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219 Protein kinase inhibition: natural and synthetic variations on a theme Susan S Taylor* and Elzbieta Radzio-Andzelm How a protein physiological solved kinase is turned function crystal subfamilies structures reveals are effective. inhibitors Although target is due to the extended inhibitors occupy. mechanisms Although or natural site, specifically high degree targeting is proving to be very successful, designing inhibitors that target kinase site can be achieved that these of the ATP binding is also wide other surfaces site latitude for of the kinases. Addresses Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0654, USA *e-mail: [email protected] Current Opinion in Chemical Biology 1997, 1:219-226 http://biomednet.com/elecref/1367593100100219 0 kinases, which are activated through complex but tightly controlled kinase cascades in response to a number of factors, including stress and cytokines, represent only one set of targets where selective inhibitors are actively being sought. as well product of the protein there by the ATP binding of specificity surface of conformations. other than the active most synthetic the active site, a remarkably which mimetic to surfaces protein that are utilized into inactive and adenine as complementarity Examination different of strategies kinases for its activity. representing a variety nature to lock protein Pseudosubstrate off is as critical as is its catalytic Current Biology Ltd ISSN 1367-5931 Abbreviations catalytic C cdk cyclin-dependent kinase MAP mitogen-activated protein myosin light chain kinase MLCK PKA CAMP-dependent protein kinase PKG cGMP-dependent protein kinase PKI protein kinase inhibitor PRS2 peripheral recognition site 2 R regulatory Introduction Fischer and Krebs [l] were the first to demonstrate, in 1955, that the reversible equilibrium between active and inactive conformations of a protein could be regulated by the post-translational addition of a phosphate moiety. Not only are there thousands of proteins that utilize this regulatory mechanism, but the number of protein kinases that catalyze these phosphoryl transfer reactions also number in the hundreds (reviewed in [Z]). It is predicted that the human genome alone encodes over 2000 protein kinases. Since these enzymes regulate almost every process in the eukaryotic cell by serving as on/off switches, it is critical that every kinase be tightly regulated physiologically. They are turned off and on by very precise signals; thus if one could develop strategies for selectively regulating a specific kinase by therapeutic intervention, one could control an entire signaling pathway. The mitogen-activated protein (MAP) Although the catalytic core of protein kinases has been evolutionarily conserved in all eukaryotic protein kinases that phosphorylate serine, threonine, and tyrosine (reviewed in [Z]), the mechanisms by which the inhibition of each kinase is achieved vary considerably. Although the details differ, a continuing theme is the utilization of modular domains for multiple functions. As the crystal structures of more protein kinases have been solved, not only of the catalytic cores but also of the cores plus their regulatory domains, a variety of motifs for regulation have been identified. These examples, summarized below, not only provide a foundation for thinking more broadly about the molecular features that regulate each kinase, but also provide wide latitude for developing inhibitors which extend beyond those obtainable using the conventional approach of designing molecules that bind to the enzyme’s active site. The purpose of this review is to summarize some of the mechanisms used physiologically to achieve protein kinase inhibition and compare them to the mechanism that is used by most known synthetic and natural product inhibitors. Three specific inhibition mechanisms will be discussed. Twitchin and CAMP-dependent protein kinase (PKA) are examples of protein kinases that are regulated by a pseudosubstrate mechanism; cdk2 and the inactive insulin receptor provide examples of inhibition that is achieved by an adenine mimetic mechanism; Hck and Src provide examples of inhibition that is achieved by locking the enzyme into an inactive conformation using surfaces other than the active site. Inhibition mechanisms are discussed in the context of the active enzyme and the requirements for activity at the active site cleft. The inhibitor domains can be part of the same polypeptide chain, as in twitchin and Hck, or part of a separate