systematic mutational analysis of camp-dependent protein kinase

THE J O U R N A L OF BIOLOGICAL CHEMISTRY (2) 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 7, Issue of March 5, pp. 4806-4814, 1992 Printed in U. S. A .

Systematic Mutational Analysis of CAMP-dependent Protein Kinase Identifies Unregulated Catalytic Subunits and Defines Regions Important for the Recognition of the Regulatory Subunit*

(Received for publication, September 11,1991)

Craig S. GibbsSg, Daniel R. Knightonll, Janusz M. Sowadskin, Susan S. Taylorq, and Mark J. ZollerS From the $Department of Protein Engineering, Genentech Inc., South San Francisco, California 94080 and the llDepartment of Chemistry, University of California, S a n Diego, La Jolln, California 92093-0654

A library of mutants of the catalytic subunit of the Saccharomyces cerevisiae CAMP-dependent protein kinase was screened in vitro for mutants defective in the recognition of the regulatory subunit. The mutations identified were mapped onto the three-dimensional structure of the mouse catalytic subunit with a peptide inhibitor. Mutations defective in the recognition of both the regulatory subunit and the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) mapped to the peptide-binding site shared by all substrates and inhibitors of the catalytic subunit and func- tionally define the binding site for the autoinhibitor sequence in the hinge region of the regulatory subunit. Mutants defective only in the recognition of the regulatory subunit identified residues that comprise additional binding sites for the regulatory subunit. The majority of these residues are clustered on the surface of the catalytic subunit in a region flanking the distal portion of the autoinhibitor/peptide-binding site. The simultaneous substitution of L Y S ’ ~ ~ , LysZs7, and LysZe’ in this region caused a 260-fold decrease in affinity for the regulatory subunit, whereas the catalytic efficiency toward Kemptide decreased by only 1.8-fold. The substitution of autophosphorylated ThrZ4l, also in this region, and the 3 residues interacting with the phosphate also caused an unregulated phenotype.

The protein kinases represent a large and structurally very diverse family of enzymes. However, with the notable excep- tion of the oncogenic members of the family, all of the protein kinases are tightly regulated so that they can be turned off in the absence of the appropriate activation signal. The CAMP- dependent protein kinase was one of the first protein kinases to be discovered, and this relatively simple protein kinase continues to serve as a prototype for the entire family. The holoenzyme is an inactive tetramer composed of two mono- meric catalytic subunits bound to a dimer of regulatory sub-

* This work was supported by the Lucille P. Markey Foundation and National Science Foundation Grants DMB-8918788 (to M. J. Z.) and DIR-88-22385 (to S. S. T.), Grants GM19301-20 and GM34921- 06 (to S. S. T.) and GM37674-01 (to J. M. S.) and Training Grants T32CA09523 and T32DK07233 (to D. R. K.) from the National Institutes of Health, American Cancer Society Grant BE-48G (to S. S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed Dept. of Protein Engineering, Genentech Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080.

units. The cooperative binding of two molecules of CAMP to each regulatory subunit decreases the affinity of the regulatory subunit for the catalytic subunit and promotes the dissociation of the holoenzyme into a dimer of regulatory subunits and two active catalytic monomers. The catalytic subunit transfers the y-phosphate from ATP to specific serine and threonine residues in selected protein substrates to mod- ify their biological activities in response to a variety of regulatory signals (reviewed by Krebs and Beavo (1979) and Taylor et al. (1990). This study probes the interactions between the regulatory (R) and catalytic (C,) subunits of the yeast CAMP-dependent protein kinase responsible for the regulation of phosphotransferase activity.

The substrate specificity of CAMP-dependent protein kinase, defined by the analysis of synthetic peptide substrates and inhibitors based on the sequences flanking the phosphorylation sites in protein substrates, is determined by a dibasic consensus sequence, RRX(S/T)Hy (Hy = hydrophobic) (Table I). The 2 arginine residues and the hydrophobic residue are of primary importance (Kemp et al., 1977; Zet- terqvist et al., 1990; Walsh et al., 1990; Denis et al., 1991). The catalytic subunit recognizes the regulatory subunit in a manner analogous to the recognition of protein substrates. The regulatory subunit has a modular domain structure composed of an N-terminal dimerization domain separated from two tandem CAMP-binding domains at the C terminus by a hinge region. The hinge region contains an “autoinhibitor site” (an autophosphorylation site or a pseudosubstrate site) that conforms to the dibasic consensus sequence (Table I) and presumably occupies the active site and acts as a compet- itive inhibitor of the catalytic subunit (reviewed by Hardie (1988) and Taylor et al. (1990)).

Several studies utilizing chemical modification (Corbin et al., 1978), limited proteolysis (Weldon and Taylor, 1985), chemical cross-linking (First et al., 1988), genetic selection (Cannon et al., 1990), and site-directed mutagenesis (Kuret et al., 1988; Durgerian and Taylor, 1989; Buechler and Taylor, 1991; Wang et al., 1991) have demonstrated the importance of the autophosphorylation site in the yeast R subunit and the mammalian type I1 regulatory (RII) subunit and the pseudosubstrate site in the mammalian type I regulatory (RI) subunit for the association of the regulatory and catalytic subunits. Similarly, the regions of the catalytic subunit that recognize the substrate sequence have been clearly identified by chemical modification studies (Miller and Kaiser, 1988; Buechler and Taylor, 1988, 1990) and site-directed mutagenesis (Gibbs and Zoller, 1991a, 1991b) and more recently confirmed at the molecular level by the solution of the three- dimensional structure of the mammalian catalytic subunit

4806

Recognition Sites for Regulatory Subunit in Protein Kinase A TABLE I

Consensus recognition sequences in Kemptide, PKZ, and autoinhibitor regions of the mammalian and yeast regulatory subunits

The recognition sequences present in inhibitors and substrates of the CAMP-dependent protein kinases conform to the dibasic consensus sequence RRX(S/T)Hy (Hy = hydrophobic). The consensus sequence is boxed. The phosphorylation site (position P) is underlined. In the bovine type I1 regulatory and yeast regulatory subunits, position P in the autoinhibitor sequence is occupied by a phosphorylatable serine residue, in which case the sequence is referred to as an autophosphorylation site. In the bovine type I regulatory subunit and PKI, this residue is replaced by alanine, in which case the sequence is referred to as a pseudosubstrate site. The 2 arginine residues at positions P-2 and P-3 that are the primary determinants of phosphorylation site specificity are in boldface twe.

