Louise N. Johnson

Active and inactive protein kinases: structural basis for regulation.

lytic groups and relief of steric blocking to allow access Protein kinases and phosphatases play pivotal roles in of substrates to the catalytic site. The activation segregulating and coordinating aspects of metabolism, ment and the control of its conformation via phosphorygene expression, cell growth, cell motility, cell differentilation plays a key role in these transformations. It can ation, and cell division. As a result, if cellular life is to be involved in recognition of regulatory subunits, in aufunction in an orderly manner, the switching on and off toinhibition of substrate binding, and in promotion of of protein kinases and phosphatases is as crucial for the correct orientation of domains and catalytic site resitheir function as their catalytic activity. dues. This review summarizes our current understandThe total number of distinct kinase domain sequences ing of control by the activation segment based on recent available is now approaching 400 (Hardie and Hanks, structure determinations of active and inactive kinases. 1995). Multiple sequence alignments have indicated that The first observation of Thr-197 phosphorylation in all protein kinases should have similar structures, and the activation segment of cAPK was reported in 1979 this has been confirmed by recent crystal structure de(Shoji et al., 1979). Although itwas speculated that phosterminations. Conserved features have been identified phorylation at discrete sites might be of physiological in 12 subdomain regions of all protein kinases, and resiimportance in the regulation of enzyme activity, it was dues from these subdomains have been implicated in not until 1990 that mutagenesis studies indicated the essential roles in enzyme structure and function. Protein significance of this site for the recognition of the regulakinases exhibit variability in other parts of the kinase tory subunit (Levin and Zoller, 1990), and not until 1993 domain, and different kinases may contain additional that it was definitively shown that phosphorylation is domains, additional subunits, or both. These features promoted by an autocatalytic event that is crucial for allow several different mechanisms for control. activation (Steinberg et al., 1993). The crystal structure Control mechanisms that have been recognized to determination of cAPK in 1991 (Knighton et al., 1991a, date include the following: control by additional subunits 1991b) showed the structural importance of Thr-197 or domains that may function in response to second phosphorylation and demonstrated possible roles of messengers (e.g., cyclic AMP binding to the regulatory phosphorylation in promotion of activation. The strucsubunit of cyclic AMP–dependent protein kinase [cAPK], ture provided a definitive model to which other kinases Ca/calmodulin binding to calmodulin-dependent could be related. Also in 1991, both the fission yeast protein kinases, and Ca and diacyl glyerol binding to cell division control kinase (cdc2) (Gould et al., 1991) N-terminal domains of protein kinase C); control by addiand the microtubule-associated protein kinase (MAPK) tional subunits whose level of expression varies de(Payne et al., 1991) were found to be activated by phospending on the functional state of the cell (e.g., cyclin phorylation on residues that mapped to a position simiregulation of the cyclin-dependent protein kinases lar to Thr-197 in cAPK. These results showed the impor[CDKs]); control by additional domains that target the tance of this site not only as an autophosphorylation kinase to different molecules or subcellular localizations site, as in cAPK, but also as a site involved in kinase (e.g., the SH2 and SH3 domains of the Src kinases); cascade activation mechanisms. For the tyrosine kicontrol by additional domains that inhibit the kinase nases, autophosphorylation of pp60 at position Tyractivity by an autoregulatory process (e.g., myosin light 416 (now known to be in the activation segment) had chain kinase [MLCK]); and control by phosphorylation been shown in the early 1980s, and its significance for and dephosphorylation by kinases and phosphatases. control in the cellular counterpart of Src kinase was Phosphorylation of specific threonine, serine, or tyrosine established by 1987 (reviewed by Hunter, 1987). The residues may occur at a number of sites. Some of these following year, trans-autophosphorylation of the insulin are located in the N-terminal or C-terminal portions of receptor tyrosine kinase (IRK) was elaborated, and the the polypeptide chain, which lie outside the kinase dosimilarity in sequence location of some of these sites main (e.g., in Src kinase and calmodulin-dependent kito that in Src kinase and its relatives and in cAPK was nase II [CaMKII]) or on othersubunits (e.g., in phosphorynoted (reviewed by White and Kahn, 1994). As more and lase kinase [PhK]). A key aspect of regulation recognized more kinases have been discovered and sequenced and in recent years is that many protein kinases are phosfurther kinase cascades established, it is recognized phorylated on a residue(s) located in a particular segthat control by phosphorylation in the activation segment in the center of the kinase domain, which is termed ment is a property of most, but not all, protein kinases