subunit, as in PKA and cdk2. In many cases these inhibitory domains serve dual functions its inactive state and binding conformation. Structural features by inhibiting the kinase in to other ligands in the active of the protein kinase core All known eukaryotic protein kinases share a conserved catalytic core, in spite of considerable diversity in size, mechanism of regulation and activation, and subcellular localization. Since the structural features of this core in its active conformation are best understood in functional terms for PKA, one of the simplest protein kinases, we shall use it as a prototype for the entire family. The 220 Next generation therapeutics Modes of inhibition Pseudosubstrate inhibition The mechanism of inhibition whereby the active site of the kinase is occupied by an inhibitor peptide that resembles a substrate was first suggested as the mechanism for inhibition of the catalytic subunit of PKA by its regulatory subunits (discussed in [14]). This strategy as a general inhibition mechanism was more fully explored by Kemp and co-workers [15] with myosin light chain kinase (hILCK). The crystal structures of PKA [4] and twitchin [16**] support this mechanism of inhibition. The crystal structure of the C subunit of PKA w;1s first solved as a binary complex with a peptide derived from the heat stable protein kinase inhibitor (PKI) (residues 5-24). The PKI sequence resembles the consensus sequence for PKA substrates. As predicted by the binding of peptide analogs to PKA [17], this pseudosubstrate occupies the active site cleft [18]. In addition to this consensus sequence, which alone is not sufficient to convey high affinity binding, PKI has an essential amphipathic helix that lies amino-terminal to the consensus site. This helix binds to a site peripheral to the active site cleft. This peptide, PKI (residues 5-2-l), binds with high affinity (Ki =0.2 nhl) in the presence of ATP [17,19]. The regulatory (R) subunits of PKA contain a similar which is thought to occupy the consensus sequence, active site cleft in a similar manner. To achieve high affinity binding, however, the R subunit interacts with the surface on the C subunit that lies carboxy-terminal to the consensus site. This region includes the essential phosphorylation site, Thr197, and the basic residues that surround this phosphate [ZO]. This site is designated as peripheral recognition site 2 (PRSZ), to distinguish it from the surface that is required by PKI [21]. Binding of these inhibitors is thus bipartite. One segment that resembles a substrate binds to the active site cleft. while a second segment. contiguous in PKI but not contiguous in R, binds to an additional site peripheral to the active site cleft. Both PKI and R are competitive inhibitors with respect to peptide substrates. The structure of twitchin (Figure 2) provides probably the most dramatic example to date of pseudosubstrate inhibition [ 16”]. In this case, the conserved kinase core is followed by an extended pseudosubstrate sequence, which occupies the active site cleft as was predicted earlier. This is followed by an IgG domain. The structure of the kinase domain and the autoinhibitor segment alone define the core and confirmed the pseudosubstrate mechanism of kinase inhibition [ZZ], while the subsequent structure of the kinase core plus the IgG domain shows the active site completely occluded by the adjacent domain [ 16”]. The pseudosubstrate is docked into the active site cleft while the IgG domain securely anchors itself on the Protein Figure kinase inhibition: natural and synthetic variations on a theme Taylor and Radzio-Andzelm 221 1 (b) .oop Active conformation of the conserved core of the catalytic subunit of PKA. (a) In the center is the conserved c 1997 Current Opmvx I” Chemical Biology core in its closed conformation wit h the two lobes (residues 40-l 27 and 126-300) shaded differently. The conserved residues (G50, G52, G55, K72, E91, Dl66, N171, D164, E206, D220 and R260, in single letter code for amino acids) are shown as balls. ATP is bound at the base of the active site cleft between the two lobes while the inhibitor peptide PKI (residues 5-241, shown in white, binds on the surface of the large lobe. (b) An expanded view of the small lobe where the central role of the glycine-rich loop is emphasized. The glycine-rich loop (residues 49-57) and the linker that joins the two lobes (residues 120-l 27) are shown in black. The structures were generated using PDB file 1 ATP. surface that lies carboxy-terminal to the active site cleft. In hlLCK. a