4807

Consensus sequence

Kemptide L R R A S L G PKI (residues 5-24) T T Y A D F I A S G R T G R R N A I H D Bovine RI subunit S P P P P N P V V K G R R R R G A I S A Bovine RII subunit E E D L D V P I P G R F D R R V S V C A Yeast R subunit (BCY1) T S T P P L P M H F N A Q R R T S V S G

bound to a peptide derived from the heat-stable inhibitor PKI' (Knighton et al., 1991b).

However, several lines of evidence suggest that there must be additional interactions between the regulatory and catalytic subunits that provide additional specificity and binding energy to the complex. The catalytic subunit binds the regulatory subunit with an affinity in the nanomolar range (Hof- mann, 1980; Kuret et al., 1988), whereas the best peptide substrates and inhibitors based on the dibasic consensus sequence have binding constants in the micromolar range (Zetterqvist et al., 1990; Walsh et al., 1990). Recently, it was demonstrated that a mutant form of the mammalian RII subunit, in which both arginine residues in the autophosphorylation site are substituted with alanine, is still capable of forming a stable complex with the catalytic subunit (Wang et al., 1991). The equivalent substitutions in a peptide substrate would have reduced the specificity by 1 X 106-fold (Kemp et al., 1977).

Previously, Levin et al. (1988) and Levin and Zoller (1990) used a genetic screen to identify ThrZ4l in the yeast catalytic subunit that appeared to be important for regulatory subunit binding but not for the recognition of a peptide substrate. "Charged-to-alanine" scanning mutagenesis of the C1 subunit was used recently to identify residues important for catalysis and substrate specificity (Gibbs and Zoller, 1991a, 1991b). In this study, the same set of mutants was used to scan the surface of the catalytic subunit for residues important for interactions with the regulatory subunit and to map the R subunit-binding determinants on the three-dimensional structure of the catalytic subunit.

EXPERIMENTAL PROCEDURES

Yeast Strains and Plasmids-Yeast strain LL8 (MATa tpk1::URAS tpk2::HZS3 tpk3::TRPI BCYI leu2 + YEp(ADE8)TPKl) contains disruptions in all three chromosomal TPK genes encoding catalytic subunits of the yeast CAMP-dependent protein kinase: viability is maintained by the presence of TPKl on a multicopy vector containing the origin of replication from the yeast 2-pm plasmid (Levin and Zoller, 1990). LL8 is the parent of all the strains discussed in this study.

YEp(LEU2)TPKI is a multicopy yeast expression vector containing the TPKl gene encoding the C1 subunit and the auxotrophic marker LEU2 (Levin and Zoller, 1990). YEp(LEU2)TPKI served as the template for site-directed mutagenesis.

YEp(ADE8)ADH-BCYI is a multicopy yeast expression vector containing the BCYI gene encoding the yeast R subunit under the control of the ADH promoter and the auxotrophic marker ADE8. A mutated version of this plasmid (YEp(ADE8)ADH-

' The abbreviations used are: PKI, protein kinase inhibitor; MOPS, 4-morpholinepropanesulfonic acid.

BCYI(T144N,S145A)) was also used, in which the BCYl gene contains two mutations in the hinge region (Thr144 + Asn and Ser 145+

Ala) that result in a regulatory subunit that binds more tightly to the catalytic subunit (Levin and Zoller, 1990). Yeast media and culture conditions were according to Sherman et al. (1982). Yeast strains were transformed by the lithium acetate procedure (Ito et al., 1983).

Site-directed Mutagenesis and Derivation of Mutant Yeast Strains-The charged amino acids in the C1 subunit were substituted with alanine by oligonucleotide-directed mutagenesis on a single- stranded, uracil-containing template of YEp(LEU2)TPKI as described previously (Gibbs and Zoller, 1991a). Yeast strain LL8 was transformed with the mutated plasmids; transformed strains were grown without selection; and strains that had lost the wild-type plasmid were selected using the auxotrophic markers, LEU2 and ADE8 (Zoller et al., 1991). Mutant yeast strains were transformed with YEp(ADE8)ADH-BCYl to overexpress the R subunit and to amplify the expression of the C1 mutant (Zoller et al., 1988). Strains were also transformed with YEp(ADEB)ADH-BCYl(Tl44N,S145A) to overexpress the R subunit tight-binding and to aid the purification of C1 mutant; that have reduced affinity for the R subunit (Levin and Zoller, 1990).

Determinution of Kinase Activity in Crude Cell Extracts-Crude, gel-filtered cell extracts were made from 10-ml cultures of yeast strains coexpressing C1 mutants from YEp(LEU2)TPKl and the yeast R subunit from YEp(ADE8)ADH-BCYI as describedpreviously (Gibbs and Zoller, 1991a). Phosphotransferase activity against a synthetic peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemp- tide)) was measured in the presence or absence of 10 p~ cAMP under V,,, conditions for the wild-type enzyme (200 p M ATP, 1000 cpm/ pmol [Y-~'P]ATP, 200 p M Kemptide, 10 mM MgClz, 50 mM MOPS (pH 7.0), 0.25 mg/ml bovine serum albumin) as described previously (Roskoski, 1983; Gibbs and Zoller, 1991a). The concentration of the C1 subunit in the extracts was determined by quantitative Western blotting as described previously (Gibbs and Zoller, 1991a).

Purification of Selected Mutant Enzymes-Selected C, mutants with reduced affinity for the yeast R subunit were coexpressed in yeast strains expressing the R subunit tight-binding mutant from YEp(ADEB)ADH-BCYI(T144N,S145A). One-liter cultures were grown and harvested; and wild-type and Cl mutants were purified by ammonium sulfate fractionation and affinity chromatography using an immobilized anti-regulatory subunit monoclonal antibody and elution with 1 mM cAMP as described previously (Zoller et al., 1988; Gibbs and Zoller, 1991b). Enzyme preparations were judged to be at least 95% pure as determined from sodium dodecyl sulfate-polyacryl- amide gels stained with Coomassie Blue. Enzyme concentration was determined by the Bio-Rad protein-dye binding assay (Bradford, 1976).

