binding domains, site A and site B, which can be distinguished by their affinity for cAMP analogs and kinetic properties. Analogs modified at the N-6 position bind ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 264,No. 28, Issue of October 5, pp. 16672-16678,1989 Printed in U.S.A.
Functional Characterizationof CAMP-binding Mutations in Type I Protein Kinase* (Received for publication, June 19,1989)
Leslay A. CorrellS, Terry A. WoodfordQ, Jackie D. Corbins, Pamela L. Mellonll, and G. Stanley McKnightSII From the #Departmentof Pharmacology, University of Washington, Seattle, Washington 98195, the §Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee37232, and TThe Salk Institute, La Jolla, California 92037
A mutant formof the typeI regulatory subunit (RI) function (21, secretion (3), and enzyme activation (4). Most of CAMP-dependentprotein kinase has been cloned and of the actions of cAMP are mediated by the activation of a sequenced (Clegg, C.H., Correll, L. A., Cadd, G.C., serinelthreonine CAMP-dependent protein kinase, which exand McKnight, G. S . (1987) J. Biol. Chem. 262, ists as a tetrameric complex of two regulatory subunits (R) 13111-131 19)which contains two point mutations inbound to two catalytic subunits (C). Holoenzyme activation the site B CAMP-binding site, a Gly to Asp at position occurs when cAMP binds to the regulatory subunits, causing 324 (B2) and an Arg to His at position 332 (Bl). In release of the catalytic subunits and the subsequent phosthis report, theeffect of each independent mutationon phorylation of target proteins. the rate of dissociation of cAMP from RI, the CAMPThe carboxyl-terminal region of R contains two CAMPmediated activationof holoenzyme and the inducibility binding domains, site A and siteB, which can be distinguished of CAMP-responsivegenes has been characterized. Dissociation of cAMP from either recombinant wild type by their affinity for cAMP analogs and kinetic properties. RI or the B1 mutant demonstrated biphasic kinetics, Analogs modified at the N-6 position bind preferentially at site A, whereas C-8 substituted analogs usually prefer site B indicating two sites with different affinitiesCAMP. for have established that dissociation of Dissociation from the B2 subunit, however, wasmon- (5).Kineticstudies site B had been cAMP from R is biphasic, occurring more rapidly from the A ophasic and very rapid indicating that destroyed and that the rate of dissociation from site A site (6, 7). It has been suggested that cAMP binds to both was increased. The cAMP activation constants (KO) of sites in a positively cooperative manner to activate the holothe wild type and B1 holoenzymes were 40 and 188 enzyme (8,9). The CAMP-binding domains of mammalian R subunits are nM, respectively, and demonstrated positive cooperativity, with Hill coefficients of 1.61 for the wild type strikingly homologous to theprocaryotic CAMP-binding proand 1.67 for B1. The B2 holoenzyme required much tein, catabolitegene activator protein(CAP),’ which regulates greater concentrationsof CAMP,4.7 PM, for half-max- the transcription of several genes involved in metabolism. imal activationand did not display positive cooperativThe structure of the CAMP-CAP complex has been deduced ity. Constitutive expression in mouse AtT2O pituitary from x-ray diffraction studies (10). Although the analogous R cells of the B1 mutant resulted in onlya small shift in subunit-CAMP complex has not been examined by x-ray the KO for kinase activation in these cells compared crystallography, based on homology with CAP, Weber et al. with B2 expression which increased the K , by more (11) have suggested apotential structure for the CAMPthan 100-fold. Transientexpression of the B1 subunit binding domain of the R subunit which predicts the amino in human JEG-3 choriocarcinoma cells inhibited for- acids involved in a CAMP-binding pocket. Also, the study of skolin activation of a CAMP-responsive promoter by R subunit mutants which prevent CAMP-binding at sites A 35%,whereas similar expressionof the B2 RI subunit or B has been useful in identifying important amino acids inhibited the response by 90%.These results suggest that the Gly to Asp mutation at amino acid 324 com- involved in cAMP binding. Recently Bubis et al. (12) showed pletely blocksCAMPbinding to site B whereas the Arg that by altering a highly conserved arginine residue, Arg-209, to His mutation at position 332 causes a more subtle cAMP binding is abolished at theA siteand thatsubstituting position 371 decreases the binding affinity alteration in cAMP binding. Expression of either mu- a Phe for the Tyr at tant RI in animal cells results ina dominant repression of cAMP and abolishes positive cooperativity of cAMP actiof CAMP-dependentprotein kinase activity and CAMP- vation between the A and B sites (13). Previously, we reported the cloning of RI cDNAs containing dependent protein kinase-mediatedprocesses. point mutations ineither site Aor B (14) isolated from CAMPresistant S49 lymphoma cell lines termed Ka mutants. These mutant cells produced equal amounts of wildtype and mutant RI proteins, yet required 5-20-fold greater concentrations of Cyclic AMP regulates a variety of intracellular events in cAMP to achieve half-maximal holoenzyme activation (15). animal cells including gene transcription (I), ion channel Peptide fragmentsof mutant RI proteins showed charge shifts compared with wild type RI peptides, and these differences by National Institutes of Health * This work was supported in part were used to map the location of the mutations (16). A cell HD23677 Research Grant GM32875 (to G. S. M.) and HD23818 and (to P. L. M.). The costsof publication of this articlewere defrayed in line containing a mutation in the site B CAMP-binding dopart by the payment of page charges. This article must thereforebe hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed.
