Proc. Nati. Acad. Sci. USAVol. 90, pp. 442-446, January 1993Biochemistry

The conserved lysine of the catalytic domain of protein kinases isactively involved in the phosphotransfer reaction and notrequired for anchoring ATP

(enzyme mechanism/protein-tyroslne kinase/phosphorylation)

ANA C. CARRERA, KIRILL ALEXANDROV, AND THOMAS M. ROBERTS*Dana-Farber Cancer Institute, Department of Cellular and Molecular Biology, Harvard Medical School, Boston, MA 02115

Communicated by Ruth Sager, October 21, 1992 (received for review September 9, 1992)

ABSTRACT The study of the various protein kinasesreveals that, despite their considerably diversity, they haveevolved from a common origin. Eleven conserved subdomainshave been described that encompass the catalytic core of theseenzymes. One of these conserved regions, subdomain II, con-tains an invariant lysine residue present in all known proteinkinase catalytic domains. Two facts have suggested that thisconserved lysine of subdomain II is essential for binding ATP:(i) several investigators have demonstrated that this residue isphysically proximal to the ATP molecule, and (ig) conservativesubstitutions at this site render the kinase inactive. However,these results are also consistent with a functional role of theconserved lysine of subdomain H in orienting or facilitating thetransfer of phosphate. To study in more detail the role ofsubdomain II, we have generated mutants of the protein-tyrosine kinase pp56k4k that have single amino acid substitu-tions within the area surrounding the conserved residue Lys-273 in subdomain II. When compared with wild-type pp56kk,these mutants displayed profound reductions in their phos-photransfer efficiencies and small differences in their affinitiesfor ATP. Further, the substitution of argnine for Lys-273resulted in a mutant protein unable to transfer the -phosphateofATP but able to bind 8-azido-ATP with an efficiency similarto that of wild-type ppS6"k. These results suggest that theregion including Lys-273 of subdomain II is involved in theenzymatic process of phosphate transfer, rather than in an-choring ATP.

Protein kinases (PKs) are phosphotransferases that catalyzethe transfer of the y-phosphate ofATP to an amino acid sidechain (for review see refs. 1-7). Sequence similarities definetwo major units in the family of PKs: a conserved catalyticcore and nonconserved flanking regions (1). The peripheralnonconserved regions flanking the catalytic core are impor-tant for functions such as regulation and subcellular local-ization (1, 2). The sequence alignment of the catalytic do-mains of PKs (1) reveals that the conservation is not uniformbut, rather, consists of alternating regions of high and lowhomology. Eleven major conserved subdomains have beenidentified (I to XI), which are separated by regions of lowerconservation (1).The first clue for localizing the ATP binding site within the

catalytic core came from studies on the cAMP-dependentkinase. Affinity labeling with the ATP analog 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA; ref. 8) inhibited theenzyme by covalently modifying Lys-72 (8), a conservedresidue of subdomain II. FSBA contains a reactive group ata position that approximates the y-phosphate of ATP. Theproximity of Lys-72 to the y-phosphate of ATP was con-firmed by other investigators (6, 7, 9, 10). The inactivation

observed in several PKs upon site-directed mutagenesis ofthis conserved lysine of subdomain 11 (11, 12), together withthe previous data, supported the idea that the conservedlysine of subdomain II is essential for the binding of ATP.However, as other investigators have pointed out (11), all theevidence presented to date is also consistent with the possibleparticipation of this subdomain in the actual mechanism ofphosphate transfer.A second area of the kinase domain that has been impli-

cated in ATP binding is the glycine-rich loop of subdomain I(10). This loop, displaying the consensus sequence Gly-Xaa-Gly-Xaa-Xaa-Gly, is close to the phosphates ofMgATP in thecrystal structure of the cAMP-dependent kinase (6). Thenearly invariant Gly-50 and Gly-52 fall within this consensussequence. This motif is part of the Rossmann fold structureassociated with many nucleotide binding sites (13). A similarmotif containing a glycine-rich loop is found in proteins asdiverse as adenylate kinase (14), GTP-binding proteins suchas p2lms (P loop; ref. 15), hexokinase (16), HSC70 (17), andactin (18). The single motif common to all these nucleotide-binding proteins is the glycine-rich motif, suggesting that itmay serve as phosphate anchor (19).The available data on the functional role of subdomains I

and II of the catalytic core of PKs do not clearly establishwhether the conserved lysine of subdomain II is essential forATP binding or whether its major role is related to the actualtransfer of phosphate. To analyze the role of subdomain II,we have studied 10 mutants with single amino acid substitu-tions in the vicinity of Lys-273 of the lymphoid protein-tyrosine kinase pp56lck.

