Proc. Nati. Acad. Sci. USA Vol. 90, pp. 442-446, January 1993 Biochemistry The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required 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 (receivedfor review September 9, 1992)

ABSTRACT The study of the various protein kinases observed in several PKs upon site-directed mutagenesis of reveals that, despite their considerably diversity, they have this conserved lysine of subdomain 11 (11, 12), together with evolved from a common origin. Eleven conserved subdomains the previous data, supported the idea that the conserved have been described that encompass the catalytic core of these lysine of subdomain II is essential for the binding of ATP. enzymes. One of these conserved regions, subdomain II, con- However, as other investigators have pointed out (11), all the tains an invariant lysine residue present in all known protein evidence presented to date is also consistent with the possible kinase catalytic domains. Two facts have suggested that this participation of this subdomain in the actual mechanism of conserved lysine of subdomain II is essential for binding ATP: phosphate transfer. (i) several investigators have demonstrated that this residue is A second area of the kinase domain that has been impli- physically proximal to the ATP molecule, and (ig) conservative cated in ATP binding is the glycine-rich loop of subdomain I substitutions at this site render the kinase inactive. However, (10). This loop, displaying the consensus sequence Gly-Xaa- these results are also consistent with a functional role of the Gly-Xaa-Xaa-Gly, is close to the phosphates ofMgATP in the conserved lysine ofsubdomain H in orienting or facilitating the crystal structure of the cAMP-dependent kinase (6). The transfer of phosphate. To study in more detail the role of nearly invariant Gly-50 and Gly-52 fall within this consensus subdomain II, we have generated mutants of the protein- sequence. This motif is part of the Rossmann fold structure tyrosine kinase pp56k4k that have single amino acid substitu- associated with many nucleotide binding sites (13). A similar tions within the area surrounding the conserved residue Lys- motif containing a glycine-rich loop is found in proteins as 273 in subdomain II. When compared with wild-type pp56kk, diverse as adenylate kinase (14), GTP-binding proteins such these mutants displayed profound reductions in their phos- as p2lms (P loop; ref. 15), hexokinase (16), HSC70 (17), and photransfer efficiencies and small differences in their affinities actin (18). The single motif common to all these nucleotide- for ATP. Further, the substitution of argnine for Lys-273 binding proteins is the glycine-rich motif, suggesting that it resulted in a mutant protein unable to transfer the -phosphate may serve as phosphate anchor (19). of ATP but able to bind 8-azido-ATP with an efficiency similar The available data on the functional role of subdomains I to that of wild-type ppS6"k. These results suggest that the and II of the catalytic core of PKs do not clearly establish region including Lys-273 of subdomain II is involved in the whether the conserved lysine of subdomain II is essential for enzymatic process of phosphate transfer, rather than in an- ATP binding or whether its major role is related to the actual choring ATP. transfer of phosphate. To analyze the role of subdomain II, we have studied 10 mutants with single amino acid substitu- Protein kinases (PKs) are phosphotransferases that catalyze tions in the vicinity of Lys-273 of the lymphoid protein- the transfer of the y-phosphate of ATP to an amino acid side tyrosine kinase pp56lck. chain (for review see refs. 1-7). Sequence similarities define two major units in the family of PKs: a conserved catalytic core and nonconserved flanking regions (1). The peripheral EXPERIMENTAL PROCEDURES nonconserved regions flanking the catalytic core are impor- Mutant Construction. To prepare