’ The abbreviations used are: CAP, catabolite gene activator protein; HPLC, high performance liquid chromatography; bp, base pairs; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
16672
CAMP-binding Mutations in Mouse RI
16673
main was used to clone R:[ cDNAs for sequence analysis. TWO Bdb were expressed in Escherichia coli (DHI) and the Rf proteins as previously described (17). Briefly, the E. coli strain DH1 G to A transitions were found in the site B-coding sequence purified was transformed with the various RI expression vectors, ampicillinof the mutantRI. The resulting changes in the mutant protein resistant colonies selected, and expression verified by 8-N3[32P]~AMP are aGly-324 to Asp (B2): and Arg-332 to His (Bl), consistent photoaffinity labeling of bacterial protein extracts. Positive colonies with the negative charge shift observed in the RI proteins weregrown in 250 ml of L-broth containing 50pg/ml ampicillin overnight a t 37 "C and then subcultured into 1 liter of L-broth plus from this cell line. Gly-324 is invariant among the other CAMP-binding regions. However, Arg-332 is not, although it ampicillin. The trcpromoter was derepressed with the addition of 0.5 M isopropyl-B-D-thiogalactoside during the early exponential phase is located within a region of homology with other CAMP- m (A550= 0.2-0.25) of bacterial growth and theRI protein isolated from binding domains. bacterial cultures in the log phase of growth. Cells were lysed by In thispaper, we have separated and expressed the two site freeze/thaw in TEM (10 mM Tris-HC1, pH 7.4, 1 mM EDTA, 5 mM B amino acid substitutions in order to examine their individ- 2-mercaptoethanol) containing protease inhibitors, followed by one ual effects on cAMP dissociation, kinase activation, and the passage through a French pressure cell a t 15,000 psi and centrifugation at 5,000 X g for 20 min. RI protein was partially purified by ability to alterbiological responses in transfected cells. fractionation on a DEAE-Sephacel (Pharmacia LKB Biotechnology Inc.) ion exchange column and used to form holoenzyme with bovine Csubunitin the presence of magnesium acetate and ATP (17). Vectors-Previously, we described the construction of the eucar- Reconstituted CAMP-dependentprotein kinase was dialyzed against yotic expression vectors MT-REV and HL-REV, bothcontaining the TEM andpurified on DEAE-Sephacel. Fractions containing holoenoriginal site B mutant (Bdb) RI cDNA. We altered the RI sequence zyme wereidentified by CAMP-dependentprotein kinase activity and in this plasmid to contain the individual site B (either B1 or B2) pooled. HPLC gel filtration using a Zorbax GF250column (Du Pont) point mutations for studies in this paper. The Bdb and B2 cDNAs was performed to remove any contaminatingCsubunit, and the were originally cloned into pUC 12. The only restriction site between purified CAMP-dependent protein kinase was obtained in a single the two point mutations is .HinfI a site one base 5' of the B1mutation. fraction. cAMP Binding and DissociationAssay~"[~H]cAMP binding activAn 86-bp HinfIlPstI fragment was removedfrom Bdb containing the B2 sequence, and the wild type RI sequence was used to replace it ity was measured by a filtrationassay described by Sugden and Corbin (21). Samples containing 15-20 nM purified RI were incubated with giving the isolated B1 mutant. (specific P activity 25-30 Ci/mmol, ICN), Bacterial expression vectors were constructed as described by a mixture of 1~ M [ ~ H ] c A M Woodford (17) by placing the entireRI cDNA into the plasmid 50 mM potassium phosphate, pH 6.8, 1 mM EDTA, 0.5 mg/ml type pKK322-2 (Amersham Corp.), containing a fused trp-lac (trc) pro- IIA histone, and 2 M NaCl at 25 "C. After 45 min, 100p~ of unlabeled moter. The mutant B1 or B2 450-bp EcoRIIPstI fragment was then cAMP (Sigma) was added and aliquots filtered on type HA, 0.45 pm filters (Millipore) at theappropriate time points. substituted in place of the wild type sequence. isolation of Stable Clones Expressing Mutant RZ Genes-AtT2O The previously constructed HL-REV plasmid contained an RI cDNA with an altered site A-coding sequence. To construct the wild cells were transfected with 10 pg of HL-REVneo by CaPO, precipitype RIand the various siteB mutants, a 500-bp BglIIIEcoRI tation asmodified byParker and Stark(22). Four h afterthe addition of CaP04/DNA,cells were glycerol-shockedin HBS (20 mM HEPES, fragment containing the wild type RI sequence and the450-bp EcoRI/ PstI fragments previously sequenced from either wild type, B1, B2, 135 mM NaC1,5 mMKC1, 10 mM NazHPOI, pH 7, 55 mM glucose)/ or Bdb cDNA clones were substituted in the RI cDNA in HL-REV. 15%glycerol 3-4 min and placed in fresh media. Forty-eight h later, A 2.2-kilobase EcoRI/BamHl fragment containing a neomycin phos- the cells were split 1:lO into medium containing 500 pg/ml G-418 photransferase gene flanked by SV40 promoter sequences and poly- (GIBCO). Within 2-3 weeks, G-418 resistant colonies appeared and adenylation signal sequence3was removedfrom the plasmid pKOneo were subcloned. mRNA Expression-To show that cell lines were expressing