EXPERIMENTAL PROCEDURESMutant Construction. To prepare substitution mutants in

subdomain II of pp56lck, we used syn-k, synthetic Ick geneencoding pp56lck (unpublished work), cloned into Gex-2Tvector (20). Mutants have been generated by replacement ofthe codons encoding Lys-269 to Lys-276 by the correspond-ing synthetic fragment containing an 8% level of sequencedegeneracy. Escherichia coli JM109 (Stratagene) coloniestransformed with the mixture of mutated constructs werepicked and screened for overexpression of full-length mole-cules. DNA preparations obtained from the bacterial coloniesproducing full-length proteins were then sequenced. Tenmutants, corresponding to single amino acid substitutionsbetween Lys-269 and Lys-276, were selected for furtheranalysis.

Analysis of Protein Production. Several parameters wereoptimized for induction of protein production to achievemaximal activity and solubility of the protein. Soluble and

Abbreviations: FSBA, 5'-(p-fluorosulfonylbenzoyl)adenosine;GST, glutathione S-transferase; PK, protein kinase.*To whom reprint requests should be addressed.

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insoluble fractions were separated by centrifugation at 13,000x g for 30 min at 40C. The amount of pp56lck protein presentin each fraction was estimated by Western blotting (as in ref.21) using anti-pp561ck antibodies (Santa Cruz Biotechnology,Santa Cruz, CA). The activity of the soluble products wasanalyzed in kinase reactions using enolase as a substrate (asbelow). The optimal conditions for enzyme solubility andactivity were obtained with overnight cultures of E. coli X90(22) diluted 1:10 and incubated for 4 hr in TY medium at 370C(in the absence of isopropyl 8-D-thiogalactopyranoside).Cells were recovered by centrifugation and suspended in 1%(vol/vol) Triton X-100 with phenyl methylsulfonyl fluoride (2mM), aprotinin (0.15 unit/ml), leupeptin (2.5 mg/ml), pep-statin (10 pg/ml), NaF (5 AM), and Na3VO4 (2 mM) at 40C.Sonication was performed by two rounds of 10-sec pulses (5min apart) at a duty cycle of70% and output control of 5 (HeatSystems Ultrasonics, Farmingdale, NY, model W225).The glutathione S-transferase (GST)-pp56lck fusion protein

(G-pp56lck) was purified as described (20). Protein concen-tration was estimated by Coomassie brilliant blue R(Sigma)staining of SDS/polyacrylamide gels, with bovine serumalbumin as standard, or by binchoninic acid (BCA) assay(Pierce). Western blot analysis was performed as reported(21). Anti-phosphotyrosine antibodies were prepared by B.Druker in our laboratory.

Kinase Reactions, Data Analysis, and Photoaffinity Label-ing. For kinase reactions, 10 ,l containing 50 ng of purifiedkinase was preincubated at 25°C for 1 min and mixed with 20,ul of 2x kinase reaction cocktail and 10 ,ul of acid-denaturedenolase (at the appropriate concentration). The cocktailcontained 50 mM Tris-HC1 (pH 7.4), 10 mM MnC12, and theappropriate dilution of the ATP stock (100 ,uM ATP, 10 ,Ciof [y-32P]ATP per ,ul, 3000 Ci/mmol, NEN/DuPont; 1 Ci =37 GBq). Reaction mixtures were incubated at 25°C for 2 min(mixed every 30 sec), and reactions were terminated byaddition of 10 ,ul of 100 mM EDTA (pH 8.0). For thecomparison of G-pp56Ick and baculoviral pp56lck (30%o pure;ref. 23), reaction mixtures were incubated for 5 min. Sub-strate and enzyme were resolved by SDS/PAGE. For thedetermination of kinetic parameters, phosphate incorporatedinto enolase was quantitated by liquid scintillation counting.Vmx and Km were estimated by graphic methods (24, 25).Photoaffinity labeling of G-pp56Ick was performed as de-scribed (26).