related family member, the pseudosubstrate peptide domain overlaps with a calmodulin binding site (discussed in [23]). When Car+ is bound to calmodulin, the CaZ+/calmodulin complex competes favorably with the inhibitory domain, thus exposing the active site of the enzyme. Twitchin is activated in a similar fashion, by binding to another protein complex, CaZ+/SlOO [El]. PKA and twitchin are assembled as fully active modules that are maintained in an inactive state by formation of a stable complex with an inhibitor. In the case of PKA, the inhibitor is a separate protein, whereas with twitchin (and IZILCK) the inhibitor is a contiguous domain of the kinase itself. In both cases, a secondary messenger causes the release of the inhibitor segment [14,23]. cyclin (see Figure 3). The carboxyl terminus of p27Q~l specifically folds over onto the small lobe of the kinase core. The glycine-rich loop, a key component of the nucleotide positioning motif, becomes a distorted and discontinuous part of the p sheet. The Tyr88 residue of p27Kipl folds into the adenine binding pocket and hydrogen bonds with the backbone carbonyl of Glu81 and the backbone amide of Leu83, both in the short linker strand that joins the two lobes. A comparison of the active site filled by ATP and the corresponding adenine binding pocket of cdkZ-cyclin-p27Kil’l filled with the Tyr88 of p27Kipl is also shown in Figure 3. How widely this mechanism is utilized by other protein kinases must await crystal structures of other inhibited enzymes. However, given the critical importance of protein-protein interactions for kinase signaling pathways. this is likely to be used Adenine mimetic repeatedly. inhibition While pseudosubstrate inhibition is very attractive and logical, it is not the only mechanism by which kinase inhibition is achieved. Another mechanism that has onlv been recognized more recently utilizes a peptide segment that mimics the adenine ring of ATP. This pepcide functions as an adenine mimetic and fills the shielded pocket that is typically occupied by the nucleotide base in the active kinase. To date, the best example of this mechanism is represented by the structure of p27Kllll bound to the cdk2-cyclin complex [ZS”]. In this structure p27KiPl spans the surface of both cdk2 and The structure of a truncated form of the insulin receptor provides another example in which a peptide sequence can fold into the active site cleft and mimic the adenine ring of ATP, thus inhibiting enzymatic activity [26]. In this case, it is Tyrl158, part of the activation loop, that folds into the adenine binding pocket. The tyrosine hydroxyl makes contacts similar to those seen in the cdk2-cyclin-p27W~~ complex. This crystal structure suggests an intriguing mechanism whereby the dephosphorylated tyrosines in the activation loop stabilize the inactive conformation of the enzyme. The full physiological relevance of this 222 Next Figure 2 generation therapeutics Autoregulation of the twitchin and Hck kinases [16”,26”]. Autoregulation (inhibition of kinase activity) is achieved using two different mechanisms that result in the active site being maintained in an inhibited state by domains that flank the core. (a) The structure of the twitchin kinase core joined to the autoinhibitor segment and the IgG domain. The autoinhibitor segment (residues 6199-6255) is locked firmly into place at the active site cleft by numerous contacts and by the positioning of the IgG domain. (b) The structure of the Hck kinase core with its amino-terminal &c-homology flanking SH2 and SH3 domains. In this case the SH2 and SH3 domains bind to Tyr526 in the carboxy-terminal tail and to the linker strand that joins the SH2 domain to the core, respectively. In this structure of the inhibited kinase, the active site cleft is fully exposed but locked into a conformation that cannot support catalysis. The structure of twitchin was generated using PDB file 1 KOA. The coordinates for Hck were kindly provided by John Kuriyan (The Rockefeller mechanism for the insulin receptor awaits a structure solution of the full-length protein, since both membrane localization and the carboxy-terminal tail may contribute to the conformation of the core. The physiological relevance of this mode of inhibition is also unclear since in the cell, where ATP concentrations are high, ATP may displace Tyr1158 and lead to structural instability. It is clear, however, that the adenine binding pocket can be an important potential target for physiological inhibition. Other mechanisms inactive for locking the kinase