the (18).The ends were filled in with Klenow and the fragment ligated to a blunt-ended Sal1 sitein, HL-REV, and HL-REVB to make HL- transfected RIgenes, RNA was prepared by digestion in 0.01 M NaAc, REV,neo and HL-REvBnt.0, respectively. To confirm that these pH 4.5, containing 0.5% sodium dodecyl sulfate, followed by phenol vectors contained the B2 point mutation, they were fixed to nitrocel- extraction, electrophoresed on formaldehyde gels, blotted to nitrocellulose, hybridized with a complementary 32P-end-labeledoligonucle- lulose as described by Uhler et al. (23), and probed with a nickotide (12-mer) probe, and washed off at various temperatures ranging translated 321-bp NcoIIStuI fragment from HBV 3"untranslated from 25 to 45 "C.The probe washed off the mutantsequence between region of the HL-REVneo vector. Molecules of RNA/cell were deter32-40 "C but remained hybridized to the wild type sequence. mined by solution hybridization as previously described (23). Each M T - R E V Bwas ~ ~ constructed from a plasmid containing the mouse RNA sample was hybridized in triplicate with a single-stranded 35SRI cDNA (MT-REV,) and contained the RI Bdb cDNA, flanked by labeled 321 nucleotide NcoIIStuI HBV cRNA (synthesized from an approximately 700 bp of the mouse metallothionein-1 (MT-1) pro- SP6 vector containing this fragment) at 68 "C for 16 h. Samples were moter sequence to regulate transcription and 630bp of the hGH digested with RNase A and T1 (Sigma) for 45 min at 37 "C. The polyadenylation signal sequence. A unique EcoRI site and ApaI site labeled hybrids were precipitated with 10% trichloroacetic acid, filare present within the siteB domain-coding sequence of the RI tered onto GFC filters (Whatman) whichwere then treated with cDNA. MT-REVB vectors containing only the B1 or B2 alterations Soluene 350 (Packard), and the radioactivity determined by liquid were constructed by subst:ituting the variant 284-bp EcoRIIApaI scintillation counting using Omnifluor/toluene (Du Pont-New Engfragment in place of the Bdb sequence in MT-REVB~~. confirm To land Nuclear). A standard curve was constructed by hybridization to the presence of the B1 or B2 mutations in MT-REVB, the 260-bp known quantities of the complementary 321-nucleotide NcoIIStuI PstIIPstI 3' fragment of the RI cDNAwas subcloned into the HBV single-stranded RNA. Molecules/cell were calculated based bacteriophage M13(mp19) and sequenced by the dideoxy method (19). upon the estimate of 6 pg of DNA/mouse cell (23). CellCulture-All cells were maintained in Dulbecco'smodified CAMP-dependent Protein Kinase Actiuity-Soluble protein exEagle's medium containing 100 units/ml penicillin G and 100 pg/ml tracts were prepared from cells by sonication in cell homogenization streptomycin sulfate (GIBCO). Cultures weregrown at 37"C in a buffer (10 mM NaPO,, pH 7.0, 1mM EDTA, 1mM dithiothreitol, 250 humidified incubator in an atmosphere of 90% air and 10% CO,. mM sucrose) and the particulate fraction removed by centrifugation Human JEG-3 choriocarcinoma cells were supplemented with 10% in a microcentrifuge at 4 "C for 15 min. Protein concentrations were newborn calf serum (GIBCO) and passaged by dissociation in Caz+, determined by Bradford assay (Bio-Rad) (24). Cyclic AMP-dependent M e free saline (Versene) containing 20 pg/ml trypsin (Sigma). Cells protein kinase activity was measured by the method of Roskoski (25) were plated a t 2-3 X lo6 cells/lOO-mrn plate 3 days before transfec- using the synthetic substrate Kemptide (Penisula Laboratories). To tion. Mouse AtT2O pituitary cells (20) weregrown in Dulbecco's obtain the Hill coefficient and half-maximal activation (K.) value modified Eagle's medium plius 10% horse serum (GIBCO) and were with purified holoenzyme, kinase activation curves in the presence of passaged by dissociation in Versene alone. Confluent cells were split 0-100 p M cAMP were constructed, and plottedas Hill plots. Maximal 1:20 1 daybefore transfection. Stable subclones were selected in kinase activity was measured at cAMP concentrations from 2 to 100 media containing 500 pg/ml G-418 (GIBCO) and maintained in 300 p M for the wild type and B1 enzymes, and at cAMP concentrations pg/ml G-418.Cellswere passaged out of G-418 when plated for from 30 to 140 p M for the B2 holoenzyme. The B2 holoenzyme, experiments. however, continued to activate at the highest cAMP concentrations RI Protein and Holoenzxme Purification-KK-REV B1, B2, and allowable in our assay, therefore, maximum activity of the enzyme is EXPERIMIZNTAL PROCEDURES
16674
CAMP-binding Mutations inMouse RI
330
323
FIG. 1. RI protein, nucleotide, and amino acid sequenceof RI site B. The RI protein is shown, indicating the positions of the two CAMP-binding domains, sites A and B. The nucleotide and amino acid sequence of the COOHterminal CAMP-binding domain (site E ) in the wild type RI is shown along with the B1 and B2 point mutations and resulting amino acid changes. The asterisks indicate amino acids identical to the CAP protein thought to bind CAMP.The HinfI restriction site used to separate the B1 andB2 mutations is underlined.