Determination of IC50 Values for Purified C1 Mutants-Purified C, mutants (1 nM) were preassociated with the purified yeast R subunit at a range of concentrations (between 5 and 5000 nM) at 30 "C for 15 min in the presence of 200 p~ ATP, 1000 cpm/pmol [Y-~'P]ATP, 10 mM MgC12, 50 mM MOPS (pH 7.0), 0.25 mg/ml bovine serum albumin. Following preassociation, reactions were initiated by the addition of the peptide substrate Kemptide to a final concentration of 100 pM (approximately K,) and incubated at 30 "C for 15 min. Reaction rates were measured in the manner described above. The 50% inhibitory concentration (If&) was determined graphically from

4808 Recognition Sites for Regulatory Subunit in Protein Kinase A

plots of percent activity uersus log concentration of the R subunit. The R subunit used in these assays was a deleted version of the yeast R subunit, in which the dimerization domain has been removed by the deletion of residues 7-95. This deleted R subunit associates with the catalytic subunit as a monomer as opposed to the wild-type dimer.' The binding of this deleted R subunit to the Cl subunit (IC, = 17 nM) is comparable to that displayed by the wild-type yeast R subunit (ICs, = 20 nM) (Levin and Zoller, 1990). The deleted yeast R subunit was expressed in Escherichia coli and purified as described previously (Johnson et al., 1987) and diluted in 10 mM MOPS (pH 7.0), 5% glycerol, 0.625 mg/ml bovine serum albumin.

Kinetic Analysis of Purified C1 Mutants-Purified Cl mutants were subjected to analysis by steady-state kinetics. Reactions were per- formed under the standard conditions described above for the determination of activity in crude cell extracts; however, the concentration of one substrate was kept constant in excess of K,, whereas the concentration of the other was varied between 5 and 0.2 X K,. The kinetic data were fitted to the Michaelis-Menten equation by the Marquardt-Levenburg nonlinear regression algorithm and used to calculate the kinetic parameters k,.,, K m ( M g A T p ) , and Km(Kemptide) and their associated standard errors.

Heat Shock Assay-Yeast strains containing C1 mutants were streaked on rich medium (YEPD) and grown for 5 days at 30 "C. Fresh replica plates were inoculated with stationary phase cells at room temperature. The plates were subjected to a heat shock at 60 "C for various times and allowed to recover at 30 "C for 3 days. YEPD medium was prepared according to Sherman et al. (1982).

RESULTS

Screening of Charged-to-Alanine Mutants of C1 Subunit for Those Defective in Recognition of Regulatory Subunit-In a previous study (Gibbs and Zoller, 1991a), a set of 62 site- directed mutants of the C1 subunit was constructed in which the charged amino acids (Asp, Glu, Arg, Lys, and His) were systematically substituted with alanine in groups of 1, 2, or 3 residues (Table 11). The charged-to-alanine mutants of the yeast C1 subunits were used to replace the plasmid-borne copy of wild-type TPKl (YEp(LEU2)TPKl) in a ,yeast strain (LL8) that contains disruptions in all three chromosomal TPK genes using a plasmid shuffle procedure (Zoller et al., 1991). One mutant (Aspzz8 + Ala) had insufficient activity to support growth (Gibbs and Zoller, 1991a); the equivalent residue in the mammalian enzyme (Asp") was proposed to be interacting with ATP through M e in the crystal structure of the mammalian enzyme (Knighton et al., 1991a). The 61 viable mutant yeast strains remaining were transformed with the plasmid YEp(ADE8)BCYl to overexpress the regulatory subunit and to enhance the expression of the C1 mutants. Crude cell extracts from strains expressing the C1 mutants were screened for activity against the peptide substrate Kemp- tide in the presence and absence of 10 PM CAMP. C1 mutants with reduced affinity for the regulatory subunit should form the holoenzyme less readily and display increased levels of specific activity in the absence of CAMP.

Most of the C1 mutants were inhibited by the regulatory subunit in the crude cell extracts (Table 11), displaying low levels of activity in the absence of cAMP comparable to that displayed by the wild-type C1 subunit (6.3% (specific activity - cAMP)/(specific activity + CAMP)). However, 14 mutants were >25% active in the absence of cAMP and were potentially defective in the recognition of the regulatory subunit. These mutants could be divided into two classes depending on whether they were also defective in the recognition of the peptide substrate Kemptide.

C1 Mutants Defective in Recognition of Regulatory Subunit and of Peptide Substrate-Most of the mutants that were defective in the recognition of the regulatory subunit also had reduced specific activity toward Kemptide, displaying (25%

J. Kuret and M. J. Zoller, unpublished data.

TABLE I1 Regulation of the specific actiuity of charged-to-alnnine

mutants of the Cl subunit The residues substituted in the charged-to-alanine mutants of the

C1 subunit are shown. The single-letter code for the amino acids is used. The nomenclature used to describe the mutants is: the amino acid substituted in the yeast Cl subunit, followed by the residue number, followed by the amino acid replacement (Wetzel, 1988). Specific activity (micromoles of phosphate transferred per minute/ milligram of C1 subunit) was measured under Vmax conditions for the wild-type enzyme in the presence and absence of 10 p M CAMP. Values cited are the means of three separate measurements. Specific activity expressed as the percentage of the wild-type activity measured in the presence of 10 p~ cAMP is reprinted from Gibbs and Zoller (1991a). Percent specific activity of -cAMP/+cAMP represents the specific activity in the absence of cAMP for each mutant expressed as a percentage of the specific activity observed in the presence of 10 p M CAMP. The residue number in the mammalian catalytic subunit equals the residue number in the yeast C1 subunit - 44.