substitution mutants in tant for functions such as regulation and subcellular local- subdomain II of pp56lck, we used syn-k, synthetic Ick gene ization (1, 2). The sequence alignment of the catalytic do- encoding pp56lck (unpublished work), cloned into Gex-2T mains of PKs (1) reveals that the conservation is not uniform vector (20). Mutants have been generated by replacement of but, rather, consists of alternating regions of high and low the codons encoding Lys-269 to Lys-276 by the correspond- homology. Eleven major conserved subdomains have been ing synthetic fragment containing an 8% level of sequence identified (I to XI), which are separated by regions of lower degeneracy. Escherichia coli JM109 (Stratagene) colonies conservation (1). transformed with the mixture of mutated constructs were The first clue for localizing the ATP binding site within the picked and screened for overexpression of full-length mole- catalytic core came from studies on the cAMP-dependent cules. DNA preparations obtained from the bacterial colonies kinase. Affinity labeling with the ATP analog 5'-(p- producing full-length proteins were then sequenced. Ten fluorosulfonylbenzoyl)adenosine (FSBA; ref. 8) inhibited the mutants, corresponding to single amino acid substitutions enzyme by covalently modifying Lys-72 (8), a conserved between Lys-269 and Lys-276, were selected for further residue of subdomain II. FSBA contains a reactive group at analysis. a position that approximates the y-phosphate of ATP. The Analysis of Protein Production. Several parameters were proximity of Lys-72 to the y-phosphate of ATP was con- optimized for induction of protein production to achieve firmed by other investigators (6, 7, 9, 10). The inactivation maximal activity and solubility of the protein. Soluble and

The publication costs of this article were defrayed in part by page charge Abbreviations: FSBA, 5'-(p-fluorosulfonylbenzoyl)adenosine; payment. This article must therefore be hereby marked "advertisement" GST, glutathione S-transferase; PK, protein kinase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 442 Downloaded by guest on September 24, 2021 Biochemistry: Carrera et al. Proc. Natl. Acad. Sci. USA 90 (1993) 443

insoluble fractions were separated by centrifugation at 13,000 A Subdomain x g for 30 min at 40C. The amount of pp56lck protein present 11 in each fraction was estimated by Western blotting (as in ref. 21) using anti-pp561ck antibodies (Santa Cruz Biotechnology, K v A V K S L K Santa Cruz, CA). The activity of the soluble products was 269 270 271 272 273 274 275 276 analyzed in kinase reactions using enolase as a substrate (as N L S A R N M V below). The optimal conditions for enzyme solubility and (-4) (-3) (-2) (-1) (MI) (+1) (+2) (+3) activity were obtained with overnight cultures of E. coli X90 (22) diluted 1:10 and incubated for 4 hr in TY medium at 370C N(M2) M(M3) (in the absence of isopropyl 8-D-thiogalactopyranoside). Cells were recovered by centrifugation and suspended in 1% B (vol/vol) Triton X-100 with phenyl methylsulfonyl fluoride (2 N C CSn+ - N N ,r C C O mM), aprotinin (0.15 unit/ml), leupeptin (2.5 mg/ml), pep- 101- statin (10 pg/ml), NaF (5 AM), and Na3VO4 (2 mM) at 40C. Sonication was performed by two rounds of 10-sec pulses (5 71- -S min apart) at a duty cycle of70% and output control of5 (Heat Systems Ultrasonics, Farmingdale, NY, model W225). The S-transferase fusion protein glutathione (GST)-pp56lck -S + (G-pp56lck) was purified as described (20). Protein concen- ~ ~ tration was estimated by Coomassie brilliant blue R(Sigma) s_ o staining of SDS/polyacrylamide gels, with bovine serum 4371-*.I. | albumin as standard, or by binchoninic acid (BCA) assay 43 L ,- (Pierce). Western blot analysis was performed as reported 3 (21). Anti-phosphotyrosine antibodies were prepared by B. Druker in our laboratory. CY ) , C Kinase Reactions, Data Analysis, and Photoaffinity Label- cE N y t N