Structural features of the protein kinase domain and targeted binding by small-molecule inhibitors

Protein kinases are key components in cellular signaling pathways as they carry out the phosphorylation of proteins, primarily on Ser, Thr, and Tyr residues. The catalytic activity of protein kinases is regulated, and they can be thought of as molecular switches that are controlled through protein–protein interactions and post-translational modifications. Protein kinases exhibit diverse structural mechanisms of regulation and have been fascinating subjects for structural biologists from the first crystal structure of a protein kinase over 30 years ago, to recent insights into kinase assemblies enabled by the breakthroughs in cryo-EM. Protein kinases are high-priority targets for drug discovery in oncology and other disease settings, and kinase inhibitors have transformed the outcomes of specific groups of patients. Most kinase inhibitors are ATP competitive, deriving potency by occupying the deep hydrophobic pocket at the heart of the kinase domain. Selectivity of inhibitors depends on exploiting differences between the amino acids that line the ATP site and exploring the surrounding pockets that are present in inactive states of the kinase. More recently, allosteric pockets outside the ATP site are being targeted to achieve high selectivity and to overcome resistance to current therapeutics. Here, we review the key regulatory features of the protein kinase family, describe the different types of kinase inhibitors, and highlight examples where the understanding of kinase regulatory mechanisms has gone hand in hand with the development of inhibitors.     

A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

The growth factor-activated AGC protein kinases RSK, S6K, PKB, MSK and SGK are activated by serine/threonine phosphorylation in the activation loop and in the hydrophobic motif, C-terminal to the kinase domain. In some of these kinases, phosphorylation of the hydrophobic motif creates a specific docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind the phosphate. Moreover, we demonstrate that RSK2, S6K1, PKBalpha, MSK1 and SGK1 contain a similar phosphate-binding pocket, which they use for intramolecular interaction with their own phosphorylated hydrophobic motif. Molecular modelling and experimental data provide evidence for a common activation mechanism in which the phosphorylated hydrophobic motif and activation loop act on the alphaC-helix of the kinase structure to induce synergistic stimulation of catalytic activity. Sequence conservation suggests that this mechanism is a key feature in activation of >40 human AGC kinases.     

Kinases and Pseudokinases: Lessons from RAF

Protein kinases are thought to mediate their biological effects through their catalytic activity. The large number of pseudokinases in the kinome and an increasing appreciation that they have critical roles in signaling pathways, however, suggest that catalyzing protein phosphorylation may not be the only function of protein kinases. Using the principle of hydrophobic spine assembly, we interpret how kinases are capable of performing a dual function in signaling. Its first role is that of a signaling enzyme (classical kinases; canonical), while its second role is that of an allosteric activator of other kinases or as a scaffold protein for signaling in a manner that is independent of phosphoryl transfer (classical pseudokinases; noncanonical). As the hydrophobic spines are a conserved feature of the kinase domain itself, all kinases carry an inherent potential to play both roles in signaling. This review focuses on the recent lessons from the RAF kinases that effectively toggle between these roles and can be “frozen” by introducing mutations at their hydrophobic spines.     

Lining the pockets of kinases and phosphatases

The regulation of the activity of kinases and phosphatases is an essential aspect of intracellular signal transduction. Recently determined structures of AGC protein kinases, including isoforms of PKB, PKC, GRK and ROCK, indicate that occupancy of a hydrophobic pocket in the kinase N-lobe by a segment of the protein immediately C terminal to the kinase domain provides a mechanism for regulating kinase activity. In addition, crystal structures of Aurora-A and Aurora-B, which are closely related to AGC family kinases, in complex with their activators, TPX2 and INCENP, respectively, show how allosteric kinase activation is achieved by the binding of the activator protein to an equivalent hydrophobic pocket. Hence, regulation of kinase activity by analogous interactions is a shared regulatory mechanism of these kinases. Two crystal structures have explained the molecular basis of PKA anchoring through its regulatory subunits by members of the AKAP family of scaffold proteins. AKAPs can also interact directly with protein kinase and phosphatase catalytic domains. The crystal structure of the PP1 catalytic subunit in complex with the targeting subunit MYPT1 indicates that there is also scope for intimate phosphatase regulation by scaffold proteins.     

Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization

Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core.     

A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCzeta, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCzeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCzeta and PKCiota, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCzeta, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCzeta. These findings indicate that the hydrophobic motif of PRK2 and PKCzeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.     

The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3-kinase-dependent manner in response to insulin/growth factors signalling. Activation entails phosphorylation of these kinases at two residues, the T-loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T-loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the 'PIF-binding pocket', plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T-loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF-binding pocket of PDK1 and their T-loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF-binding pocket of PDK1 is not required for the phosphorylation of PKBalpha by PDK1. The PIF-binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.     

Molecular dynamics analysis of conserved hydrophobic and hydrophilic bond-interaction networks in ErbB family kinases

The EGFR (epidermal growth factor receptor)/ErbB/HER (human EGFR) family of kinases contains four homologous receptor tyrosine kinases that are important regulatory elements in key signalling pathways. To elucidate the atomistic mechanisms of dimerization-dependent activation in the ErbB family, we have performed molecular dynamics simulations of the intracellular kinase domains of three members of the ErbB family (those with known kinase activity), namely EGFR, ErbB2 (HER2) and ErbB4 (HER4), in different molecular contexts: monomer against dimer and wild-type against mutant. Using bioinformatics and fluctuation analyses of the molecular dynamics trajectories, we relate sequence similarities to correspondence of specific bond-interaction networks and collective dynamical modes. We find that in the active conformation of the ErbB kinases, key subdomain motions are co-ordinated through conserved hydrophilic interactions: activating bond-networks consisting of hydrogen bonds and salt bridges. The inactive conformations also demonstrate conserved bonding patterns (albeit less extensive) that sequester key residues and disrupt the activating bond network. Both conformational states have distinct hydrophobic advantages through context-specific hydrophobic interactions. We show that the functional (activating) asymmetric kinase dimer interface forces a corresponding change in the hydrophobic and hydrophilic interactions that characterize the inactivating bond network, resulting in motion of the αC-helix through allostery. Several of the clinically identified activating kinase mutations of EGFR act in a similar fashion to disrupt the inactivating bond network. The present molecular dynamics study reveals a fundamental difference in the sequence of events in EGFR activation compared with that described for the Src kinase Hck.     

PDK2: the missing piece in the receptor tyrosine kinase signaling pathway puzzle

Activation of members of the protein kinase AGC (cAMP dependent, cGMP dependent, and protein kinase C) family is regulated primarily by phosphorylation at two sites: a conserved threonine residue in the activation loop and a serine/threonine residue in a hydrophobic motif (HM) near the COOH terminus. Although phosphorylation of these kinases in the activation loop has been found to be mediated by phosphoinositide-dependent protein kinase-1 (PDK1), the kinase(s) that catalyzes AGC kinase phosphorylation in the HM remains uncharacterized. So far, at least 10 kinases have been suggested to function as an HM kinase or the so-called "PDK2," including mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MK2), integrin-linked kinase (ILK), p38 MAP kinase, protein kinase Calpha (PKCalpha), PKCbeta, the NIMA-related kinase-6 (NEK6), the mammalian target of rapamycin (mTOR), the double-stranded DNA-dependent protein kinase (DNK-PK), and the ataxia telangiectasia mutated (ATM) gene product. However, whether any or all of these kinases act as a physiological HM kinase remains to be established. Nonetheless, available data suggest that multiple systems may be used in cells to regulate the activation of the AGC family kinases. It is possible that, unlike activation loop phosphorylation, phosphorylation of the HM site in the different AGC family kinases is mediated by distinct kinases. In addition, phosphorylation of the AGC family kinase at the HM site could be cell type, signaling pathway, and substrate specific. Identification and characterization of the bonafide HM kinase(s) will be essential to verify these hypotheses.     