Mutant Mutation in yeast C1 Specific activity

Mutant/wild t w e -cAMP/+cAMP subunit

1 2

4 3

5 6

8 7

10 9

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28X 28Y 29 30 31 32 33x 34x 34Y 35 36 38 39 40 41 42 43 44 45 46 47 48 49 50 51

53 52

54 55 56 57 58 59 60

Wild t m e

E4A, E5A

D15A, D16A, R17A K12A

E20A, E21A, K24A E26A, E29A, R30A

E40A. K42A E31A. E36A

E45A, K46A, E47A E50A. E53A, K54A K56A, H59A E65A, E66A, K69A R76A K81A, D86A R91A RlOOA H102A, R105A R107A, H108A K116A K119A, K120A, E121A R125A, K127A E130A, H131A D134A, R136A D134A, E135A, R136A H144A, R149A D156A D165A E168A, E171A R177A, K178A R181A K188A, E193A E199A, H202A K204A, D205A R209A D210A K212A E214A D219A, K220A H223A, K225A K233A, D237A D247A E252A

D264A K257A. K261A

E274A D284A, K289A E292A, K293A E298A, R300A E307A, D308A K310A, D311A, R315A R319A, D320A R324A E333A. D334A K336A, H338A

E347A, K348A, R352A K342A, E343A

E355A. E359A D368A D373A, K374A E377A, E378A, D379A E387A, D388A D392A, R395A, D396A

51.5 35.6 45.6 57.6 84.3 55.8

101.2 39.5

47.6 46.5 44.4 58.5 66.5 14.4

38.9 27.1

40.2 0.3

16.2 42.7 22.6 34.6 0.3

108.2 42.1

50.8

135.1 2.3

109.1 40.4 70.6 93.2 10.5 0.4 0.3 1.7

81.4 68.6

119.8 27.5

133.5 0.9

95.1

90.1 3.7

82.1

102.2 80.9

60.3 35.9

109.3 5.8

103.7 16.8

127.3 89.7

25.0 58.4

134.7 7.5

100.0 31.8

% 5.7 3.4 5.3 4.5 8.0 4.2 5.2

6.4 1.2

6.7 5.1

10.6 7.5

20.9 19.0 12.0 7.2

12.5 9.1

80.7 4.7

13.8 20.8 10.0 1.3 3.7

25.4 37.1 4.5 9.1 5.2

92.6 4.6

27.6 27.7 49.1 5.1

34.0 7.2

43.6 54.8 34.5 7.9

36.9 6.1 7.4 6.3 4.3 8.6

53.3 7.1

17.2 1.8

5.8 3.9

33.9 6.7

14.6 6.5

3.7 3.7 6.3

Recognition Sites for Regulatory Subunit in Protein Kinase A 4809

of the specific activity of the wild-type enzyme. Cell extracts containing these mutants were analyzed by steady-state kinetics to determine the effect of the mutation on the kinetic parameters hat, Km(Kemptide)) and &(M~ATP). The catalytic efficiency (kCat/Km) of these mutants toward Kemptide was reduced by 12-1000-fold (Table 111).

Mutants involving the substitution of Glu171, Glu214, Asp247, and G ~ u ~ ~ ~ are defective mainly in Km(Kemptide). In a previous study, using site-directed mutagenesis, Glu171, Glu214, and G ~ u ~ ~ ~ were predicted to be involved in specific electrostatic interactions with the 2 arginine residues (at positions P-2 and P-3) that comprise the consensus recognition sequence (Gibbs and Zoller, 1991b). The subsequent solution of the three- dimensional structure of the binary complex of the mammalian catalytic subunit with a peptide inhibitor derived from the naturally occurring inhibitor (PKI) (Knighton et al., 1991a) 1991b) defined the peptide-binding site in a cleft in the C-terminal domain of the molecule and confirmed the earlier predictions. The residue in the mammalian enzyme equivalent to Asp247 (GluZo3) interacts with the arginine at position P-6 in PKI. This position is not present in Kemptide, which only extends to position P-4; however, it was suggested that GluZo3 could interact with the arginine at position P-2 if the arginine position at P-6 was not present (Knighton et al., 1991b). Thus, these residues interact directly with the peptide substrate and are likely to be involved in the recognition of the autoinhibitor region of the regulatory subunit.

Mutants involving the substitution of Aspz1', GluZ5', and A r e display large defects in kat Asp2l0 is likely to be a residue that is directly involved in catalysis. The equivalent residue in the mammalian enzyme (Asp'66) points toward the alanine residue at the phosphorylation site of the inhibitor in the crystal structure. This residue may act as a general base to enhance the nucleophilic properties of the hydroxyl group when the alanine is replaced by a serine in a substrate. The residues equivalent to GluZ5' and Arp?24 interact with each other in the mammalian catalytic subunit (G1uZo8 and A r p , respectively) and appear to play an important structural role in linking two distant portions of the polypeptide chain and buttressing the peptide-binding site (Knighton et al., 1991b).

The remaining mutants in this class display complex kinetic defects. Mutants involving the substitution of Glu13', Hid3', ArgW, and Lys212 are defective in K,,,(M~AT~) as well as kcat and &(Kemptide). The residues in the mammalian enzyme equivalent to Arg20' and Lys212 flank the putative catalytic base (Asp"') on the catalytic loop. The residues equivalent to

Glu13' and His'31 are located near the ATP-binding site in the structure (Knighton et al., 1991a). The primary defect in these mutants may be the recognition of MgATP. The binding of MgATP has been shown to precede peptide substrate binding in the kinetic mechanism (Whitehouse and Walsh, 1983; Whitehouse et al., 1983); consistent with this, C1 mutants defective in the recognition of MgATP are also defective in the recognition of the peptide substrate (Gibbs and Zoller, 1991a) and consequently may also be defective in the recognition of the regulatory subunit. Ringheim and Taylor (1990) have demonstrated that the binding of MgATP to the mammalian catalytic subunit is a prerequisite for reassociation with the type I regulatory subunit. Alternatively, the substitution of these residues may perturb the structure of the catalytic subunit in a region that interacts with the regulatory subunit.