RESULTSMutant Construction. To distinguish between a passive role

of subdomain II in anchoring ATP or a catalytic role in thephosphorylation reaction, we chose to analyze the kineticconsequences of introducing single amino acid substitutions(conservative or nonconservative) at residues located be-tween Lys-269 and Lys-276 of pp56Ick. Wild-type and mu-tated genes encoding pp56Ick were cloned in p-Gex-2T tofacilitate production of mutant proteins referred to asG-pp561ck in bacteria and subsequent purification. Fig. 1Aillustrates the area of subdomain II of pp56lck examined, aswell as the various single amino acid substitutions selectedfor the study.

G-ppS6I-k Protein Production, Purification, and EnzymaticCharacterization. To compare mutants in subdomain II withwild-type G-pp56lck, we first optimized the expression of thewild-type enzyme in bacteria (see Experimental Procedures).The optimal conditions yielded maximal activity (see belowfor comparison with baculoviral pp56Ick) and solubility(=90%). Using these conditions, we compared the lysates ofnontransformed E. coli X90 (C3) with lysates ofX90 bacteriaexpressing wild-type fusion protein G-pp561ck (WT), mutatedG-pp561ck (see nomenclature in Fig. lA), a GST-Ser/Thrkinase Raf-1 fusion protein (Cl), and GST (C2). Analysis of

A Subdomain 11

K269N(-4)

v270L(-3)

A271S(-2)

V K S L272 273 274 275A R N M(-1) (MI) (+1) (+2)

N(M2) M(M3)

K276V

(+3)

BN C - N NCSn+ ,r C C O

101-

71- -S

-S ~ ~ +

43L,-I..4371-* | s_ o

3

D0 - E1 01- -1

743-

CY cE N y)

t, N C

a

FIG. 1. Comparison of E. coli lysates containing wild-type ormutated G-ppS6lck: (A) Representation of the positions of wild-typepp5ock that have been substituted (upper line) and the correspondingamino acids introduced (lower line). Numbers in parentheses rep-resent the positions of the residues relative to Lys-273. Substitutionsof Lys-273 were named Ml (Lys-273 -. Arg), M2 (Lys-273 -. Asn),and M3 (Lys-273 -+ Met). (B-D) E. coli X90 bacteria were trans-formed with constructs encoding wild-type (WT) or single-substitution mutants of G-pp56ock (named as in A), GST-Ser/Thrkinase Raf-1 fusion protein (Cl), or GST (C2) or with medium alone(C3). Bacteria were induced and lysed, and soluble proteins wererecovered by centrifugation. Fifteen microliters of each lysate wasloaded on an SDS/10%o polyacrylamide gel and analyzed by Westernblotting using anti-pp561ck antibodies (B), Coomassie blue staining(C), or Western blotting using anti-phosphotyrosine antibodies (D).MW, molecular weight markers (Mr x 10-3 at left).

15 pul of each lysate by Western blotting with anti-pp56Ickantibodies revealed that bacteria transformed with the vari-ous G-pp561ck constructs, but not the controls, containedsimilar amounts of G-pp561ck (Fig. 1B, an 82-kDa bandcorresponding to 26 kDa ofGST fused to 56 kDa of pps6lck).Further, a similar protein composition was found (Fig. 1C)when the same volume of the various preparations of X90bacterial lysates (containing the various constructs) wereanalyzed by Coomassie blue staining. In contrast, differentintensities of phosphotyrosine signal were detected whensimilar volumes of the lysates were compared by anti-phosphotyrosine Western blotting (Fig. 1D). These resultsindicate that the different mutants display different kinaseactivities.