ing. For kinase reactions, 10 ,l containing 50 ng of purified D0 - E kinase was preincubated at 25°C for 1 min and mixed with 20 1 01- -1 a ,ul of 2x kinase reaction cocktail and 10 ,ul of acid-denatured enolase (at the appropriate concentration). The cocktail contained 50 mM Tris-HC1 (pH 7.4), 10 mM MnC12, and the 743- appropriate dilution of the ATP stock (100 ,uM ATP, 10 ,Ci of [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 by addition of 10 ,ul of 100 mM EDTA (pH 8.0). For the FIG. 1. Comparison of E. coli lysates containing wild-type or comparison of G-pp56Ick and baculoviral pp56lck (30%o pure; mutated G-ppS6lck: (A) Representation of the positions of wild-type ref. 23), reaction mixtures were incubated for 5 min. Sub- pp5ock that have been substituted (upper line) and the corresponding amino acids introduced (lower line). Numbers in parentheses rep- strate and enzyme were resolved by SDS/PAGE. For the resent the positions ofthe residues relative to Lys-273. Substitutions determination of kinetic parameters, phosphate incorporated of Lys-273 were named Ml (Lys-273 -. Arg), M2 (Lys-273 -. Asn), into enolase was quantitated by liquid scintillation counting. and M3 (Lys-273 -+ Met). (B-D) E. coli X90 bacteria were trans- Vmx and Km were estimated by graphic methods (24, 25). formed with constructs encoding wild-type (WT) or single- Photoaffinity labeling of G-pp56Ick was performed as de- substitution mutants of G-pp56ock (named as in A), GST-Ser/Thr scribed (26). kinase Raf-1 fusion protein (Cl), or GST (C2) or with medium alone (C3). Bacteria were induced and lysed, and soluble proteins were recovered by centrifugation. Fifteen microliters of each lysate was RESULTS loaded on an SDS/10%o polyacrylamide gel and analyzed by Western blotting using anti-pp561ck antibodies (B), Coomassie blue staining Mutant Construction. To distinguish between a passive role (C), or Western blotting using anti-phosphotyrosine antibodies (D). of subdomain II in anchoring ATP or a catalytic role in the MW, molecular weight markers (Mr x 10-3 at left). phosphorylation reaction, we chose to analyze the kinetic consequences of introducing single amino acid substitutions 15 pul of each lysate by Western blotting with anti-pp56Ick (conservative or nonconservative) at residues located be- antibodies revealed that bacteria transformed with the vari- tween Lys-269 and Lys-276 of pp56Ick. Wild-type and mu- ous G-pp561ck constructs, but not the controls, contained tated genes encoding pp56Ick were cloned in p-Gex-2T to similar amounts of G-pp561ck (Fig. 1B, an 82-kDa band facilitate production of mutant proteins referred to as corresponding to 26 kDa of GST fused to 56 kDa of pps6lck). G-pp561ck in bacteria and subsequent purification. Fig. 1A Further, a similar protein composition was found (Fig. 1C) illustrates the area of subdomain II of pp56lck examined, as when the same volume of the various preparations of X90 well as the various single amino acid substitutions selected bacterial lysates (containing the various constructs) were for the study. analyzed by Coomassie blue staining. In contrast, different G-ppS6I-k Protein Production, Purification, and Enzymatic intensities of phosphotyrosine signal were detected when Characterization. To compare mutants in subdomain II with similar volumes of the lysates were compared by anti- wild-type G-pp56lck, we first optimized the expression of the phosphotyrosine Western blotting (Fig. 1D). These results wild-type enzyme in bacteria (see Experimental Procedures). indicate that the different mutants display different kinase The optimal conditions yielded maximal activity (see below activities. for comparison with baculoviral pp56Ick) and solubility G-pp56Ick was purified as described (20). This procedure (=90%). Using these conditions, we compared the lysates of yielded apparently pure wild-type or mutated G-pp561ck as nontransformed E. coli X90 (C3) with lysates