Evolution of the eukaryotic protein kinases as dynamic molecular switches

Protein kinases have evolved in eukaryotes to be highly dynamic molecular switches that regulate a plethora of biological processes. Two motifs, a dynamic activation segment and a GHI helical subdomain, distinguish the eukaryotic protein kinases (EPKs) from the more primitive eukaryotic-like kinases. The EPKs are themselves highly regulated, typically by phosphorylation, and this allows them to be rapidly turned on and off. The EPKs have a novel hydrophobic architecture that is typically regulated by the dynamic assembly of two hydrophobic spines that is usually mediated by the phosphorylation of an activation loop phosphate. Cyclic AMP-dependent protein kinase (protein kinase A (PKA)) is used as a prototype to exemplify these features of the PKA superfamily. Specificity in PKA signalling is achieved in large part by packaging the enzyme as inactive tetrameric holoenzymes with regulatory subunits that then are localized to macromolecular complexes in close proximity to dedicated substrates by targeting scaffold proteins. In this way, the cell creates discrete foci that most likely represent the physiological environment for cyclic AMP-mediated signalling.     

The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells

BACKGROUND: Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the 'T-loop' of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1) activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown. RESULTS: Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted were viable. In PDK1(-/-) ES cells, PKB, p70 S6 kinase and p90 Rsk were not activated by stimuli that induced strong activation in PDK1(+/+) cells. Other AGC kinases - namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) - had normal activity or were activated normally in PDK1(-/-) cells. The insulin-like growth factor 1 (IGF1) induced PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1(-/-) cells. IGF1 did not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1(-/-) cells. CONCLUSIONS: PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1(-/-) cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase.     

A Catalytically Disabled Double Mutant of Src Tyrosine Kinase Can Be Stabilized into an Active-Like Conformation

Tyrosine kinases are enzymes playing a critical role in cellular signaling. Molecular dynamics umbrella sampling potential of mean force computations are used to quantify the impact of activating and inactivating mutations of c-Src kinase. The potential of mean force computations predict that a specific double mutant can stabilize c-Src kinase into an active-like conformation while disabling the binding of ATP in the catalytic active site. The active-like conformational equilibrium of this catalytically dead kinase is affected by a hydrophobic unit that connects to the hydrophobic spine network via the C-helix. The αC-helix plays a crucial role in integrating the hydrophobic residues, making it a hub for allosteric regulation of kinase activity and the active conformation. The computational free-energy landscapes reported here illustrate novel design principles focusing on the important role of the hydrophobic spines. The relative stability of the spines could be exploited in future efforts to artificially engineer active-like but catalytically dead forms of protein kinases.     

Protein structure: discovering selective protein kinase inhibitors

A plethora of important targets for therapeutic intervention occurs in the protein kinase superfamily, one of the most thoroughly investigated groups of drug targets. Kinases have a deep hydrophobic ATP binding site that has been successfully exploited with the discovery of potent ATP-competitive drugs. However, most features of this pocket are well conserved in all protein kinases, which explains why kinase inhibitors typically exhibit a fairly indiscriminate spectrum of activity. Crystal structures of various protein kinases bound to their ligands are described, which begin to explain the observed selectivity profiles of kinase inhibitors. The insights gained from these structures suggest several approaches to improve inhibitor specificity and these approaches are summarized. The exciting potential of new high-throughput methods in structure determination that enable the systematic atomic-resolution investigation of large numbers of inhibitors bound to their various kinase targets will be discussed.     

The hydrophobic phosphorylation motif of conventional protein kinase C is regulated by autophosphorylation.