G-pp56Ick was purified as described (20). This procedureyielded apparently pure wild-type or mutated G-pp561ck asjudged by Coomassie blue staining (Fig. 2A). Purified wild-type G-pp56lck was highly active as estimated by autophos-phorylation (Fig. 2B). The Gex-2T-syn-k construct encodingG-pp561ck includes in its sequence a protease site (thrombin)

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A

c:

cs

B

a:Cf)Is-

--2i_8

- 2 0 8 -

-1701 - 1i1-

-4 3 -

-2 8 -

-1 8-

FIG. 2. SDS/PAGE analysis of purity and autokinase activity ofpurified G-pp,6Ick: Wild-type (WT) and Lys-273 -. Arg mutant(K273R) G-pp56Ick proteins were produced in E. coli X90. The fusionprotein present in the bacterial lysates was purified by using glu-tathione-Sepharose beads (200 ng of pure protein obtained from 400/kg of the total soluble protein fraction). Purified WT and K273RG-pp561ck were analyzed by SDS/PAGE followed by Coomassie bluestaining (A). Samples were also tested for their autophosphorylatingactivity in vitro and resolved by SDS/PAGE. The resulting gel wasanalyzed by autoradiography (B). MWM, molecular weight markers(Mr X 10-3 at right in A).

located between the GST fragment and pp5sck. We alsocompared the phosphotransfer activity of the fusion proteinG-pp56Ick with (i) similar amounts of bacterial pp56lck ob-tained upon cleavage of the GST fragment with protease (asin ref. 20) and (ii) similar amounts of G-pp561ck immunopu-rified by using anti-pp56lCk antibodies (as in ref. 21). Thespecific kinase activity present in the various preparationswas comparable (data not shown). Unfortunately, proteasetreatment caused a significant amount of a pp56ick breakdownproduct, and immunopurification failed to purify pp56ck tohomogeneity. Purification of the wild-type and mutantG-pp561ck proteins using glutathione beads yielded =50 ng ofpure G-pp561ck from 100 pug of total soluble bacterial protein.To determine the enzymatic parameters of G-pp561ck, the

concentration of purified enzyme was estimated by SDS/PAGE followed by Coomassie blue staining. Fifty nanogramsof G-pp561ck was mixed with various amounts of ATP andenolase and subjected to kinase reaction. A time course ofthereaction revealed that the incorporation of phosphate waslinear at least for the first 5 min (data not shown). Therefore,for all the assays, 2-min incubations were used to remain inthe linear range. To measure the Km of G-pp561ck for ATP,enolase concentration was fixed at 5.5 puM and ATP concen-tration was varied from 0.25 to 10 ,M (corresponding to<0.5x to >5x Km of G-pp561ck for ATP). To calculate the Kmfor enolase, ATP concentration was fixed at 5 A&M andenolase was varied from 0.34 to 22 ,uM (corresponding to<0.1 X to >3 X Km of G-pp56Ick for enolase). To evaluate thephosphotransfer activity, we determined the apparent Vnfor enolase phosphorylation in the presence of excess ATP (5,uM ATP, corresponding to Sx K.n). Enzyme and substratewere resolved by SDS/PAGE. To estimate initial velocity,

the amount of phosphate incorporated into enolase wasmeasured by liquid scintillation counting. The data wereevaluated by Eisenthal-Cornish-Bowden (24), and Line-weaver-Burk (25) approximations, which yielded similarvalues in every case.The values of apparent Km and V,: obtained for bacterial

G-pp561ck using enolase as a substrate were as follows: KmATP = 0.97 + 0.20 ,uM (mean ± SD), Km enolase = 5.58 ±0.87 AuM, and Vma,, = 22.74 + 1.56 nmol/(min-mg) (Table 1).The G-pp561ck preparation had an apparent Km for ATPsimilar to that of purified baculoviral pp56Ick (D. Winkler,personal communication). With regard to the apparent Vmadifferent values have been reported even for the same en-zyme preparation, depending on the substrate analyzed(highest values have been obtained with T-cell receptor{-chain peptides; ref. 27). However, when compared underthe same reaction conditions, baculoviral pp56lck and bacte-rial G-pp561ck displayed similar phosphotransfer activities(Fig. 3).