ofX90 bacteria judged by Coomassie blue staining (Fig. 2A). Purified wild- expressing wild-type fusion protein G-pp561ck (WT), mutated type G-pp56lck was highly active as estimated by autophos- G-pp561ck (see nomenclature in Fig. lA), a GST-Ser/Thr phorylation (Fig. 2B). The Gex-2T-syn-k construct encoding kinase Raf-1 fusion protein (Cl), and GST (C2). Analysis of G-pp561ck includes in its sequence a protease site (thrombin) Downloaded by guest on September 24, 2021 444 Biochemistry: Carrera et al. Proc. Natl. Acad. Sci. USA 90 (1993) the amount of phosphate incorporated into enolase was A B measured by liquid scintillation counting. The data were evaluated by Eisenthal-Cornish-Bowden (24), and Line- c: weaver-Burk (25) approximations, which yielded similar a: Cf) Is- values in every case. cs The values of apparent Km and V,: obtained for bacterial --2i_8 G-pp561ck using enolase as a substrate were as follows: Km ATP = 0.97 + 0.20 ,uM (mean ± SD), Km enolase = 5.58 ± - 2 0 8 - 0.87 AuM, and Vma,, = 22.74 + 1.56 nmol/(min-mg) (Table 1). The G-pp561ck preparation had an apparent Km for ATP -1701 - 1i1- similar to that of purified baculoviral pp56Ick (D. Winkler, personal communication). With regard to the apparent Vma different values have been reported even for the same en- zyme preparation, depending on the substrate analyzed -4 3 - (highest values have been obtained with T-cell receptor {-chain peptides; ref. 27). However, when compared under the same reaction conditions, baculoviral pp56lck and bacte- rial G-pp561ck displayed similar phosphotransfer activities (Fig. 3). - 2 8 - Analysis of pp563& Mutants Containing Single Amino Acid Substitutions in Subdomain H. To determine whether the decreased kinase activity ofthe single amino acid substitution mutants in residues Lys-269 to Lys-276 (Fig. 1) was due to a decrease in the binding ofATP or, alternatively, to a decrease -1 8- in the efficiency of the phosphotransfer reaction, we evalu- ated the kinetic parameters of the mutants in vitro. Mutants FIG. 2. SDS/PAGE analysis of purity and autokinase activity of were purified (as above) and the Km for ATP and Vm,, for purified G-pp,6Ick: Wild-type (WT) and Lys-273 -. Arg mutant enolase were determined (as above). In agreement with (K273R) G-pp56Ick proteins were produced in E. coli X90. The fusion previous studies performed with pp6Osrc and epidermal protein present in the bacterial lysates was purified by using glu- growth factor receptor (11, 12, 28), mutations at the position tathione-Sepharose beads (200 ng of pure protein obtained from 400 /kg of the total soluble protein fraction). Purified WT and K273R 273 of pp5sck rendered the kinase inactive. Substitutions in G-pp561ck were analyzed by SDS/PAGE followed by Coomassie blue all of the other positions, between 269 and 276, yielded staining (A). Samples were also tested for their autophosphorylating partially active proteins. The Km for ATP of each of the activity in vitro and resolved by SDS/PAGE. The resulting gel was mutants was similar to the Km for ATP of wild-type pp5s6ck analyzed by autoradiography (B). MWM, molecular weight markers (Table 1). In contrast, every substitution yielded a pps6Ick (Mr X 10-3 at right in A). protein with lower phosphotransfer activity than wild-type G-pp561ck. located between the GST fragment and pp5sck. We also The similarity of the Km for ATP of conservative and compared the phosphotransfer activity of the fusion protein nonconservative substitution mutants within subdomain II G-pp56Ick with (i) similar amounts of bacterial pp56lck ob- suggests that this area is not likely to be responsible for ATP tained upon cleavage of the GST fragment with protease (as binding. In addition, the fact that the enzyme phosphotrans- in ref. 20) and (ii) similar amounts of G-pp561ck immunopu- fer efficiency is significantly altered when residues in the rified by using anti-pp56lCk antibodies (as in ref. 21). The