Biol. Chem. 266, 14139-14142 9. Flotow, H., Graves, P. R., Wang, A,, Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 266, 14264-14269 10. Ramakrishna, S., DAngelo, G., and Benjamin, W. B. (1990) Biochemistry 29, 7617-7624 11. Fiol, C. J., Haseman, J. H., Wang, Y., Roach, P. J., Roeske, R. W.,Kowalezuk, M., and DePaoli-Roach, A. A. (1988)Arch. Biochem. Biophys. 267,797402 12. Park, I. IC, and DePaoli-Roach, A. A. (1991) FASEB J. 5, (Abstr. 26871, 833 13. Woodgett, J. R. (1990) EMBO J. 9, 2431-2438 14. Puziss, J. W., Hardy, T. A,, Johnson, R. B., Roach, P. J., and Hieter, P. (1994) Mol. Cell. Biol. 14, 831-839 15. Ruel, L., Bourouis,M., Heitzler, P., Pantesco, V., and Simpson,P. (19921Nature 362,557-559 16. Wang, Q. M., Roach, P. J., and Fiol, C. J. (1994)Anal. Biochem. 220,397-402 17. Montminy, M. R., Sevarino, K. A,, Wagner, J. A., Mandel, G., and Goodman, R. H.(1986) Proc. Natl. Acad. Sci. U.S. A . 83,6682-6686 18. Quinn, P. (1993) J . Biol. Chem. 268, 16999-17009 19. Masson, N., Ellis, M., Goodbourn, S., and Lee, K. (1992) Mol. Cell. Biol. 12, 1096-1106 20. Meyer, H. E., Hoffmann-Posorske, E., Korte, H., and Heilmeyer, L. M. G., Jr. (1986) FEES Lett. 204, 61-66 21. Fiol, C. J . , Wang, A,, Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 266, 60614065 22. Wang, Q . M., Fiol, C. J., De Paoli-Roach, A., and Roach, P. J. (1994) J. Biol. Chem. 269,14566-14574 23. Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078 24. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130 25. Colbran, J. L., Roach, P. J., Fiol, C. J., Dixon, J. E., Andrisani, 0.M., and Corbin, J. D. (1992) Biochem. Cell Biol. 70, 1277-1279 26. Olsen, D. B., and Eckstein, F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 14511455 27. Andrisani, 0.M., Hayes, T.E., Roos, B., and Dixon, J. E. (1987) Nucleic Acids Res. 15, 57154728 28. Berkowitz, L. A., and Gilman, M. Z. (1990) Proc. Natl. Acad. Sci. U. S. A . 87, 5258-5262 29. Pothier, F.,Quellet, M., Julien, J. P., and Guerin, S. (1992) DNA Cell Biol. 11, 83-90 30. Gorman, C. M., Moffat, L. E, and Howard, B. H. (1982) Mol. Cell. Biol. 2, 104P1051 31. Sheng, M., Thompson, M. A., and Greenberg, M. E. (1991) Science 262,14271430 32. Hughes, K., Nikolakaki, E., Plyte, S. E., Totty, N. F., and Woodgett, J . R. (1993) EMBO J. 12. 803-808 33. Plyte, S. E., Hughes, K., Nikolakaki, E., Pulverer, B. J., and Woodgett, J. R. (1992)Biochim. Biophys. Acta 1114, 147-162 34. Sutherland, C., Leighton, I. A,, and Cohen, P. (19931 Biochem. J. 296, 15-19 35. Sutherland, C., and Cohen, P. (1994) FEES Lett. 338, 37-42