Analysis of pp563& Mutants Containing Single Amino AcidSubstitutions in Subdomain H. To determine whether thedecreased kinase activity ofthe single amino acid substitutionmutants in residues Lys-269 to Lys-276 (Fig. 1) was due to adecrease in the binding ofATP or, alternatively, to a decreasein the efficiency of the phosphotransfer reaction, we evalu-ated the kinetic parameters of the mutants in vitro. Mutantswere purified (as above) and the Km for ATP and Vm,, forenolase were determined (as above). In agreement withprevious studies performed with pp6Osrc and epidermalgrowth factor receptor (11, 12, 28), mutations at the position273 of pp5sck rendered the kinase inactive. Substitutions inall of the other positions, between 269 and 276, yieldedpartially active proteins. The Km for ATP of each of themutants was similar to the Km for ATP of wild-type pp5s6ck(Table 1). In contrast, every substitution yielded a pps6Ickprotein with lower phosphotransfer activity than wild-typeG-pp561ck.The similarity of the Km for ATP of conservative and

nonconservative substitution mutants within subdomain IIsuggests that this area is not likely to be responsible for ATPbinding. In addition, the fact that the enzyme phosphotrans-fer efficiency is significantly altered when residues in thevicinity of Lys-273 are substituted suggests that Lys-273/subdomain II is involved in the process ofphosphate transfer.Comparison of the ATP-Binding Ability of Wild-Type

G-pp56'C and Lys-273 -+ Arg Substitution Mutant. The ki-netic analysis ofthe single-substitution mutants ofsubdomainII suggested that this subdomain is not directly involved inanchoring ATP. If this is the case, the inactive mutant atposition 273 should be able to bind ATP with similar effi-ciency compared with wild-type pp56lck. To determinewhether this hypothesis was correct, we chose to use ATP

Table 1. Analysis of the kinetics of subdomain II mutantsof G-pp56Ick

Km,utMVK.,AM Y~~~~max.Substitution* ATP Enolase nmol/(min-mg)Wild type 0.97 ± 0.20 5.58 ± 0.87 22.74 ± 1.56K273Xt ND ND 0V272A (-1) 1.92 ± 0.36 1.86 ± 0.28 0.50 ± 0.02S274N (+1) 2.06 ± 0.53 5.61 ± 0.75 2.90 ± 0.16A271S (-2) 0.95 ± 0.35 10.12 ± 3.58 5.68 ± 4.56L275M (+2) 0.83 ± 0.25 4.77 ± 1.59 3.66 ± 0.76V270L (-3) 1.61 ± 0.25 7.74 ± 0.71 18.95 ± 2.54K276V (+3) 1.08 ± 0.59 9.00 ± 2.29 6.46 ± 0.74K269N (-4) 1.28 ± 0.42 2.92 + 0.58 6.24 + 0.72*See Fig. 1A.tX = R (Arg), M (Met), or Asn (N).

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A

0 .0CM , >Ye g

B

MWM

c:

CNBe

.X0I

CD)-.D

._l

to'__

- 2 0 8

-10101 _ _

-7 1

IN _ a-43.

FIG. 3. Comparison of the activity of bacterial arpp5oCk: Bacterial wild-type (G-lck) and Lys-273 --

(K273) G-ppS61ck proteins were produced and purifiedBaculoviral pp,56ck (bv-lck) was produced and purified(21, 23). Fifty nanograms of baculoviral or bacterial ccated with arrows) was tested for the ability to pacid-denatured enolase (band at 43 kDa). After the kirsamples were resolved by SDS/10% PAGE andCoomassie Blue staining (A) or autoradiography (B). Nular weight markers (M, x 10-3).

analogs containing a crosslinking group. FSBA N

because it contains the crosslinking group in Ianalogous to the y-phosphate (8) whose transferpositioning are probably regulated by Lys-273.used 8-azido-[y-32P]ATP whose crosslinking grolon carbon 8 ofthe adenosine. When similar amoui

A

0d

_ a

'S.