BACKGROUND: A growing number of kinases are now known to be controlled by two phosphorylation switches, one on a loop near the entrance to the active site and a second on the carboxyl terminus. For the protein kinase C (PKC) family of enzymes, phosphorylation at the activation loop is mediated by another kinase but the mechanism for carboxy-terminal phosphorylation is still unclear. The latter switch contains two phosphorylation sites - one on a 'turn' motif and the second on a conserved hydrophobic phosphorylation motif - that are found separately or together in a number of other kinases. RESULTS: Here, we investigated whether the carboxy-terminal phosphorylation sites of a conventional PKC are controlled by autophosphorylation or by another kinase. First, kinetic analyses revealed that a purified construct of the kinase domain of PKC betaII autophosphorylated on the Ser660 residue of the hydrophobic phosphorylation motif in an apparently concentration-independent manner. Second, kinase-inactive mutants of PKC did not incorporate phosphate at either of the carboxy-terminal sites, Thr641 or Ser660, when expressed in COS-7 cells. The inability to incorporate phosphate on the hydrophobic site was unrelated to the phosphorylation state of the other key phosphorylation sites: kinase-inactive mutants with negative charge at Thr641 and/or the activation-loop position were also not phosphorylated in vivo. CONCLUSIONS: PKC betaII autophosphorylates at its conserved carboxy-terminal hydrophobic phosphorylation site by an apparently intramolecular mechanism. Expression studies with kinase-inactive mutants revealed that this mechanism is the only one responsible for phosphorylating this motif in vivo. Thus, conventional PKC autoregulates the carboxy-terminal phosphorylation switch following phosphorylation by another kinase at the activation loop switch.     

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.     

Serine/threonine protein kinases and apoptosis

Over the past decade, our understanding of apoptosis, or programmed cell death, has increased greatly, with the identification of some of the major components of the apoptotic programme and the processes regulating their activation. Although apoptosis is an intrinsic process present in all cells, it can be regulated by extrinsic factors, including hormones, growth factors, cell surface receptors, and cellular stress. The actions of both pro- and antiapoptotic factors are often affected by modulation of the phosphorylation status of key elements of the apoptotic process. This minireview will focus on the role of protein kinases in apoptosis. Apoptosis is a multistep process and protein kinases have been implicated both in the upstream induction phase of apoptosis and in the downstream execution stage, as the direct targets for caspases. Due to the space constraints of this review it is not possible to discuss all of the kinases involved in the apoptotic process and we have focused here on the role of the serine/threonine protein kinases. The kinases of this family that have been suggested to play a role in apoptosis are the mitogen-activated protein kinase (MAPK) family, specifically p42/44 ERK, p38 MAPK and c-Jun N-terminal kinase (JNK), cyclic AMP-dependent protein kinase (PKA), protein kinase B (PKB), or Akt and protein kinase C (PKC). We have also considered briefly the potential for the regulation of these kinases by tyrosine protein kinases, such as c-abl.     

Regulation of protein kinases in insulin,growth factor and Wnt signalling

Protein kinase cascades provide the regulatory mechanisms for many of the essential processes in eukaryotic cells. Recent structural and biochemical work has revealed the basis of phosphorylation regulation of three consecutive protein kinases - phosphoinositide-dependent kinase 1 (PDK1), protein kinase B (PKB)/Akt and glycogen synthase kinase 3beta (GSK3beta) - which transduce signals generated by insulin and/or growth factors binding to cell surface receptors. PDK1 and PKB are both AGC family kinases. Whereas PKB is positively regulated via its phosphorylated C-terminal hydrophobic motif, the activity and specificity of PDK1 are determined by equivalent hydrophobic motifs of substrate AGC kinases. In a contrasting mechanism, GSK3beta is negatively regulated by competitive autoinhibition by its phosphorylated N terminus. GSK3beta also functions in the developmental Wnt signalling pathway, but without cross-talk with the PDK1-PKB/Akt pathway. Structural studies of GSK3beta complexes are contributing to our understanding of the phosphorylation-independent mechanism that insulates the Wnt and insulin/growth factor pathways.     

Conserved spatial patterns across the protein kinase family

Protein kinases are a large family of enzymes heavily involved in signal transduction, regulation of metabolism, and control of cell growth and differentiation. These functions require precise recognition of widely diverse signals and substrates, and very detailed control of protein kinase activity. Large molecules interact primarily through recognition of surface features. Comparison of surfaces is complicated by both sequence diversity and conformational variability, including multiple possible rotameric states of side chains. We used a recently developed method of protein surface comparison to compare different serine/threonine and tyrosine kinases. As we have shown, two hydrophobic cores inside a protein kinase molecule are connected by a unique formation, called the "spine". It exists only in the active conformation of protein kinases and is dynamically disassembled during the inactivation process. Detection of such structures by any other method was not possible as the residues which comprise the spine do not form any sequence or 3D motifs in a traditional sense.