C1 Mutants Defective Only in Recognition of Regulatory Subunit-Three mutants (28X, 38, and 41) were defective in the recognition of the regulatory subunit, yet retained full activity against the peptide substrate Kemptide. These mutants were identified by screening for those that displayed high levels of unregulated activity in vitro (Fig. 1 and Table 11). The residues substituted in these mutants are candidates for those that recognize a site on the regulatory subunit apart from the autoinhibitor region. Alternatively, they could recognize residues in the autoinhibitor region of the R subunit that are not present in Kemptide. These mutants were expressed in yeast and purified using an immobilized anti- regulatory subunit antibody. The affinity of the C1 mutants for the regulatory subunit was determined from the concentration of the purified regulatory subunit required to achieve half-maximal inhibition of the C1 mutant at 1 nM (IC5' value). In addition, the purified C1 mutants were analyzed by steady- state kinetics to determine the kinetic parameters kc , ,

Mutants 28X (Arg177 + Ala, Lys17' + Ala), 38 (LYs '~~ + Ala, Asp237 + Ala), and 41 (LYs '~~ + Ala, Lys261 + Ala) displayed IC5' values increased by 4.2-, 9.6-, and 7.9-fold, respectively. The catalytic efficiency toward Kemptide ( kcat/ Km(Kempt,de)) for mutants 38 and 41 was essentially unaffected, decreasing by 1.1- and 1.2-fold, respectively, suggesting again that these mutants involve a binding site for the regulatory subunit distinct from the peptide substrate-binding site (Table IV). The catalytic efficiency of mutant 28X was decreased by 2.3-fold due to an increased Km(Kemptide), suggesting that the residues substituted in this mutant may be implicated

&(Kemptide), and &(MgATP).

TABLE I11 C1 mutants defective in the recognition of the regulatory subunit and of Kemptide

Kinetic parameters were determined by steady-state kinetic analysis of the mutant enzymes in crude cell extracts as described by Gibbs and Zoller (1991a). The estimates of kcat for these mutants determined in crude cell extracts have additional error arising from the estimation of enzyme concentration by Western blotting.

yeast C1 subunit Mutation in

in murine C subunit Equivalent residues

(-cAMP/+cAMP) Specific activity

t a t K m (MgATPl

% E130A, H131A E86, H87 80.7 E168A, E171A" P124, E127 25.4 R209A" R165 92.7 D210A" Dl66 K212A" K168

27.6 27.7

E214A" E170 49.1 D247A E203 E252A"

43.9 E208

E274A" E230 54.8

R324A" R280 36.9

Wild type" 53.3 6.3

Kinetic data were from Gibbs and Zoller (1991a).

S-1

9.79 1.09 3.18 0.05 0.34 6.19

13.33 0.24 2.65 0.37

16.87

PM

128 61

135 60 98 18 13 21 16 27 39

Km IKemptldel

m M

0.700 5.361 0.257 0.230 2.149 8.496 0.761 1.508 2.705 0.085 0.096

S"/mM

13.99 0.20

12.37 0.22 0.16 0.73

0.16 0.98

17.5

4.35 175

4810 60

50

40

30

20

10

T Recognition Sites for Regulatory Subunit in Protein Kinase A

1

L - ~ m v n w ~ m m o - ~ m a n w ~ m ~ o - ~ m ~ n w ~ ~ ~ m o - ~ ~ ~ ~ n w m m o - ~ ~ v n ~ ~ ~ m o - ~ m ~ n ~ ~ m ~ o

- - - - - - e - - - N N N N N N N N m m N ~ m m m ~ v m ~ m m ~ a v ~ a a ~ ~ v u n n n ~ n n n n n n w ~ N N m m m

Mutant FIG. 1. Regulation of specific activity of charged-to-alanine mutants of C1 subunit. Mutants that

display high levels of unregulated activity identify those that are defective in the recognition of the regulatory subunit, but not in activity against Kemptide. Percent unregulated activity is the ratio of the activity of each C1 mutant in the absence and presence of CAMP multiplied by the specific activity of each mutant expressed as the percentage of the specific activity of wild type. See Table I for data and residues substituted.

in peptide substrate binding as well as in the recognition of the regulatory subunit.

These mutants each involve the simultaneous substitution of two charged amino acids with alanine; the individual substitution of each of these 6 residues also affected the recognition of the regulatory subunit (Table IV). The IC50 values for these single-substitution mutants were increased by 2.1- 4.2-fold, whereas the catalytic efficiency against Kemptide did not decrease at all and in fact increased due to increases of up to %fold in kcat. The amino acid substitutions were also combined in multiple-substitution mutants, in which 4 and 6 residues were substituted simultaneously. The values for mutants 28X+38, 28X+41, and 38+41, each containing four substitutions, were increased by 205-, 350-, and 260-fold, respectively (Table IV). These mutants displayed only small decreases in their catalytic efficiency against Kemptide of 2.1-, 2.3-, and 1.8-fold, respectively, strengthening the sugges- tion that these residues are involved in contacts with the regulatory subunit additional to those that are made with the peptide substrate. All 6 residues identified in this study were simultaneously substituted in mutant 28X+38+41 (Table IV); this mutant could not be purified. The purification strategy involves the coexpression of the C1 mutant with a tight- binding mutant of the regulatory subunit (BCYI- (T144N,S145A)) and purification by immunoaffinity chromatography using an immobilized antibody directed against the regulatory subunit. The multiple-substitution mutant 28X+38+41 presumably retains insufficient affinity for the regulatory subunit to be purified in this way. Thus, the affinity of this mutant for the regulatory subunit is likely to be decreased beyond the 200-350-fold observed for the multiple-

substitution mutants that could be purified. This mutant was coexpressed with the regulatory subunit, and the kinetic parameters were determined for the enzyme in crude cell extracts (Table IV). This mutant was completely uninhibited by the regulatory subunit, displaying 97% of its activity in the absence of CAMP; however, the catalytic efficiency of this mutant against Kemptide was decreased by only 6.8-fold. The K m ( M g ~ ~ ~ ) for all these mutants was not significantly different from that observed for the wild-type enzyme.