6

*4

I +

N¢4 N

XX X

MWM a y

-208- L

-1 01- l

- 7 1 -

U~~~~~~~~~~

-~~~~~~~~~~~~

FIG. 4. Photoaffinity labeling of wild-type and L)mutant G-ppS6Ick with 8-azido-ATP: One hundred Xpurified control GST protein (Gex-2T), wild-type G-pp.mutated G-pp561ck (K273R) was mixed with 8-azido-I40C. Photoaffinity labeling was achieved when theirradiated (+). After 5 min, samples were boiled in L.and resolved by SDS/1Oo PAGE. Proteins wereCoomassie blue staining (A) and autoradiography (B)lecular weight markers (Mr x 10-3).

of purified wild-type or Lys-273 -* Arg mutant G-pp561ckwere analyzed, the mutant at position 273 bound an amountof 8-azido-ATP similar to the amount bound by the wild-typeenzyme (Fig. 4B). In contrast, GST incubated under similarconditions yielded a small background signal (Fig. 4B). The

MWM small signal obtained when wild-type G-pp561ck was incu-- 2 0 8 bated in the absence of UV light (Fig. 4B) corresponds to the

residual phosphotransfer activity of pp56lck at the tempera-ture of incubation (40C). The signals of 8-azido-[32P]ATP

-1 01 incorporated into wild-type and mutated pp56Ick were due tospecific ATP binding, as judged by the decrease in these

7 1 signals observed upon addition of EDTA, absence of Mn2 ,

or addition of excess nonradioactive ATP (data not shown).Upon subtraction of the background signal, the ratio ofmutant to wild-type signal (cpm/cpm) was calculated from

4 3 five different experiments. The value obtained, 0.98 + 0.33,indicates that a similar amount ofATP reacts with wild-typeenzyme and with the Lys-273 -* Arg mutant.

DISCUSSIONThe results indicate that single amino acid substitutions in thearea of Lys-273 affect the ability of pp56lck to transfer

id eukaryotic phosphate to a protein substrate but do not significantly alterArg mutant its ability to bind ATP.as in Fig. 2. Two motifs have been classically implicated in the regu-as described lation of the ATP binding: the glycine-rich loop (10), and theenzyme (indi- conserved lysine of subdomain II (Lys-72 of cAMP kinase,hosphorylate Lys-273 of pp56lck; refs. 8 and 9). Initially these sites wereiase reaction, defined by labeling of the cAMP-dependent kinase withaWM molec- acetic anhydride: Lys-47 (next to the Gly-Xaa-Gly-Xaa-Xaa-

Gly motif) and Lys-72 were protected by MgATP againstmodification with acetic anhydride (10). These elegant stud-

was avoided ies provided information about which areas of a kinase werethe position proximal to the ATP, but did not delineate the specific roleand correct of each of these areas. Mutagenesis revealed that bothandc e regions were required for the kinase to be active (10, 11, 28,Instead, we 29), but again did not establish specific roles.

Up is located To study the functional role of the conserved lysine ofnts (Fig. 4A) subdomain II (Lys-273 of pp56ock), we chose to prepare

random mutations in each of the residues located betweenB Lys-269 and Lys-276 of pp561ck. The central interest of our

analysis was to distinguish between a passive role in ATP+ binding and an active role for Lys-273 in the phosphotransfer

+ reaction. However, this was not the only residue mutated,since previous reports have demonstrated that mutations inthis residue inactivate the kinase (11, 12, 28), making enzy-matic determinations impossible. To evaluate the affinities ofthe mutants for ATP and their kinase activities, we measured

w^ the apparent Km for ATP and Vmax for enolase phosphory-lation. In addition, we compared the ability of wild-typepp56lck and the inactive mutant Ml (Lys-273 - Arg) to bind

A an ATP analog (8-azido-ATP, an ATP analog with thecrosslinking group at carbon 8 of the adenosine). From thesestudies, we learned that single amino acid substitutions in themicroenvironment of Lys-273 induced a decrease in thekinase activity without significantly altering the affinity forATP. In addition, the inactive Ml mutant (Lys-273 -+ Arg)bound 8-azido ATP as efficiently as wild-type pp56lck, indi-cating that the conserved lysine of subdomain II was notessential for binding of ATP. In agreement with our obser-vations, substitution of Ala for Lys-116 of the yeast cAMP-

as7oArg dependent kinase (corresponding to Lys-72 of the bovinenanograms of enzyme) generated an enzyme with only residual phos-5[1k (W]T), or photransfer activity (103 times lower kcat) but with a similar