*
*
WT
TTT
*
338
Ala CCT
AlaValThr CGG GCT
*
P h e Gly
Glu Ile Ala Leu Leu Met Asn Arg Pro Arg GGT GAA ATT GCC CTG CTG ATG AAT CGT
*
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A
Asw
considered to he the maximum activity attainable in theassay. Transient Transfection of JEG-3 Cells and Luciferase Assay-The reporter plasmid a-168 luciferase contains the coding sequence of bacterial luciferase regulated by the promoter from the human asubunit glycoprotein hormone gene (33).' This promoter sequence contains two tandem CRE sequences and a tissue-specific element which confers placental specific expression, thus directing high levels of expression in the presence of cAMP in JEG-3 cells (26). Cells were transfected with 0.5 pg of a-168-luciferase, 4 pg of RSV-8-galactosidase (27), and 10 pgof MT-REV, or MT-REVe by the method of Capo4 precipitation described previously (28). The DNA was added to 0.5 ml of 2 X HBS, and an equal volume of 0.25 M CaCL wasadded while vortexing. After allowing 5-10 min for precipitation to occur, the precipitate was added directly to the medium on the cells and incubated for 5 h. The medium was then replaced with Dulbecco's modified Eagle's medium plus 2.5% newborn calf serum. Between 2426 h later,cells were treated with 10 p~ forskolin with 100 p~ IBMX (Sigma) for 14-15 h and then collected. The cells were collected in 200 p1 of 100 mM KPO,, pH 7.8, 1 mM dithiothreitol, and soluble protein was obtained by freeze/thaw (29). Five pgof protein were assayed for luciferase activity by the method of de Wet et al. (30). Briefly, protein was added to a mixture containing 100 mM KPO,, 20 mM ATP, and 15 mM MgSO,. The sample was placed in a monolight 2001 (Analytical Luminescence Laboratory, Inc.), which added Dluciferin (Analytical Luminescence Laboratory, Inc.) and measured the emitted light. In order to normalize luciferase activity with the transfection efficiency of each sample, &galactosidase activity was measured as described by Edlund et al. (27). Twenty pgof protein were assayed with the substrate o-nitrophenyl-8-galactopyranoside (Sigma) a t 37 'C. When the samples reached a pale yellow color, the reaction was stopped with NaHC03 and the absorbance at 420 nm measured. The ratio of luciferase to @-galactosidasewas calculated for each sample. RESULTS
The RI Protein and Site B Nucleotide and Amino Acid Sequences-Fig. 1 shows the RI protein and the location of the two CAMP-bindingdomains. The wild type RI nucleotide sequence is shown and theposition of two G to A transitions in cDNAs isolated from S49 lymphoma Ka mutant cells is indicated. The resulting mutant proteins contained an Arg to His substitution at position 332 in the B1 mutant anda Gly to Asp replacement at position 324 in theB2 mutant protein. Dissociation of PHICAMPfrom RI Subunit Proteins-To compare the properties of the purified mutant proteins and holoenzymes, the various RI cDNAs were subcloned into the bacterial expression vector, pKK322-2, as diagrammed in Fig. 2A. Transcription from this vector is regulated by a fusion trp-lac promoter. Wild type and mutant RI proteins were partially purified for in vitro studies by DEAE chromatography as described by Woodford et al. (17).
'P. L. Mellon, L. A. Correll, T. A. Woodford, J. D. Corbin, and G. S. McKnight, manuscript in preparation.
A . Ptrc
RI cDNR UGR
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' I
82
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0
'
10
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o
.
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30 TIME (rnin)
20
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40
50
FIG. 2. The dissociation of bound ['HICAMP from wild type,
B1 a n d B2 mutant RI subunits. A, the bacterial expression vector, pKK322-2, containing a trp-lac fusion promoter ( P t r c ) and the RI cDNAwas constructed as described under "Experimental Procedures." EcoRIIPstI fragments containing the RI wild type, or the B1 or B2 point mutations, were ligated into the plasmid. The asterisk indicates the location of the mutantsequences. B, purified RI protein (2-25 pg/ml) was incubated with 1p~ [3H]cAMPat 25 'C for 45 min ( B e ) . Excess unlabeled cAMP was then added to initiate exchange and aliquots removed at the appropriate time points ( B )to measure cAMP binding as described under "Experimental Procedures."