vicinity of Lys-273 are substituted suggests that Lys-273/ specific kinase activity present in the various preparations subdomain II is involved in the process ofphosphate transfer. was comparable (data not shown). Unfortunately, protease Comparison of the ATP-Binding Ability of Wild-Type treatment caused a significant amount of a pp56ick breakdown G-pp56'C and Lys-273 -+ Arg Substitution Mutant. The ki- product, and immunopurification failed to purify pp56ck to netic analysis ofthe single-substitution mutants ofsubdomain homogeneity. Purification of the wild-type and mutant II suggested that this subdomain is not directly involved in G-pp561ck proteins using glutathione beads yielded =50 ng of anchoring ATP. If this is the case, the inactive mutant at pure G-pp561ck from 100 pug of total soluble bacterial protein. position 273 should be able to bind ATP with similar effi- To determine the enzymatic parameters of G-pp561ck, the ciency compared with wild-type pp56lck. To determine concentration of purified enzyme was estimated by SDS/ whether this hypothesis was correct, we chose to use ATP PAGE followed by Coomassie blue staining. Fifty nanograms of G-pp561ck was mixed with various amounts of ATP and Table 1. Analysis of the kinetics of subdomain II mutants enolase and subjected to kinase reaction. A time course ofthe of G-pp56Ick reaction revealed that the incorporation of phosphate was KmK.,,utMVAM linear at least for the first 5 min not Y~~~~max. (data shown). Therefore, Substitution* ATP Enolase nmol/(min-mg) for all the assays, 2-min incubations were used to remain in the linear range. To measure the Km of G-pp561ck for ATP, Wild type 0.97 ± 0.20 5.58 ± 0.87 22.74 ± 1.56 enolase concentration was fixed at 5.5 puM and ATP concen- K273Xt ND ND 0 tration was varied from 0.25 to 10 ,M (corresponding to V272A (-1) 1.92 ± 0.36 1.86 ± 0.28 0.50 ± 0.02 <0.5x to >5 x Km of G-pp561ck for ATP). To calculate the Km S274N (+1) 2.06 ± 0.53 5.61 ± 0.75 2.90 ± 0.16 for enolase, ATP concentration was fixed at 5 A&M and A271S (-2) 0.95 ± 0.35 10.12 ± 3.58 5.68 ± 4.56 enolase was varied from 0.34 to 22 ,uM (corresponding to L275M (+2) 0.83 ± 0.25 4.77 ± 1.59 3.66 ± 0.76 <0.1 X to >3 X Km of G-pp56Ick for enolase). To evaluate the V270L (-3) 1.61 ± 0.25 7.74 ± 0.71 18.95 ± 2.54 phosphotransfer activity, we determined the apparent Vn K276V (+3) 1.08 ± 0.59 9.00 ± 2.29 6.46 ± 0.74 for enolase phosphorylation in the presence of excess ATP (5 K269N (-4) 1.28 ± 0.42 2.92 + 0.58 6.24 + 0.72 ,uM ATP, corresponding to Sx K.n). Enzyme and substrate *See Fig. 1A. were resolved by SDS/PAGE. To estimate initial velocity, tX = R (Arg), M (Met), or Asn (N). Downloaded by guest on September 24, 2021 Biochemistry: Caffera et al. Proc. Natl. Acad. Sci. USA 90 (1993) 445

of purified wild-type or Lys-273 -* Arg mutant G-pp561ck A B were analyzed, the mutant at position 273 bound an amount of 8-azido-ATP similar to the amount bound by the wild-type c: enzyme (Fig. 4B). In contrast, GST incubated under similar .X CD) 0 -. conditions yielded a small background signal (Fig. 4B). The 0 .0 CN I D CM , > MWM Be MWM small signal obtained when wild-type G-pp561ck was incu- Ye g ._l - 2 0 8 bated in the absence of UV light (Fig. 4B) corresponds to the - 2 0 8 residual phosphotransfer activity of pp56lck at the tempera- to' ture of incubation (40C). The signals of 8-azido-[32P]ATP __ -10101 __ -1 01 incorporated into wild-type and mutated pp56Ick were due to specific ATP binding, as judged by the decrease in these 7 1 signals observed upon addition of EDTA, absence of Mn2 , -7 1 or addition of excess nonradioactive ATP (data not shown). Upon subtraction of the background signal, the ratio of mutant to wild-type signal (cpm/cpm) was calculated from IN _ a-43. 4 3 five different experiments. The value obtained, 0.98 + 0.33, indicates that a similar amount of ATP reacts with wild-type enzyme and with the Lys-273 -* Arg mutant.