s-PaTple wat affinity for ATP (4-fold higher Km; ref. 30).aemmli buffer The kinase reaction includes three steps: (i) binding ofATPvisualized by and protein substrate, (ii) delivery of phosphate from the1. MWM, mo- ATP molecule to the protein substrate, and (iii) release of

reaction products. The decreased kinase activity (Vma,) ofthe

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substitution mutants in the microenvironment of Lys-273might be caused by a direct inhibition of the phosphatetransfer or, alternatively, by a decreased ability to release theproducts (ADP and phosphorylated protein substrate). How-ever, since the conserved lysine seems to interact primarilywith the phosphate that is transferred during the kinasereaction (see below), we favor the hypothesis that the alteredphosphotransfer activity of these mutants is due to a directinhibition of the phosphate transfer. This inhibition could bedue to the alteration of Lys-273 positioning or, alternatively,to a direct role in catalysis for residues surrounding Lys-273.The latter possibility is very unlikely, since the strongestinhibitory effect was found when residues adjacent to Lys-273 were substituted. These residues are expected to beburied within the protein core (6), and internal residuesgenerally do not participate in catalysis. The altered trans-ference ofthe y phosphate by the mutants might be caused bya direct effect on the interaction between Lys-72 and they-phosphate or, alternatively, be the consequence of analtered interaction of the kinase with the a and f phosphates,which in turn would affect the y-phosphate positioning.One interpretation of our data is that the glycine-rich loop

is the motif primarily responsible for ATP anchoring. Thatthe Kd of the cAMP-dependent kinase for adenosine is only3- to 4-fold greater than its Kd for ATP (9) suggests that theadenosine, anchored by the glycine-rich motif, is the princi-pal area of the ATP involved in the binding to the kinase. Thefunction of the conserved lysine of subdomain II might thenbe to orientate appropriately the y-phosphate and/or facili-tate its transfer. When the side chain of this residue (substi-tution of arginine for lysine) or its physical location in theglobular protein (mutations in the adjacent residues) is al-tered, the mutated enzyme still binds ATP with a similaraffinity (Table 1 and Fig. 4), but its ability to transferphosphate is impaired (Table 1 and Figs. 2 and 4).Our results can be rationalized by considering previous

data. First, while the glycine-rich loop is found to interactwith nontransferable phosphates in the nucleotide (14),Lys-72 of cAMP kinase seems to be located closer to thephosphate that is transferred (y phosphate; refs. 6, 7, and 9).Second, several lines of evidence suggest that Lys-72 con-tacts may differ somewhat in the presence and absence ofATP, as would be expected for a residue involved in catal-ysis. Studies performed with dicyclohexyl carbodiimide (31)indicate that in the absence of ATP, Lys-72 seems to interactprimarily with Asp-184. In addition, the structure of the PKAcatalytic domain confirms that, in the absence of ATP, theseresidues are localized in close proximity (6). In contrast, inthe presence of ATP, Lys-72 seems to interact primarily withthe -y-phosphate since (i) Lys-72 reacts with the crosslinkinggroup of FSBA (located at a position similar to the y-phos-phate; ref. 8); (ii) analysis of the cocrystal of the cAMPcatalytic domain with a peptide and ATP has localized theATP molecule in a cleft formed between the C-terminal lobe(containing Asp-184) and the N-terminal lobe (containingLys-72), and in this complex, Asp-184 and Lys-72 are foundto be close to the y-phosphate (6, 7); and (iii) studies withBeuzoadenosine 5'-triphosphate have confirmed the proxim-ity of Lys-72 and the -phosphate in the presence of ATP (9).

In summary, the observations presented here demonstratethat the conserved lysine of subdomain II is not directlyinvolved in anchoring ATP. Our results also indicate thatalterations in the positioning or side chain of this conservedlysine diminish the kinase activity of the enzyme, suggestingthat this residue may have an active role in the mechanism ofphosphate transfer.

We thank Dr. D. Oprian for his help and advice in the mutantpreparation and Drs. V. Calvo, H. Paulus, N. Williams, R. Kolodner,L. Ling, C. Gee, H. Paradi, and A. West for their comments on themanuscript. This work was supported by Public Health ServiceGrant CA43803 (to T.M.R.). A.C.C. was supported by the ConsejoSuperior Investigaciones Cientificas of Spain.

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