Dissociation of [3H]cAMP from the wild type, B1 and B2 RI subunits was measured at 25 "C.The wild type RI protein showed a biphasic dissociation rate aspreviously reported (5). Dissociation from the A site occurred rapidly with a T* of about 5 min whereas dissociation from the B site proceeds with a Tlh of approximately 25 min. Like the nativeRI, dissociation from the B1protein was also biphasic, with very similar Tahvalues to thewild type protein. In contrast,dissociation of cAMP from the B2 subunit was much more rapid ( Tlh= 15 s) than would be expected if the A site were intact
CAMP-binding Mouse inMutations
RI
16675
expression vector, HL-REV (14). In this plasmid, transcription initiates in the long terminal repeat of the Harvey sarcoma virus and a polyadenylation signal is provided by the 3”untranslated sequences from hepatitis B virus. The gene for neomycin phosphotransferase, flanked 5’ and 3’ by SV40 promoter and polyadenylation sequences, was also ligated into the HL-REV plasmid (Fig. 4A) to permit selection of stable transformants in (3-418. The resulting vector is designated HL-REVBneo. Mouse AtT20 pituitary cells were transfected with the HLREVBneovectors in orderto test the ability of the B1 and B2 substitutions to affect CAMP-dependent protein kinase activity in animal cells. Cell extracts from transfected clones surviving in G-418 were assayed for relative levels of CAMPdependent protein kinase activity at 5 pM CAMP, since wild type CAMP-dependent protein kinase is maximally activated at thisconcentration (32).Reduced amounts of kinase activity were found in 80-90% of transformants (10 transformants of each type, B1, B2, or Bdb). Kinase activity in cells expressing the B1 mRNA ranged from 66-85%, whereas cells expressing the B2 substitution had much lower CAMP-stimulated kinase, ranging from as low as 7% up to 50%, depending upon the amount of B1 orB2 mRNA expressed (data not shown). Expression of the transfected RI cDNA was confirmed by using a radiolabeled probe specific for the HBV polyadenylation region of HL-REV to probe Northern blots of total RNA isolated from stable transformants. The results showed a band at thepredicted message size of about 1.5 kilobases in cells with reduced kinase activity. Messenger RNA levels were quantitated by solution hybridization with an SP6 riboprobe directed against the 3’ sequence from hepatitis B virus. The level of inhibition of CAMP-dependent protein kinaseactivity was compared with the amountof mRNA expressed as shown in Fig. 4. Expression of the B1 mRNA at 15-20 molecules/ cell, as in B1.l and B1.2, resulted in a slight shift in the activation curve to higher cAMP concentrations. Similar levels of B2 expression in B2.1 greatly repressed kinase activity and shifted the activation curve to very high cAMP concentrations. For comparison the endogenous level of wild type RI mRNA is approximately 180 molecules/cell in AtT2O cells emphasizing the dominant effects of these RI mutants (see “Discussion”). When 50 molecules/cell of the B2 or the Bdb mRNA were expressed as in B2.2 and Bdb.1, respectively, enzyme activation remained low even at 5-50 pM CAMP. It was not possible to obtain 100% activation of CAMP-dependent protein kinase in the mutant extractsdue to competition between high levels of cAMP and ATP. These results indicated that both the Gly to Asp mutation at amino acid 324 and theArg to His mutationat amino acid 332 cause discernible effects on kinase activation in stable, transfectedcells. A sensitive assay for the biological effects of mutant RI expression was used to compare the B1 and B2 mutations as described below. Transient Expression of Either Bl or B2 in JEG-3 Cells I I Interfered with CAMP-mediated Gene Induction-Transient expression in JEG-3cells was performed with another series of vectors referred to as MT-REV, and MT-REVB in which -L 0 1 2 3 4 5 transcription is regulated by the MT-1promoter as shown in LOG I c R M P I n M Fig. 5A. Vectors containing RI cDNAs with the B1 or B2 FIG. 3. Hill plots generated from cAMP activation data. The point mutations were constructed by substituting theapprocAMP dependence of activation of the reconstituted wild type ( W T ) , priate EcoRIIApaI fragment in MT-REVBdb. A sufficient level B1 and B2 mutant holoenzymes was measured as described under of RI proteinwas constitutively expressed from the MT-REV “Experimental Procedures.” Data were then plotted as Hill plots to obtain the Hill coefficient and KOof activation for each holoenzyme. constructs in JEG-3cells such that Zn induction of the MT1promoter was unnecessary for these experiments. The activity at each cAMP concentration divided by the maximum A reporter gene, a-168 luciferase, containing the coding activity attainable for the holoenzyme isrepresented by y. The holoenzyme concentrations used were 10-15 nM. region of the bacterial enzyme luciferase preceded by a 168(Fig. 2B). These results indicated that cAMP binding to the A sitewas dramatically alteredby the Gly to Asp mutation in site B. This alteration (B2) was the dominant mutation, since Bdb (containing both the B1 and B2 mutations) behaved essentially like B2 (data not shown). Based on these observations, B1 