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The Conserved Lysine of the Catalytic Domain of Protein Kinases Is 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).
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