DISCUSSION The results indicate that single amino acid substitutions in the area of Lys-273 affect the ability of pp56lck to transfer FIG. 3. Comparison of the activity of bacterial arid eukaryotic phosphate to a protein substrate but do not significantly alter pp5oCk: Bacterial wild-type (G-lck) and Lys-273 -- Arg mutant its ability to bind ATP. (K273) G-ppS61ck proteins were produced and purified as in Fig. 2. Two motifs have been classically implicated in the regu- Baculoviral pp,56ck (bv-lck) was produced and purified as described lation of the ATP binding: the glycine-rich loop (10), and the (21, 23). Fifty nanograms of baculoviral or bacterial cenzyme (indi- conserved lysine of subdomain II (Lys-72 of cAMP kinase, cated with arrows) was tested for the ability to phosphorylate Lys-273 of pp56lck; refs. 8 and 9). Initially these sites were acid-denatured enolase (band at 43 kDa). After the kiriase reaction, defined by labeling of the cAMP-dependent kinase with samples were resolved by SDS/10% PAGE and Coomassie Blue staining (A) or autoradiography (B). NaWM molec- acetic anhydride: Lys-47 (next to the Gly-Xaa-Gly-Xaa-Xaa- ular weight markers (M, x 10-3). Gly motif) and Lys-72 were protected by MgATP against modification with acetic anhydride (10). These elegant stud- analogs containing a crosslinking group. FSBA Nwas avoided ies provided information about which areas of a kinase were because it contains the crosslinking group in Ithe position proximal to the ATP, but did not delineate the specific role of each of these areas. revealed that both to the whose transfer and ccorrect Mutagenesis analogous y-phosphate (8) and e regions were required for the kinase to be active (10, 11, 28, positioning are probably regulated by Lys-273. Instead, we 29), but again did not establish specific roles. used 8-azido-[y-32P]ATP whose crosslinking grolUp is located To study the functional role of the conserved lysine of on carbon 8 ofthe adenosine. When similar amouints(Fig.4A) subdomain II (Lys-273 of pp56ock), we chose to prepare random mutations in each of the residues located between A B Lys-269 and Lys-276 of pp561ck. The central interest of our analysis was to distinguish between a passive role in ATP

I + + binding and an active role for Lys-273 in the phosphotransfer N¢4 N + reaction. However, this was not the only residue mutated, XX X 0d MWM a y since previous reports have demonstrated that mutations in this residue inactivate the kinase (11, 12, 28), making enzy- -208- L matic determinations impossible. To evaluate the affinities of the mutants for ATP and their kinase activities, we measured _ a 6 -1 01- l w ^ the apparent Km for ATP and Vmax for enolase phosphory- 'S. *4 lation. In we the of - 7 1 - addition, compared ability wild-type pp56lck and the inactive mutant Ml (Lys-273 - Arg) to bind A an ATP analog (8-azido-ATP, an ATP analog with the crosslinking group at carbon 8 of the adenosine). From these U~~~~~~~~~~ studies, we learned that single amino acid substitutions in the microenvironment of Lys-273 induced a decrease in the kinase activity without significantly altering the affinity for

-~~~~~~~~~~~~ ATP. 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 not essential for binding of ATP. In agreement with our obser- vations, substitution of Ala for Lys-116 of the yeast cAMP- FIG. 4. Photoaffinity labeling of wild-type and L) dependent kinase (corresponding to Lys-72 of the bovine mutant G-ppS6Ick with 8-azido-ATP: One hundred Xnanogramsas7oArgof enzyme) generated an enzyme with only residual phos- purified control GST protein (Gex-2T), wild-type G-pp.5[1k (W]T), or photransfer activity (103 times lower kcat) but with a similar mutated G-pp561ck (K273R) was mixed with 8-azido-I for ATP ref. 40C. Photoaffinity labeling was achieved when the s-PaTplewat affinity (4-fold higher Km; 30). irradiated (+). After 