Crystallographic Studies of the Activity of Hen Egg-White Lysozyme

The chemical evidence for the enzymic activity of lysozyme will be discussed in detail by other speakers at this meeting, but in order to describe our crystallographic studies of the interactions between the enzyme and its substrates it is necessary to summarize briefly what was known about them at the beginning of our work. Simultaneously with his discovery of lysozyme Fleming (1922) discovered a Gram-positive species of bacteria, Micrococcus lysodeikticus, which is particularly susceptible to the action of the enzyme. It was not until much later, however, that Salton (1952) demonstrated that the substrate is located entirely within the bacterial cell wall and it is only very recently that its chemical constitution has been established. Valuable early experiments (for example, by Meyer, Palmer, Thomson & Khorazo 1936; Meyer, Hahnel & Steinberg 1946; and by Epstein & Chain 1940) showed that lysozyme releases N-acetyl-amino sugars from M. lysodeikticus, but the first indication of the type of linkage attacked by lysozyme came when Berger & Weiser (1957) showed that lysozyme also degrades chitin, the linear polymer of N-acetylghicosamine.

Structural changes in glycogen phosphorylase induced by phosphorylation

A comparison of the refined crystal structures of dimeric glycogen phosphorylase b and a reveals structural changes that represent the first step in the activation of the enzyme. On phosphorylation of serine-14, the N-terminus of each subunit assumes an ordered helical conformation and binds to the surface of the dimer. The consequent structural changes at the N- and C-terminal regions lead to strengthened interactions between subunits and alter the binding sites for allosteric effectors and substrates.

The Structural Basis for Control of Eukaryotic Protein Kinases

Eukaryotic protein kinases are key regulators of cell processes. Comparison of the structures of protein kinase domains, both alone and in complexes, allows generalizations to be made about the mechanisms that regulate protein kinase activation. Protein kinases in the active state adopt a catalytically competent conformation upon binding of both the ATP and peptide substrates that has led to an understanding of the catalytic mechanism. Docking sites remote from the catalytic site are a key feature of several substrate recognition complexes. Mechanisms for kinase activation through phosphorylation, additional domains or subunits, by scaffolding proteins and by kinase dimerization are discussed.

Protein kinase inhibitors: contributions from structure to clinical compounds.

Protein kinases catalyse key phosphorylation reactions in signalling cascades that affect every aspect of cell growth, differentiation and metabolism. The kinases have become prime targets for drug intervention in the diseased state, especially in cancer. There are currently 10 drugs that have been approved for clinical use and many more in clinical trials. This review summarises the structural basis for protein kinase inhibition and discusses the mode of action for each of the approved drugs in the light of structural results. All but one of the approved compounds target the ATP binding site on the kinase. Both the active and inactive conformations of protein kinases have been used in strategies to produce potent and selective compounds. Targeting the inactive conformation can give high specificity. Targeting the active conformation is favourable where the diseased state has arisen from activating mutations, but such inhibitors generally target several protein kinases. Drug resistance mutations are a potential risk for both conformational states, where drug-binding regions are not directly involved in catalysis. Imatinib (Glivec), the most successful of protein kinase inhibitors, targets the inactive conformation of ABL tyrosine kinase. Newer compounds, such as dasatinib, which targets the ABL active state, have been developed to increase potency and have proved effective for some, but not all, drug-resistant mutations. The first epidermal growth factor receptor (EGFR) inhibitors in clinical use [gefitinib (Iressa) and erlotinib (Tarceva)] targeted the active form of the kinase, and this proved advantageous for patients whose cancer was caused by mutations that resulted in a constitutively active EGFR kinase domain. Newer approved compounds, such as lapatinib (Tykerb), target the inactive conformation with high potency. A further compound that forms a covalent attachment to the kinase has been found to overcome one of the major drug resistance mutations, where the effectiveness of the drug in vivo is dependent on its ability to compete successfully in the presence of cellular concentrations of ATP. Inhibitors of vascular endothelial growth factor receptor (VEGFR) kinase against cancer angiogenesis show the advantage of some relaxation in specificity. Sorafenib, originally developed as RAF inhibitor, is now in clinical use as a VEGFR inhibitor. Temsirolimus (a derivative of rapamycin) is the only example of a drug in clinical use that does not target the kinase ATP site. Instead rapamycin, when in complex with the protein FKBP12, effectively targets mTOR kinase at a site located on a domain, the FRB domain, that appears to be involved in localisation or substrate docking.