core in an conformation The recently solved structures of two nonreceptor tyrosine kinases, Src and Hck, illustrate another novel mechanism for locking the catalytic core into an inactive conformation [27”,28**]. These structures (of the core plus the contiguous Src-homology domains, SH2 and SH3,) also emphasize the importance of having a full length protein structure as opposed to structures of isolated domains. It is very easy to overinterpret the significance of crystal packing in isolated domains when the natural partner, in this case the kinase core, is missing. In the case of Src and Hck, the finely tuned inhibition mechanism was only revealed when the structures of the proteins containing all three domains were solved [27**,28”]. The orientation of the Hck kinase core relative to the SH2 and SH3 domains is shown in Figure 2b. This conformation is stabilized by two specific interactions: the phosphorylated Tyr at the carboxy-terminus, Tyr527, University, New York). is hydrogen bound to the adjacent SH2 domain, and the proline-rich segment (which links the SH2 domain to the kinase core) serves as an intramolecular docking site for the SH3 domain. Consequently, the kinase core is physically constrained in a conformation that cannot support catalysis. In addition to restricting the conformational flexibility of the core, two specific regions of the kinase core are either incorrectly aligned or disordered. The activation loop is disordered because it is not phosphorylated [B”]. Activation of Src correlates directly with the phosphorylation of Tyr416 in its activation loop [29]. In addition, the C helix in the small lobe is twisted so that the conserved Glu310 is facing away from Lys295, its partner to the in the active enzyme. Instead Glu310 is exposed solvent and faces the activation loop, where it actually forms an ion pair with the highly conserved Arg385, which immediately precedes the catalytic loop. Following phosphorylation of Tyr416, this same arginine helps to assemble the activation loop into its correct and active form (in the inhibited conformation the same residue helps to stabilize the inactive enzyme). Correct phosphorylation of this loop is essential for full activity of most protein kinases (reviewed in [30’]). In PKA, lack of this essential phosphate (on Thr197) results in a decrease in the efficiency of phosphoryl transfer and an increase in the Km for ATP [31]. In spite of its distance from the active site cleft, this phosphate is linked by an extensive network of interactions to most of the residues involved in recognition of both ATP and peptide substrates, as well as those involved in catalysis. Protein kinase inhibition: natural and synthetic variations on a theme Taylor and Radzio-Andzelm 223 Figure 3 fb) Beta 3 Linker p27(84-93) Linker ’ ATP Structural views of the cdk2 kinase bound to an inhibitor, p2i’W [25”1. (a) This structure shows cdk2-cyclin A bound to an inhibitor peptide, p2Wpt, and reveals a novel mechanism of inhibition. p27W flanks the surface of both cdk2 and cyclin A with the p27W carboxy-terminal tail binding to the adenine binding pocket of cdk2. (b) A view of the adenine pocket with Tyr88 hydrogen bonding to the linker strand and Lys33 of the kinase core. In this conformation the glycine loop is very distorted. (c) The corresponding view of this region when ATP is bound, rather than p27W. to the cdk2-Cyclin A complex. Amino acid residues are designated using single letter code in the figure. The coordinates for cdk were kindly provided by Nikola Paveltich (Memorial Sloan-Kettering Cancer Center, New York). The role of the SH3 domain in maintaining the kinase core in an inactive conformation was demonstrated clearly by a kinetic analysis of the effect of Nef [32], a small, (25-27 kDa) protein required for infection by HIV. It binds with high affinity to the SH3 domain of Hck. Nef is a potent activator of the Src kinase by a mechanism that specifically involves displacement of the SH3 domain specificity. Because the nucleotide is deeply embedded in the active site cleft and extends over such a large surface area, there are potentially many ways to achieve specificity. As seen in Figure 1, the entire nucleotide-including the adenine, ribose, and triphosphate moieties -is specifically embraced by the glycine-rich loop. Crystal structures of several of these synthetic inhibitors have now been solved, from the regulation and these structures reveal at least one common All of these inhibitors target the adenine binding kinase core. This of Src activity-a that is