appeared similar to the wild type RI subunit. Cyclic AMP Activation. of Purified Wild Type and Mutant Holoenzymes-All of the mutant RI proteins formed a stable holoenzyme complex at 4 “C which was purified by DEAESephacel chromatography and a gel filtration sizing column as described (17). Activation by cAMP was determined using CAMPconcentrations of 1 nM to 140 p M in each assay, depending on the R subulnit type, and varying the holoenzyme concentration from 0.1 to 10 nM in different experiments. To account for any competition between ATP and cAMP for binding to theC subunit, as previously reported (31), the ATP concentration was incre,ased in the kinase assay. The wild type and B1 holoenzymes reached maximal activation at much lower cAMP concentrations (1and 5 p ~ respectively) , than the B2 enzyme, which continued to activate with increasing cAMPconcentrations up to 140 pM. At this point, since competition between cAMP and ATP in the kinase assay made the results difficult to interpret, thisvalue was considered as the maximum activity attainable with the B2 holoenzyme. The activation data for holoenzymes prepared from wild type mouse RI, B1, and B2 are plotted as Hill plots in Fig. 3 in order to obtain the K,of activation and the Hill coefficient. The K,, observed for the reconstituted wild type CAMP-dependent protein kinase was 40 nM, similar to that previously reported for the native bovine enzyme (17). The His-substituted holoenzyme, B1, required approximately 4fold higher cAMP for activation, ( K , = 188 nM), whereas the Asp-substituted holoenzyme, B2, required 100-fold more CAMP,with a K, value in excess of 4 p ~Positive . cooperativity between the A site and theB site in the wild type and B1 holoenzymes was indicated by Hill coefficients of 1.61 and 1.66, respectively. The B2 holoenzyme had a Hill coefficient of about 0.6, indicating nopositive cooperativity as predicted, since the B site could presumably no longer bind CAMP. Constitutive Expression of Site BMutations Repressed CAMP-stimulated Protein Kinase Activity in AtT20 Pituitary Cells-To obtain a constitutive level of expression of altered RI proteins in stable tcansformants of mouse AtT2O cells, fragments containing the B1, B2, and Bdb mutations were used to replace the wild type RI sequences in a eucaryotic
”I /.Y
CAMP-binding Mutations in Mouse RI A. HaLTR
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8 w t . l 81.2 82.2
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FIG. 4. PKA activation in cells expressing theB2 or Bdb RI cDNAs. A, HL-REV expression vectors were constructed as described under “Experimental Procedures” and used in stable transfections of AtT20 cells. Restriction sites inparentheses indicate sites in the original plasmid and cDNA whichhave been lost due to Klenow and blunt-end ligation reactions in the construction of HL-REV. Restriction site abbreviations are as follows: Eco RI (E),BglII (Bg), PstI ( P ) ,ApaI (A), and EarnHI ( E ) . B, subclones surviving in G418 were assayed for HL-REV expression. Cells weretransfected with the wild type ( B w t . I ) , B1 (EI.1, E1.2), B2 (B2.1, E2.2), and Bdb ( B d b . l ) cDNAs. Cultures were grown to confluence, harvested, and RNA prepared for measuring HL-REV mRNA expression by solution hybridization. A probe containing 321 bases of the 3’ hepatitis B virus polyadenylation signal sequence was hybridized to mRNA samples in triplicate. C,kinase activity was measured in parallel cultures. Cells were centrifuged a t 1000 rpm for 5 min and frozen at -70 “C until the time of assay. Cell pellets were then homogenized and the soluble protein diluted to 2 mg/ml in cell homogenization buffer. Extracts were assayed for protein kinase activity in triplicate at each cAMP concentration and the activity was compared with the activity of Bwt.1 at 5 p~ cAMP (maximum activity).
0
1
2.5 5 7.5 PLASMID (ug)
10
FIG. 5. Transient expressionof the B1 and B2 mutations in JEG-3 cells. A, the construction of MT-REV is described under “Experimental Procedures.” Restriction sites in parenthesesindicate sites in the original plasmid and cDNA which have been lost due to Klenow and blunt-end ligation reactions in the construction of MTREV. Restriction sites are abbreviated as follows: KpnI ( K ) ,EamHI ( B ) , XbaI ( X ) ,BglII (Bg), EcoRI ( E ) , PstI ( P ) ,and ApaI ( A ) .B, MT-REV containing the wild type RI cDNA and either MT-REVB1, B2, or Bdb plasmids were cotransfected into JEG-3cells together with a reporter gene containing a CAMP-induciblepromoter fused to the bacterial luciferase gene. The amount of MT-REV plasmid was held constant a t 10 pg/culture dish, while the ratio of B1 or B2 to the wild type RI varied. Transcription of luciferase was induced by incubation with 10 p~ forskolin/100 p~ IBMX for 14-15 h. Maximum luciferase activity attained with 10 pg of MT-REV, was measured and compared with the luciferase activity determined following cotransfection with increasing amounts of B mutant plasmids. The data shown, calculated as described under “Experimental Procedures,” was the average of duplicates in a representative experiment and was corrected for unstimulated luciferase activity in the absence of forskolin/IBMX.