Localization of Residues Important for Recognition of Regu- latory Subunit on Three-dimensional Structure of Mammalian Catalytic Subunit-The amino acid sequence of the yeast C1 subunit was aligned with the sequence of the mammalian catalytic subunit (Hanks et al., 1988). The residues identified in this study as being important for the recognition of the regulatory subunit are conserved in the mammalian enzyme apart from LYS'~', which is conservatively substituted with arginine, and Asp237, which is substituted with glycine. These residues were mapped onto the three-dimensional structure of the mammalian catalytic subunit bound to a peptide inhibitor derived from the naturally occurring, heat-stable inhibitor PKI (Knighton et al., 1991a, 1991b). The 6 residues identified as important only for the recognition of the regulatory subunit map to two regions of the structure.

The residues in the mammalian enzyme equivalent to Arg177 and Lys17' (Arg133 and Arg134, respectively) are located at the C-terminal end of a-helix D near the peptide-binding site (Knighton et al., 1991a). These residues and their relationship to the peptide inhibitor are indicated in Fig. 2 ( A and B ) . Also indicated are the 3 conserved glutamate residues (GlulZ7, Glu17', and GluZ3O in the mammalian enzyme) that contact the

Recognition Sites for Regulatory Subunit in Protein Kinase A TABLE IV

Kinetic parameters and regulatory subunit affinities of C1 mutants defective only in the recognition of the regulatory subunit

Kinetic parameters and the 50% inhibitory concentrations (ICm values) were determined using purified mutant enzvmes as described in the text. The multide-substitution mutant 28X+38+41 was unable to be purified, thus,

4811

an iC,, value could not be determined for this mutant. However, the kinetic parameters were deteimined for the mutant enzyme in crude cell extracts. The standard errors in the kinetic parameters are 4 0 % .

Mutant Mutation in yeast C1 subunit residue in murine C subunit Lt Km (M~ATP) K m (Kemptide) kdKm ( a m p t i ) IC,

Wild type Single amino acid substitutions

28V R177A 28W K178A 38X K233A 38Y D237A 41X K257A 41Y K261A Y240A Y240A V238A V238A

28X R177A, K178A 38 K233A, D237A 41 K257A, K261A

Multiple amino acid substitutions 28X+38 R177A, K178A, K233A, D237A 28X+41 R177A. K178A. K257A. K261A

Double amino acid substitutions

R133 R134 K189 G193 K213 K217 W196 R194

8-1 P M

6.5 39

13.4 39 13.9 55 10.0 37 10.9 38 10.5 46 15.9 36 4.1 38 6.3 37

6.2 49 5.4 41 6.2 43

6.7 55 12.8 57

mM

0.096

0.130 0.137 0.086 0.101 0.092 0.162 0.087 0.122

0.217 0.085 0.112

0.211 0.440

s"lmM 68

103 101 116 108 114 98 47 52

29 64 55

32 29

nM 17

53 43 36 53 42 72 36 44

73 164 134

3500 6000

38+41 K233A; D237A; K257A; K261A 5.1 50 0.138 37 4400 28X+38+41 R177A, K178A, K233A, D237A, K257A, K261A 4.2 53 0.400 10 >>E1000

2 arginine residues at positions P-2 and P-3 that constitute the dibasic recognition sequence (Knighton et al., 1991b). The side chain of Arg'33 interacts with the side chain of GluZ3' and with the backbone carbonyl of the threonine residue at position P-5 in the peptide inhibitor. Thus, Arg'33 could contribute to the recognition of the regulatory subunit by contacting the polypeptide backbone at this position, but would not be involved in the recognition of Kemptide, the N terminus of which only extends to position P-4.

The residues in the mammalian enzyme equivalent to LYS'~~, Asp237, LysZs7, and LysZ6l (LyslS9, Glylg3, Lys213, and Lys217, respectively) cluster to define a binding site for the regulatory subunit that is adjacent to but separate from the peptide-binding site. Fig. 2 ( B and C ) shows the location of this Fegion in the larger C-terminal domain just distal to the peptide-binding site. Lysla9 and Glylg3 are located on &strand 9 and on a surface loop that extends from the C-terminal end of @-strand 9. Glylg3 is at the apex of the loop, whereas Lysla9 is in the @-strand itself. The face of this loop constitutes most of the surface on the larger lobe that lies just beyond the cleft between the two lobes of the enzyme. Lys213 and Lys217 are located nearby on an extended chain that precedes a-helix F. This segment is sandwiched beneath the loop containing L ~ S " ~ and Glylg3 and the loop that connects helices H and I. Only the outer edge of this strand is exposed to the surface. Lys217 appears to make a direct contact with the backbone carbonyl of Glylg3 in the upper loop and thus serves to stabilize the upper loop. In contrast, the side chain of Lys213 in the binary complex is exposed to the solvent. The solvent-acces- sible lysines were mapped previously by their reactivity to acetic anhydride, and these results confirm that Lys213 is highly reactive, whereas Lys217 reacts poorly (Buechler et al., 1989). The side chains of 2 additional residues (Arglg4 and TrpIg6) were highly exposed in this region of the structure. The equivalent residues in the yeast C1 subunit (Val238 and

respectively) are uncharged and therefore were not mutated in the original charged-to-alanine scan. These residues were substituted with alanine, and the mutant enzymes

(V238A and Y240A) were purified and analyzed. These mutants displayed similar phenotypes to the other single-substitution mutants from the same region (Table IV). The affinity of these C1 mutants for the regulatory subunit as determined from the ICs0 value was decreased by 2.6- and 2.1-fold, respectively, whereas the catalytic efficiency against Kemptide was decreased by 1.3- and 1.5-fold.