quite Synthetic distinct represents strategy from the active a new strategy for that targets a region site. and natural inhibitors A number of synthetic and natural product kinase inhibitors have now been identified, and so far all compete with ATP for binding. Thus, like p27Kip1, they have a subsite that mimics the binding of adenine. Despite being competitive inhibitors with respect to ATP, however, and in contrast to earlier predictions, a number of these inhibitors show exquisite specificity. Particularly impressive examples are the pyridinylimidazole inhibitors that specifically target ~38, a MAP kinase [33]. Other examples are the pyrrolo- and pyrazoloquinazolines that are specific for the epidermal growth factor (EGF) receptor (reviewed in [34]; [35]). Thus, in spite of a highly conserved core, a few critical differences allow for high feature. pocket whereby one of the rings mimics the planar adenine ring, fills the pocket, and also forms hydrogen bonds to the linker strand that joins the small and large lobes. This hydrogen bonding is to the same backbone carbonyl and amide that are complemented by the N6 and Nl nitrogens of the adenine ring of ATP. A comparison of the binding of two such inhibitors is shown in Figure 4. H7, H8, and H89 are isoquinoline sulfonamide inhibitors of PKA, PKI, and PKG. H89 shows the highest specificity for PKA (Ki =O.SnM) [36]. By solving the crystal structures of each of these inhibitors in complex with the C subunit of PKA, Engh et a/.[37-l showed that the isoquinoline ring mimics the adenine ring of ATP in the ternary complex of CATP.PKI (5-24). The nitrogen in the isoquinoline ring hydrogen bonds to the 224 Next generation therapeutics linker strand, mimicking the hydrogen bond made by the Nl nitrogen of the adenine ring of ATI? The higher degree of selectivity in H89, compared to H7 and H8, is due to an extended glycine-rich region, including a bromine, loop, resulting in distortion that interacts of the loop. the The crystal structures of cdk2 bound to three different inhibitors also reveal a common mechanism of inhibition [38,39]. All have rings that occupy the adenine binding pocket, and most also form hydrogen bonds with the linker strand. The orientation of the rings differs, however. Olomoucin, a highly specific inhibitor, actually contains a purine ring like ATP, but differs because there is a bulky substitution on the N6 nitrogen. As a consequence of this bulky substituent, the olomoucine purine ring is oriented in a configuration that is reversed compared to the purine ring of ATP [39]. In this case, the hydrogen bonding involves the N3 and N9 nitrogens in the purine ring which replace the N6 and Nl nitrogens in ATI? The indolines are another group of highly specific ATP-based inhibitors. Some of these compounds contain an oxindole core, and are specific for the fibroblast growth factor (FGF) receptor tyrosine kinase [40*]. The structure of this complex reveals that the oxindole ring binds to the adenine pocket of the FGF receptor and hydrogen bonds to the backbone of the linker strand. Specific inhibition of the ~38 kinase by pyridinylimidazoles is one of the best examples where the molecular basis for the specificity of this inhibition is understood. These compounds were first identified by screening for inhibitors of the lipopolysaccharide-induced immune response. In a classic search for the target of these inhibitors, Lee et al. [33] identified ~38, a member of the MAP kinase family. The selectivity of these inhibitors has been well documented [41]; however the actual basis for the selectivity remained unclear until crystal structures were solved. The crystal structure of ~38 is similar to that of ERK-2, another MAP kinase [42,43]. It was the structure of ~38 bound to VK-1911, a pyridinylimidazole, however, that clearly correlated specificity with a single amino acid difference in the ATP-binding pocket [44*-l. The structure-based prediction was confirmed by mutagenesis of this amino acid [44**]. Conclusions Protein kinases are clearly important targets for drug design. Earlier predictions that achieving specificity would be difficult given the highly conserved nature of the kinase core are clearly not valid. So far most synthetic inhibitors target the adenine binding pocket at the active site cleft and compete with ATP for binding. Despite this, high specificity can be achieved by this strategy [33,34,36,40*]. As the structures of more inhibitor-kinase complexes are solved, rules will hopefully be defined which will allow rational design of