bp sequence isolated from the human chorionic gonadotropin a-gene promoter was constructed. Transient expression of this plasmid in human JEG-3 choriocarcinoma cells in the presence of 10 PM forskolin alone or in the presence of M resulted in a10-20-fold induction forskolin and 100 ~ L IBMX of luciferase activity as measured in cell extracts.When cotransfected with a cDNA coding for the catalytic subunit of CAMP-dependent protein kinase, luciferase activity was induced by more than 15-fold in the absence of forskolin. Also, forskolin-stimulated luciferase activity can be inhibited by coexpression of an RI subunit cDNA containing mutations in the CAMP-binding regions (33). This system was used to demonstrate the ability of either the B1 or B2 mutations to directly affect a CAMP-stimulated biological effect in cell culture. Increasing amounts of either MT-REV B1 or B2were cotransfected into JEG-3 cells together with 01-168luciferase. A constant amount of RI expression vector was transfected
CAMP-bindingMutations in Mouse R l
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into each sample, with varying ratios of wild type to mutant negative charge provided by the Asp substitution, may alter RI cDNAs, to give a direct comparison of the effects of the the interaction between Glu-325 in site B and thecorrespondB1 and B2 mutations. Transcription of a-168 luciferase was ing Arg-334 in theabsence of cAMP binding. The role of Arg-332 in cAMP binding is less predictable, induced by incubation in thepresence of 10 PM forskolin and 100 PM IBMX. As a control, 10 pgof Mt 8-globin (34), a since Arg-332 is a variable amino acid among the otherCAMPplasmid coding for a protein that is not involved in cAMP binding domains and the substitution of a His residue conregulation, was transfected and did not effect the expression serves the positive charge at this position. Despite this, the of the reporter gene. There was also no effect of forskolin on His substitution alters the K, of holoenzyme activation by a non-CAMP regulated promoter linkedto theluciferase gene, CAMP, which probably reflects a change in binding affinity pRSV-Luciferase (30) (data not shown). Fig. 5 demonstrates at site B. There are apparently no major changes in intrathat the B1 mutation is biologically effective and represses subunit conformation, since the A and B sites remaincoupled. cAMP induction of gene expression by about 40%. Transfec- This change in cAMP binding affinitymay result from steric tion of either Bdb or B2 vectors inhibited CAMP-stimulated hindrance, due to thepresence of the bulkier, five-membered luciferase gene transcription by 90%,consistent with the side chain ring of His replacing the more flexible side chain relative levels of cAMP activation of CAMP-dependent pro- ofArg-332. Since this B1 mutation has a relatively weak effect on cAMP activation we speculate that this mutation tein kinase noted for these holoenzymes in cell extracts. occurred first in the S49 cell population probably prior to DISCUSSION selection in high concentrations of cAMP analogs. This B1 We have characterized two amino acid substitutions in the mutation might confer a slight growth advantage compared site B CAMP-binding dlomain of RI which have independent with wild type since cAMP inhibits growth of S49 cells. effects on cAMP binding, holoenzyme activation, and biolog- Resistance to high concentrations of dibutyryl CAMP would ical responses. The amino acid substitutions, Arg-332 to His then have occurred with the subsequent B2 mutation chang(Bl) and Gly-324 to Asp (B2) were previously identified in ing Gly-324 to Asp. A comparison of the effects of the B2 and site A substitucDNAs isolated from a variant S49 lymphoma cell line resistant to the lethal effects of high concentrations of dibutyryl tions demonstrates the dominant role of the B site in holocAMP (15). Cyclic AMP-binding studieswith purified recom- enzyme activation as previously suggested byOgreid and binant RIproteins indicate that theB2 sequence (Gly-324 to Doskeland (37). In the A mutant, even though the sites are Asp) eliminates cAMP binding to site B, causes a dramatic uncoupled, the K,, for activation of reconstituted holoenzyme increase in the rate of cAMP dissociation from site A, and is only 4.5-fold higher than wild type (17), indicatingthat site shifts the K, for holoenzyme activation to more than 4 PM. In Balone is effective inactivating the enzyme. When the contrast, biphasic dissociation kinetics are still observed in homologous mutation is present in site B (B2), cAMP actithe B1 subunit (Arg-332to His), and the rates of dissociation vation of the enzyme is greatly impaired and the K, is inare only slightly altered compared with the wild type. How- creased more than 100-fold compared with wild type. Both the B1 and B2 mutant R subunits produce a dominant ever, the concentration of cAMP required for half-maximal activation of the B1 holoenzyme is 188 nM compared with 40 CAMP-resistant phenotype when expressed in animal cells nM for the reconstituted wild type RI. Both thewild type and although the effect of B12 is much stronger. Constitutive B1 holoenzymes show positive cooperativity for cAMP acti- expression of B2 in mouse AtT2O pituitary cells shifts the K, vation whereas the B2 mutant enzyme has lost this property for kinase activation in cytoplasmic extracts by more than 100-fold compared with wild type RI whereas B1 expression since cAMP can nolonger bind to siteB. Gly-324, which is mutated to an Asp in the B2 construct, results in only aslight (-%fold) increase in the K,,. As is highly conserved amlong other CAMP-binding regions in all mentioned earlier,the levels of expression of either the B1 or isoforms of RI, RII, and thebacterial CAP protein. Recently, B2 