C1 Mutants Defectiue in Recognition of Regulatory Subunit Are Heat Shock-sensitiue-Yeast cells respond to nutrient limitation by arresting in the G1 phase of the cell cycle and assuming a physiological state in which they can, among other things, survive elevate? temperatures (Pringle and Hartwell, 1981). Yeast strains with a defective regulatory subunit (bcyl-) fail to respond to nutrient limitation and exhibit heat- shock sensitivity. This phenotype can be suppressed by mutations that decrease the activity of the catalytic subunit (Cameron et al., 1988). The C1 mutants defective in the recognition of the regulatory subunit that retain wild-type levels of activity, identified in this study, are analogous to a bcyl- mutant and should display the heat shock-sensitive phenotype. Fig. 3 illustrates that yeast strains containing double (28X, 38, and 41) and multiple (28X+38, 28X+41, 38+41, and 28X+38+41) amino acid substitutions in the C1 subunit that disrupt the recognition of the regulatory subunit are sensitive to a heat shock at 60 "C for 50 min.

DISCUSSION

The autoinhibitor model, in which a pseudosubstrate or autophosphorylation site in the regulatory subunit binds to the active site of the catalytic subunit in a manner analogous to a substrate, provides the basis for the mechanism of inhibition of the catalytic subunit by the regulatory subunit (reviewed by Hardie (1988) and Taylor et al. (1990)). However, several lines of evidence suggested that there must be interactions between the catalytic and regulatory subunits additional to those made between the active and autoinhibitor


n

Control Heat Shock

60%, 50 minutes

28X 28x38 28X+38

38 28x41 28x41

41 3041 3841

WT 28X+3&41 WT 28X+3841

WT WT

FIG. 3. Heat shock sensitivity of C1 mutants defective in recognition of regulatory subunit. Yeast strains expressing C, mutants containing multiple amino acid substitutions that decrease the affinity for the regulatory subunit were used to inoculate plates that were subjected to a heat shock at 60 'C for 50 min. After cooling, the plates were incubated at 30 "C for 3 days. WT, wild type.

sites. In this study, site-directed mutagenesis was used to define two additional binding regions for the regulatory subunit on the catalytic subunit of the CAMP-dependent protein kinase from yeast.

The binding determinants in the catalytic subunit for the consensus recognition sequence in peptide substrates have been clearly identified by a variety of approaches described earlier. This consensus recognition sequence, shared by all substrates and inhibitors of cAMP-dependent protein kinase, is defined in Table I. The location of this autoinhibitor/ peptide substrate-binding region in the three-dimensional structure of the mammalian enzyme is shown in Figure 2. In this study, we demonstrate that this region is also important for the recognition of the regulatory subunit as predicted by the autoinhibitor model.

Additional determinants on the C1 subunit important for binding the regulatory subunit were identified in this study by screening the charged-to-alanine mutants of the C1 subunit for those that were defective in the recognition of the regulatory subunit yet retained high levels of activity against the peptide substrate Kemptide. Two residues identified as being important for regulatory subunit binding but not for Kemp- tide binding (Arg'" and LYS"~) mapped to a region near the proximal side of the autoinhibitor/peptide-binding site. These residues are probably important for recognizing the residues present in the autophosphorylation site of the yeast R subunit that are not present in Kemptide.

A second major recognition site was identified that presum-

FIG. 2. Localization of residues important for recognition of regulatory subunit. Residues are highlighted on a space-tllhg model of the structure of the catalytic subunit of the mammalian CAMP-dependent protein kinase bound to a peptide inhibitor derived from PKI (Knighton et d, 1991b). The enzyme is colored blue; the peptide inhibitor is highlighted in red; and residues that are important for the recognition of the regulatory subunit are highlighted in yelbw. A illustrates the peptide-binding site; the 3 glutamate residues (Glum, Glu"O, and Glum') that are involved in the recognition of the dibasic consensus sequence are highlighted in pwpk. Arg'" and Arg'", residues that are important for binding of the regulatory subunit, are highlighted in yellow. The side chain of Arg'" points toward and interacts with the peptide backbone at position P-5 and interacts with GI@'. B illustrates a rotation of 90' about the vertical axis and demonstrates that the additional binding site for the regulatory mbunit identi€ied in this study is located on the opposite face of the molecule from the peptide-binding site. C represents a further 90' rotation about the vertical axis and illustrates this additional site distal to the C terminus of the peptide inhibitor. Residues important for the recognition of the regulatory subunit are highlighted in yellow. Clockwise from the bottom: LysZ1', Trp", and Lys'@ and clustered together, Arg'", Gly'88, and Lys"'.

Recognition Sites for Regulatoly Subunit in Protein Kinase A 4013

ably recognizes a portion of the regulatory subunit that lies distal to the consensus autoinhibitor site. Mutants involving the substitution of L y P , Asp237, LYS*'~, and Lysml were also defective in the recognition of the regulatory subunit, but not in the recognition of Kemptide. When the equivalent residues in the m '. enzyme were mapped on the three-dimensional S t ~ ~ t ~ r e , they were clustered together on the surface of the C-terminal domain distal to the C terminus of the inhibitor peptide that occupies the peptide-binding site (Fig. 2, B and C). The side chains of 2 residues in this region (kg'% and TIP'%) were highly exposed and the equivalent residues in the yeast C1 subunit (Valzas and TYPO, respectively) were shown to be important for the recognition of the regulatory subunit.

A dominant feature of this distal recognition region is Thra4', a residue in the yeast C1 subunit that was identified previously in a genetic screen as being important for interactions with the regulatory subunit. The substitution of this residue with alanine causes a 25-fold decrease in affinity for the yeast R subunit (Levin et aL, 1988; Levin and Zoller, 1990). This threonine residue ( T h P 7 in the mammalian enzyme) is one of two stable phosphorylation sites in the catalytic subunit (Shoji et d., 1979) and is phosphorylated auto- catalytically in uiuo (Yonemoto et d., 1991). The negative charge on the phosphate is presumably important since substitution with aspartate or glutamate can partially restore affinity for the regulatory subunit (Levin and Zoller, 1990). The phosphatase resistance of this threonine suggested that it may contribute to the structural stability of the enzyme (Shoji et d, 1979), and this prediction is consistent with the crystal structure. The ligands of the phosphate group identified from the crystal structure are His", Ar&=, and Lysl= (Fig. 4) (Knighton et d., 1991a). Lysl= also does not react with acetic anhydride, indicating independently that it is sequestered and inaccessible to solvent (Buechler et d, 1989). The replacement of each of these ligands produces an enzyme with an unregulated phenotype. However, only the substitution of the residue equivalent to Lysl= (LysZas) causes defective recognition of the R subunit without impaired catalytic activity (Table IV). The residues equivalent to Hiss7 and Arglm in the yeast C1 subunit (His13' and ArgW, respectively) were also mutated to alanine. These mutants were likewise defective in regulatory subunit binding, but also displayed defects in kt, Km(~emptida) and K m ( ~ A ~ ) (Table III). Although the molecular basis for each of these unregulated phenotypes remains to be established, the phosphothreonine is clearly an important structural determinanG and the substitution of threonine itself, the residues coordinated to the phosphate, or other residues in close proximity is apparently sufficient to perturb the structure of this region in such a way that interactions with the regulatory subunit are diminished.