inhibitors that will target a specific protein kinase. We are rapidly moving in that direction. The conserved nature of the kinase core also makes it an excellent candidate for homology modeling, whereby the Figure 4 (a) Structure (b) of synthetic inhibitors bound to various protein kinases reveal a common mechanism the active site cleft [37-l. (a) Shows ATP binding to the C subunit of PKA superimposed with thick line. and ATP is shown as a thin line. This structure was generated using PDB file 1YDS. the C subunit of PKA superimposed with ATP bound to the same pocket. In this case, H89 is line. This structure was generated using PDB file 1 YDT. for binding to the adenosine binding pocket at H8 binding to the same pocket. H8 is shown as a (b) Shows H89 binding to the active site clefi of shown as a thin line and ATP is shown as a thtck Protein sequence of the conserved kinase inhibition: core of any protein natural kinase and synthetic can be modeled onto a three-dimensional structural template [15]. Once again, as more structures are solved, the accuracy of the modeling also will improve. It is also variations on a theme Taylor and Radzio-Andzelm 225 subunit of CAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry 1993, 32:2154-2161. 11. Hemmer W, McGlone ML, Taylor SS: The role of the glycine triad in the ATP-binding site of CAMP-dependent protein kinase. J B/o/ Chem 1997, 272:16946-l 6954. clear, however, that the design of specific inhibitors need not target only the active site cleft. The C helix, the 12. Bossemeyer D: The glycine-rich sequence of protein kinases: a multifunctional element Trends Biochem Sci 1994, 19:201-205. activation loop or the segments that flank the kinase core are equally valid targets for drug design, as is targeting the ligand binding sites at the active site. Although designing inhibitors that bind to the active kinase is one strategy, designing or screening compound libraries for inhibitors that prevent kinase activation is an equally plausible strategy - one that has been explored by nature but yet to be exploited by combinatorial chemistry. 13. Herberg F, Zimmermann B, McGlone M, Taylor SS: Importance of the A-helix of the cat&tic subunit of CAMP-deoendent orotein inase for stability and f& orienting subdomain; at the ileft interface. Protein Sci 1997, 6:569-579. 14. Taylor SS, Buechler JA, Yonemoto W: CAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem 1990, 59:971-l 005. 15. Kniahton DR. Pearson RB. Sowadski JM. Means AR. Ten Evck LF. Taylor SS, Kemp BE: Structural basis for intrasteric regulation’ of myosin light chain kinase. Science 1992, 258:130-135. 16. .. Acknowledgements SW is supported by grants from the National Institutes of Health and the American Cancer Society. ER-A was supported in part by National Institutes of Health ‘liaining Grant I’HS ‘1‘32 DK07233. The authors wsh to thank N Narayana and T Diller for helpful comments and discussions. References and recommended core. 1 7. Walsh DA, Angelos KL, Van Patten SM, Glass DB, Garetto LP: The inhibitor protein of the CAMP-dependent protein kinase. In Peptides and Protein Phosphorylation. Edited by BE Kemp. Boca Raton: CRC Press, Inc.; 1990:43-84. 18. Knighton DR, Zheng J, Ten Eyck LF, Xuong N-h, Taylor SS, Sowadski JM: Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphatedependent protein kinase. Science 1991, 253:414-420. 19. Herberg FW, Taylor SS: Physiological inhibitors of the catalytic subunit of CAMP-dependent protein kinase: effect of MgATP on protein/protein interaction. Biochemisrry 1993, 32:1401514022. 20. Gibbs CS, Knighton DR, Sowadski JM, Taylor SS, Zoller MJ: Systematic mutational analysis of CAMP-dependent protein kinase identifies unregulated catalytic subunits and defines regions important for the recognition of the regulatory subunit J Biol Chem 1992, 267:4806-4614. reading Papers of particular interest, published within the annual period of review, have been highlighted as: . .. Kobe B. Heierhorst J. Feil SC. Parker MW. Benian GM. Klaudiusz RW, Kemp BE: Giant protein kinases: domain interactions and structural basis of autoregulation. EMBO J 1996, 15:6810-6821. Describes the structure of the klnase domam of twttchin plus the pseudosubstrate region and the IgG domain that lie carboxy-terminal to the conserved of special interest of outstanding intt,est 1. Fischer EH, Krebs EG: Conversion of phosphorylase b to phosphorylase a in muscle extracts. J Biol Chem 1955, 216:121-132. 2. Hanks SK, Hunter T: Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J 1995, 9576-596. 