mRNAs in these cells were significantly lower than the Woodford et al. (17) demonstrated that a mutation in the endogenous expression from the wild type RI genes. Solution homologous Gly in site A (Gly-200 to Glu) uncoupled site A hybridizations indicate 180 molecules/cell of wild type RI and site B with respect to cAMP binding and holoenzyme mRNA versus about 15-20 molecules/cell of mutant mRNA activation and increae,ed the rate of dissociation of cAMP in either clone B1.2 or B2.1. Nevertheless, the dominanteffect from the nonmutatedEl site. When the same Gly substitutions of this low level of expression of the B2 mRNA as shown in were made in the ratIt1 cDNA by site-directed mutagenesis Fig. 4C indicates that sufficient mutant R is being produced (35), the purified proteins only bound 1 molecule of CAMP/ to bind most or all of the C subunit produced in these AtT20 monomer. Taken together, these data demonstrate that the cells. The shiftin K,, for the B1-transfected AtT2O cells shown homologous glycines at amino acid positions 200 and 324 are in Fig. 4C can be compared with the 4.7-fold change in K, essential for cAMP birding at siteA and site B, respectively, expected for pure B1 mutant holoenzyme as shown in Fig. 3. and that the mutation of glycine 200 or 324 to eithera The smaller shift in K,, in the AtT2O cells is probably due to glutamic or aspartic acid residue abolishes cAMP binding to the presence of an excess of wild type RI and RII andcomthe mutated site andincreases the rate of cAMP dissociation petition between them for C subunit. It is important to note from the nonmutated site. that atambient levels of cAMP in cells, only the B2 mutant By analogy with CAP (36), Gly-324 is located at animpor- would be expected to have a strong competitive advantage in tant position in the CAMP-binding pocket. The Gly to Asp C subunit binding compared with wild type RI andRII. substitution (B2) disrupts the structureof a @-turnfollowing Transient expression of either the B1 or B2 proteins to@strand 6. The negatively charged side chain of Asp mayalso gether with a CAMP-dependent protein kinase-inducible rerepel the negative charges in the phosphate moiety of cAMP porter gene, a-168 luciferase, inhibits theforskolin-inducible thought to be positioned near Gly-324. In addition, the ho- luciferase activity by 35 and 90% for B1 andB2, respectively. mologous Glyat position 71 in CAP is adjacent to a negatively These results demonstrate that although the B1 mutant excharged Glu, as is Gly-324 in site B. This Glu in CAP interacts hibits a relatively weak effect on CAMP-dependent protein with the positively charged side chain of Arg-123 on a-helix kinase activation, it is sufficient to produce a significant C. The B2 alteration, by increasing the concentration of inhibition of CAMP-mediated gene induction. It is likely that
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a much higher level of mutant protein is being produced in the transientsystem compared with the stable transformants of AtT20 cells described above. Expression of the mutant forms of RI affects the levels of both type I and I1 CAMP-dependent protein kinase in cell culture. AtT20 pituitary cells contain RI and RII proteins, both of which form holoenzyme. Whenseparatedon an HPLC-DEAE column, 60% of total kinase activity is in the form of type I and the remaining 40% is type 11. When cell extracts from HL-REVB expressing clones were analyzed by this procedure, they containedreduced amounts of both types of kinase, rather than a selective loss of type I kinase (data not shown). This result is expected since the mutant RI should bind tightly to C and sequester it in an inactive tetramer. Competition between the mutant RI and wild type RI and RII would cause a shift in the distribution of C into inactive mutant holoenzyme. We have observed a similar decrease or elimination of both type I and I1 kinase in transformants expressing RI mutantprotein in other cell types including Y1 mouse adrenal cells and T84 human colon carcinoma cells. Stable expression of the mutant RI genes produces subclones with various levels of CAMP-dependent protein kinase activity which can beused to investigate the role of cAMP in signal transduction. In AtTZO cells, for example, secretion of pro-opiomelanocortin is stimulated by corticotropin-releasing factor which is thought to be coupled to the cAMP second messenger system (38). Preliminary data on AtT20 clones expressing mutant RI indicate that the secretory response to corticotropin-releasing factor is altered whereas the cells continue to secrete in response to Ca2+ionophores or depolarization (data not shown). Many intracellular events such as gene induction and ion channel regulation are mediated by multiple pathways involving CAMP, Ca2', inositol trisphosphate, and diacylglycerol. By introducing a specific lesion in the cAMP pathway, the role of this second messenger system and theinteractions between different pathwayscan be more clearly understood. Acknowledgments-We are indebted to Thong To for excellent technical assistance. We thank Drs. Chris Clegg and Wendy Ran for helpful discussions and suggestions in preparationof this manuscript. REFERENCES 1. Roesler, W. J., Vandenbark, G. R., and Hanson, R. W. (1988) J. Biol. Chem. 263,9063-9066 2. Costa, M. R. C., and Catterall, W. A. (1984) J. Biol. Chem. 2 5 9 , 8210-8218 3. Litvin, Y., PasMantier, R., Fleischer, N., and Erlichrnan, J. (1984) J. Biol. Chem. 2 5 9 , 10296-10302 4. Krebs, E. G. (1985) Biochem. SOC. Trans.13,813-820 5. Corbin, J. D., Rannels, S. R., Flockart, D. A., Robinson-Steiner, A. M., Tigani, M. C., Doskeland, S. O., Suva, R. H., and Miller, J. P. (1982) Eur. J. Biochem. 125,259-266
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