The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation

The positive transcription elongation factor b (P‐TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26° of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK–cyclin interactions. The CycT1 C‐terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C‐terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis.

The structural basis for substrate recognition and control by protein kinases1

Protein kinases catalyse phospho transfer reactions from ATP to serine, threonine or tyrosine residues in target substrates and provide key mechanisms for control of cellular signalling processes. The crystal structures of 12 protein kinases are now known. These include structures of kinases in the active state in ternary complexes with ATP (or analogues) and inhibitor or peptide substrates (e.g. cyclic AMP dependent protein kinase, phosphorylase kinase and insulin receptor tyrosine kinase); kinases in both active and inactive states (e.g. CDK2/cyclin A, insulin receptor tyrosine kinase and MAPK); kinases in the active state (e.g. casein kinase 1, Lck); and kinases in inactive states (e.g. twitchin kinase, calcium calmodulin kinase 1, FGF receptor kinase, c‐Src and Hck). This paper summarises the detailed information obtained with active phosphorylase kinase ternary complex and reviews the results with reference to other kinase structures for insights into mechanisms for substrate recognition and control.

Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity.

We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) fromSaccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-Å resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.

The crystal structure of the human polo-like kinase-1 polo box domain and its phospho-peptide complex.

Human polo‐like kinase Plk1 localizes to the centrosomes, kinetochores and central spindle structures during mitosis. It plays an essential role in promoting mitosis and cytokinesis through phosphorylation of a number of different substrates. Kinase activity is regulated by a conserved C‐terminal domain, termed the polo box domain (PBD), which acts both as an autoinhibitory domain and as a subcellular localization domain. We have determined the crystal structure of Plk1 PBD (residues 367–603) to 2.2 Å resolution and the structure of a phospho‐peptide–PBD (residues 345–603) complex to 2.3 Å resolution. The two polo boxes of the PBD exhibit identical folds based on a six‐stranded β‐sheet and an α‐helix, despite only 12% sequence identity. The phospho‐peptide binds at a site between the two polo boxes. It makes a short antiparallel β‐sheet connection and critical contacts to residues Trp414, Leu490, His538 and Lys540. Most of these residues had been shown to be important for biological activity through mutational studies. The results provide an explanation for phospho‐peptide recognition and create the basis for new functional studies.

The regulation of protein phosphorylation.

Phosphorylation plays essential roles in nearly every aspect of cell life. Protein kinases regulate signalling pathways and cellular processes that mediate metabolism, transcription, cell-cycle progression, differentiation, cytoskeleton arrangement and cell movement, apoptosis, intercellular communication, and neuronal and immunological functions. Protein kinases share a conserved catalytic domain, which catalyses the transfer of the gamma-phosphate of ATP to a serine, threonine or tyrosine residue in protein substrates. The kinase can exist in an active or inactive state regulated by a variety of mechanisms in different kinases that include control by phosphorylation, regulation by additional domains that may target other molecules, binding and regulation by additional subunits, and control by protein-protein association. This Novartis Medal Lecture was delivered at a meeting on protein evolution celebrating the 200th anniversary of Charles Darwins birth. I begin with a summary of current observations from protein sequences of kinase phylogeny. I then review the structural consequences of protein phosphorylation using our work on glycogen phosphorylase to illustrate one of the more dramatic consequences of phosphorylation. Regulation of protein phosphorylation is frequently disrupted in the diseased state, and protein kinases have become high-profile targets for drug development. Finally, I consider recent advances on protein kinases as drug targets and describe some of our recent work with CDK9 (cyclin-dependent kinase 9)-cyclin T, a regulator of transcription.