A potentially phosphorylatable threonine or tyrosine residue can be found in this region in almost all the protein kinases, except for the calcium/calmodulin-dependent subfamily (Table V) (Hanks et d, 1988). A phosphotyrosine residue in this region was shown to be important for the kinase activity of P130&"g''t" and pp6OC-" (Weinmaster et d., 19& Kmiecik and Shalloway, 1987), and the autophosphorylation of 2 tyrosine residues in this region is correlated with the activation of the insulin receptor kinase upon insulin binding (Zhang et d, 1991). In p34-, the equivalent phosphothreonine 167 is required for kinase activity (Booher and Beach, 1986), and phosphorylation of this residue may be required for cyclin-mediated activation of cdc2 kinase (Du- commun et al., 1991). In addition, one of the ligands of phosphothreonine 197 in mammalian CAMP-dependent pro-

Fro. 4. Localization of phosphothreonine 197 and residues that coordinate phosphate group. The a-carbon backbone of the catalytic subunit is shown in blue, the peptide inhibitor is shown in red. Phosphothreonine 197 (green) is located in the binding site for the regulatory subunit distal to the C terminus of the peptide. The side chains that coordinate the phosphate group (His@', Arg'=, and Lys'") are shown in yellow along with the other residues initially identified ae beiig important for the recognition of the regulatory subunit (Gly'Bg, Lysa13, and LyP').

tein kinase is Hiss7; this residue is equivalent to the threonine residue in the PSTAIR sequence that is conserved among the cdc2-related protein kinases (Hanks et d, 1988). This sequence has also been suggested as being important for the association of cdc2 with cyclins (Ducommun et d, 1991). It is possible that the region identified in our study is utilized by a variety of protein kinases for interactions with regulatory molecules or domains.

The presence of additional binding sites for the regulatory subunit on the catalytic subunit indicates that the determinants for the recognition of the regulatory subunit extend beyond the consensus autoinhibitor site, particularly on the C-terminal side. The importance of the au to ib i to r site in the hinge region of the regulatory subunit for the binding of the catalytic subunit has been clearly demonstrated. The region responsible for the high affinity binding of PKI resides primarily on the N-terminal side of the consensus sequence (Scott et d, 1986; Cheng et d., 1986; Glass et d, 1989; Walsh et d., 1990; Knighton et d, 1991b). However, proteolysis of the mammalian RI subunit just N-terminal to the consensus sequence produces a fragment capable of forming a high affinity complex with the catalytic subunit (Weldon and Taylor, 1985; Bubis and Taylor, 1987), suggesting that the region of the regulatory subunit on the C-terminal side of the autoinhibitor site is important for binding the catalytic subunit. This would be consistent with the location of the additional binding site in the catalytic subunit identified in this study distal to the C terminus of the inhibitor peptide. C A M P - binding site A must also communicate directly or indirectly with the sites of contact between the regulatory and catalytic subunits since C A M P still promotes the dissociation of a regulatory subunit mutant in which the autoinhibitor site is disrupted (Wang et d, 1991) or when CAMP-binding site B is deleted (Ringheim and Taylor, 1990). In addition, mutations in the CAMP-binding domains of the mammalian RI and yeast R subunits have been shown to affect holoenzyme formation (Woodford et d, 1989; Neitzel et d, 1991; Cannon et d, 1990; Dostmann and Taylor, 1991).


TABLE V Conservation of phosphorylated residues in the vicinity of threonine 197 in the mammalian catalytic subunit

The region between conserved subdomains VI1 (DFG) and VI11 (APE) is aligned according to Hanks et al. (1988). Phosuhorvlated residues are indicated in boldface tme.

Protein kinase Seauence ~ ~~~ ~ ~~~

Mammalian C D F G F A K R V K G R T - - - - - W T L C G T P E Y I = Yeast C1 D F G F A K Y V P D V T - - - - - Y T L C G T P D Y I E

pp60""" ~ 1 3 0 8 " ~ ~ " - D F G M S R E E A D G V Y A A S G G L R Q V P V K W T A P E Insulin receptor D F G M T R D I Y E T D Y Y R K G G K G L L P V R W M A P E

p34""2 E L A R S F G V P L R N Y - - T H E I V T L W Y R A P E - D F G L A R L I E D N E Y T A R - Q G A K F P I K W T A P E

The C1 mutants identified in this study as being defective in R subunit recognition at a site separate from the consensus sequence-binding site have a conditional lethal phenotype in vivo (heat shock sensitivity). The heat shock sensitivity of the unregulated C1 mutant (ThP41 +Ala) could be suppressed by the overexpression of a regulatory subunit mutant (Ser"5 + Ala) that binds the catalytic subunit 10-fold more tightly (Kuret et al., 1988); Thus, the selection of suppressing mutations in the gene for the regulatory subunit may allow the identification of the region of the R subunit that interacts with the additional binding region on the catalytic subunit identified in this study.

Acknowledgments-We would like to thank Mark Vasser and Peter Ng for synthesizing and purifying the oligonucleotides used in this study. We would also like to thank Karen Johnson for the expression and purification of the deleted yeast R subunit, Mark Montella for the photography used in this manuscript, and Wes Yonemoto for useful discussions regarding this manuscript.

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",I._ _""_

systematic mutational analysis of camp-dependent protein kinase

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