3. Zheng J, Knighton DR, Xuong N-h, Taylor SS, Sowadski JM, Ten Eyck LF: Crystal structures of the myristylated catalytic subunit of CAMP-dependent protein kinase reveal open and closed conformations. Protein Sci 1993, 2:1559-l 573. 21. Knighton DR, Zheng J, Ten Eyck LF, Ashford VA, Xuong N-h, Taylor SS, Sowadski JM: Crystal structure of the catalytic subunit of CAMP-dependent protein kinase. Science i991( 253:407-414. Gibson RM, Ji-Buechler Y, Taylor SS: Identification of electrostatic interaction sites between the regulatory and catalytic subunits of cyclic AMP-dependent protein kinase. Protein Sci 1997, in press. 22. Hu S-H, Parker MW, Lei JY, Wilce MCJ, Benian GM, Kemp BE: Insights into autoregulation from the crystal structure of twitchin kinase. Nature 1994, 369:578-581. 23. Kemp BE, Faux MC, Means AR, House C, Tiganis T, Hu S-H, Mitchelhill KI: Structural aspects: pseudosubstrate and substrate interactions. In Protein Kinase. Edited by JR Woodgett. Oxford: IRL Press at Oxford University Press; 1994:30-67. 24. Heierhorst J, Kobe B, Feil SC, Parker MW, Benian GM, Weiss KR, Kemp BE: Ca2+/SlOO regulation of giant protein kinases. Nature 1996, 380:636-639. 4. 5. Grant BD, Adams JA: Pre-steady-state analysis of CAMPdependent protein kinase using rapid quench-flow techniques. Biochemistry 1996, 35:2022-2029. 6. Lew J, Taylor SS, Adams JA: Identification of a partially ratedetermining step in the catalytic mechanism of CAMPdependent protein kinase: a transient kinetic study using stopped-flow fluorescence spectroscopy. Biochemistry 1997, 36:6717-6724. 25. .. Narayana N, Cox S, Shumel S, Taylor SS, Xuong N: Crystal structure of the poly-histidine tagged recombinant catalytic subunit of CAMP-dependent protein kinase complexed with the peptide inhibitor, PKl(5-24) and adenosine. Biochemistry 1997, 36:4438-4448. This paper gives an in-depth description of the nucleotide binding site in the catalytic subunit of CAMP-dependent protein kinase (PKA). Russo AA, Jeffrey PD, Patten AK, Massague J, Pavletich NP: Crystal Structure of the p27Kipl cyclin-dependent-kinase inhibitor bound to the cyclin A-CdkP complex. Nature 1996, 382:325-331, The structure of ~27~lP’ bound to cdk2-cyclin A reveals an adenine mimetic mechanism of kinase inhibition whereby the carboxyl terminus of p27KlPl occupies the adenine binding pocket of cdk2. 8. Hon W-C, McKay GA, Thompson PR, Sweet RM, Yang DSC, Wright GD, Berghuis AM: Structure of an enzyme required for aminoglycoside antibiotic resistance reveal hom&ogy to eukaryotic protein kinases. Cell 1997, 89:887-895. 26. Hubbard SR, Wei L, Ellis L, Hendrickson WA: Crystal structure of the tyrosine kinase domain of the human insulin receptor?. Nature 1994, 372:746-754. 27. 9. Bossemeyer D, Engh RA, Kinzel V, Ponstingl H, Huber R: Phosphotransferase and substrate binding mechanism of the CAMP-dependent protein kinas? catalytic subunit from porcine heart as deduced from the 2.0A structure of the complex with Mnz+ adenyl imidodiphosphate and inhibitor peptide PKl(5-24). EM50 J 1993, 12:649-859. Xu W, Harrison SC, Eck MJ: Three-dimensional structure of the tyrosine kinase c-Src. Nature 1997, 385:595-599. see annotation [28**]. z . 10. Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong N-h, Taylor SS, Sowadski JM: Crystal structure of the catalytic Aase 28. Sicheri F, Moarefi I, Kuriyan J: Crystal structure of the Src family .. tyrosine kinase Hck Nature 1997, 385:602-609. These two papers [27”,28”1 describe the structures of two nonreceptor tyrosine kinases. Both include the contiguous SH2 and SH3 domains and provide excellent examples of an inhibitory mechanism that is distinct from a pseudosubstrate-based mechanism. 226 29. Next generation therapeutics Boemer RJ, Kassel DB, Barker SC, Ellis B, DeLacy P, Knight WB: Correlation of the phosphorylation states of pp60c-src with tyrosine kinase activity: the intramolecular pY530-SH2 complex retains significant activity if Y419 is phosphorylated. Biochemistry 1996, 35:9519-9525. 30. Johnson LN, Noble ME, Owen DJ: Active and inactive protein . kinases: structural basis for regulation. Cell 1996, 85:149-l 56. An excellent review of activation loops in protein kinases that explains why phosphorylation of this region is typically essential for activity. 31. Adams JA, McGlone ML, Gibson R, Taylor SS: Phosphorylation modulates catalytic function and regulation in CAMPdependent protein kinase. Biochemistry 1995, 34:2447-2454. 32. Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C-H. Kurivan J. 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