5 min, samples were boiled in L. aemmli buffer The kinase reaction includes three steps: (i) binding ofATP and resolved by SDS/1Oo PAGE. Proteins were visualized by and protein substrate, (ii) delivery of phosphate from the Coomassie blue staining (A) and autoradiography (B)1. MWM, mo- ATP molecule to the protein substrate, and (iii) release of lecular weight markers (Mr x 10-3). reaction products. The decreased kinase activity (Vma,) ofthe Downloaded by guest on September 24, 2021 446 Biochemistry: Carrera et al. Proc. Natl. Acad. Sci. USA 90 (1993) substitution mutants in the microenvironment of Lys-273 We thank Dr. D. Oprian for his help and advice in the mutant might be caused by a direct inhibition of the phosphate preparation and Drs. V. Calvo, H. Paulus, N. Williams, R. Kolodner, transfer or, alternatively, by a decreased ability to release the L. Ling, C. Gee, H. Paradi, and A. West for their comments on the products (ADP and phosphorylated protein substrate). How- manuscript. This work was supported by Public Health Service ever, since the conserved lysine seems to interact primarily Grant CA43803 (to T.M.R.). A.C.C. was supported by the Consejo with the phosphate that is transferred during the kinase Superior Investigaciones Cientificas of Spain. reaction (see below), we favor the hypothesis that the altered phosphotransfer activity of these mutants is due to a direct 1. Hanks, S. K., Quin, A. M. & Hunter, T. (1986) Science 241, inhibition of the phosphate transfer. This inhibition could be 42-52. due to the alteration of Lys-273 positioning or, alternatively, 2. Taylor, S. S., Buecher, J. A. & Yonemoto, W. (1990) Annu. to a role Rev. Biochem. 59, 971-1005. direct in catalysis for residues surrounding Lys-273. 3. Kemp, B. E. & Pearson, R. B. (1990) Trends Biochem. Sci. 15, The latter possibility is very unlikely, since the strongest 342-346. inhibitory effect was found when residues adjacent to Lys- 4. Lindberg, R. A., Quin, A. M. & Hunter, T. (1992) Trends 273 were substituted. These residues are expected to be Biochem. Sci. 17, 114-119. buried within the protein core (6), and internal residues 5. Cooper, J. A. (1990) in Peptides and Protein Phosphorylation, generally do not participate in catalysis. The altered trans- eds. Kemp, B. & Alewood, P. F. (CRC, Boca Raton, FL), pp. ference ofthe y phosphate by the mutants might be caused by 85-113. a direct effect on the interaction between Lys-72 and the 6. Knighton, D. R., Zheng, J., Ten-Eyck, L. F., Ashford, V. A., y-phosphate or, alternatively, be the consequence of an Xuong, N.-H., Taylor, S. S. & Sowadsky, J. M. (1991) Science altered interaction ofthe kinase with the a and f phosphates, 253, 407-414. which in turn would affect the y-phosphate positioning. 7. Knighton, D. R., Zheng, J., Ten-Eyck, L. F., Xuong, N.-H., One interpretation of our data is that the glycine-rich loop Taylor, S. S. & Sowadsky, J. M. (1991) Science 253, 414-420. 8. Kamps, M. P., Taylor, S. S. & Sefton, B. M. (1984) Nature is the motif primarily responsible for ATP anchoring. That (London) 310, 589-592. the Kd of the cAMP-dependent kinase for adenosine is only 9. Bhatnager, D., Hartl, F. T., Roskoski, R., Jr., Lessor, R. A. & 3- to 4-fold greater than its Kd for ATP (9) suggests that the Leonard, N. J. (1984) Biochemistry 23, 4350-4357. adenosine, anchored by the glycine-rich motif, is the princi- 10. Buecher, J. A., Vedvick, T. A. & Taylor, S. S. (1989) Bio- pal area ofthe ATP involved in the binding to the kinase. The chemistry 28, 3018-3024. function of the conserved lysine of subdomain II might then 11. Kamps, M. P. & Sefton, B. M. (1986) Mol. Cell. Biol. 6, be to orientate appropriately the y-phosphate and/or facili- 751-757. tate its transfer. When the side chain of this residue (substi- 12. Snyder, M., Bishop, J. M., Mc Grath, J. & Levinson, A. (1985) tution of arginine for lysine) or its physical location in the Mol. Cell. Biol. 5, 1772-1779. globular protein (mutations in the adjacent residues) is al- 13. Rossmann, M. G., Moras, D. & Olsen, K. (1974) Nature tered, the mutated enzyme still binds ATP with a similar (London) 250, 194-199. affinity (Table 1 and Fig. 4), but its to transfer 14. Saraste, M., Sibbald, P. R. & Wittinghofer, A. (1990) Trends ability Biochem. Sci. 15, 430-434. phosphate is impaired (Table 1 and Figs. 2 and 4). 15. deVos, A. M., Tong, L., Milburn, M. V., Matias, P. M. & Our results can be rationalized by considering previous Jankarik, J. (1989) Science 239, 888-893. data. First, while the glycine-rich loop is found to interact 16. Anderson, C. M., Zucker, F. H. & Steitz, T. A. (1979) Science with nontransferable phosphates in the nucleotide (14), 204, 375-380. Lys-72 of cAMP kinase seems to be located closer to the 17. Flaherty, K. M., Flaherty, D. L. & McKay, D. B. (1990) phosphate that is transferred (y phosphate; refs. 6, 7, and 9). Nature (London) 346, 623-628. Second, several lines of evidence suggest that Lys-72 con- 18. Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. & Holmes, tacts may differ somewhat in the presence and absence of C. (1990) Nature (London) 347, 37-42. ATP, as would be expected for a residue involved in catal- 19. Hol, W. G. J. (1985) Prog. Biophys. Mol. Biol. 45, 149-153. ysis. Studies performed with dicyclohexyl carbodiimide (31) 20. Smith, D. B. & Johnson, K. S. (1988) Gene 67, 31-40. indicate that in the absence of ATP, Lys-72 seems to interact 21. Carrera, A. C., Li, P. & Roberts, T. M. (1991) Internat. primarily with Asp-184. In addition, the structure ofthe PKA Immunol. 7, 673-682. in these 22. Pallas, D. C., Schley, C., Mahoney, M., Harlow, E., Schaff- catalytic domain confirms that, the absence of ATP, hausen, B. S. & Roberts, T. M. (1986) J. Virol. 60, 1075-1084. residues are localized in close proximity (6). In contrast, in 23. Ramer, S. E., Winkler, D. G., Carrera, A. C., Roberts, T. M. the presence of ATP, Lys-72 seems to interact primarily with & Walsh, C. T. (1991) Proc. Nat!. Acad. Sci. USA 88, 6254- the -y-phosphate since (i) Lys-72 reacts with the crosslinking 6258. group of FSBA (located at a position similar to the y-phos- 24. Eisenthal, R. & Cornish-Bowden, A. (1974) Biochem. J. 139, phate; ref. 8); (ii) analysis of the cocrystal of the cAMP 715-720. catalytic domain with a peptide and ATP has localized the 25. Lineweaver, H. & Burk, D. (1934) J. Am. Chem. Soc. 56, ATP molecule in a cleft formed between the C-terminal lobe 658-670. (containing Asp-184) and the N-terminal lobe (containing 26. Sanghera, J. S., Paddon, H. B. & Pelech, S. L. (1991) J. Biol. Lys-72), and in this complex, Asp-184 and Lys-72 are found Chem. 266, 6700-6707. to be close to the y-phosphate (6, 7); and (iii) studies with 27. Watts, J. D., Wilson, G. M., Ettahadieh, E., Clark-Lewis, I., Beuzoadenosine 5'-triphosphate have confirmed the proxim- Kubanek, C.-A., Astel, C. R., Marth, J. D. & Aebershold, R. ity of Lys-72 and the -phosphate in the presence of ATP (9). (1992) J. Biol. Chem. 267, 901-907. 28. Russo, M. W., Lukas, T. J., Cohen, S. & Staros, J. V. (1985) In summary, the observations presented here demonstrate J. Biol. Chem. 260, 5205-5208. that the conserved lysine of subdomain II is not directly 29. Odawara, M., Kadowaki, T., Yamamoto, R., Shibasaki, Y. & involved in anchoring ATP. Our results also indicate that Kobe, K. (1989) Science 245, 66-68. alterations in the positioning or side chain of this conserved 30. Gibbs, C. S. & Zoller, M. J. (1991) J. Biol. Chem. 266, 8923- lysine diminish the kinase activity of the enzyme, suggesting 8931. that this residue may have an active role in the mechanism of 31. Buechler, J. A. & Taylor, S. S. (1989) Biochemistry 28, 2065- phosphate transfer. 2070. Downloaded by guest on September 24, 2021