Protein-protein interactions in the allosteric regulation of protein kinases

Protein-protein interactions involving the catalytic domain of protein kinases are likely to be generally important in the regulation of signal transduction pathways, but are rather sparsely represented in crystal structures. Recently determined structures of the kinase domains of the mitogen-activated protein kinase Fus3, the RNA-dependent kinase PKR, the epidermal growth factor receptor and Ca(2+)/calmodulin-dependent protein kinase II have revealed unexpected and distinct mechanisms by which interactions with the catalytic domain can modulate kinase activity.     

Structures of Src-family tyrosine kinases

The crystal structures of three Src-family tyrosine kinases have been determined recently. The structure of the catalytic domain of Lck has been determined in the active autophosphorylated state. The structures of larger constructs of c-Src and Hck, containing the SH3, SH2 and catalytic domains, as well as a C-terminal regulatory tail, have been determined in the down-regulated state, phosphorylated in the C-terminal tail. A comparison of these structures leads to an unanticipated mechanism for the regulation of catalytic activity by cooperative interactions between the SH2, SH3 and catalytic domains.     

Structural modes of stabilization of permissive phosphorylation sites in protein kinases: distinct strategies in Ser/Thr and Tyr kinases

Protein kinases phosphorylate several cellular proteins providing control mechanisms for various signalling processes. Their activity is impeded in a number of ways and restored by alteration in their structural properties leading to a catalytically active state. Most protein kinases are subjected to positive and negative regulation by phosphorylation of Ser/Thr/Tyr residues at specific sites within and outside the catalytic core. The current review describes the analysis on 3D structures of protein kinases that revealed features distinct to active states of Ser/Thr and Tyr kinases. The nature and extent of interactions among well-conserved residues surrounding the permissive phosphorylation sites differ among the two classes of enzymes. The network of interactions of highly conserved Arg preceding the catalytic base that mediates stabilization of the activation segment exemplifies such diverse interactions in the two groups of kinases. The N-terminal and the C-terminal lobes of various groups of protein kinases further show variations in their extent of coupling as suggested from the extent of interactions between key functional residues in activation segment and the N-terminal alphaC-helix. We observe higher similarity in the conformations of ATP bound to active forms of protein kinases compared to ATP conformations in the inactive forms of kinases. The extent of structural variations accompanying phosphorylation of protein kinases is widely varied. The comparison of their crystal structures and the distinct features observed are hoped to aid in the understanding of mechanisms underlying the control of the catalytic activity of distinct subgroups of protein kinases.     

Enhancement of ABL kinase catalytic efficiency by a direct binding regulator is independent of other regulatory mechanisms

ABL family tyrosine kinases are tightly regulated by autoinhibition and phosphorylation mechanisms. These kinases maintain an inactive conformation through intramolecular interactions involving SH3 and SH2 domains. RIN1, a downstream effector of RAS, binds to the ABL SH3 and SH2 domains and stimulates ABL tyrosine kinase activity. RIN1 binding to the ABL2 kinase resulted in a large decrease in Km and a small increase in Vmax toward an ABL consensus substrate peptide. The enzyme efficiency (k(cat)/Km) was increased more than 5-fold by RIN1. In addition, RIN1 strongly enhanced ABL-mediated phosphorylation of CRK, PSTPIP1, and DOK1, all established ABL substrates but with unique protein structures and distinct target sequences. Importantly RIN1-mediated stimulation of ABL kinase activity was independent of activation by SRC-mediated phosphorylation. RIN1 increased the kinase activity of both ABL1 and ABL2, and this occurred in the presence or absence of ABL regulatory domains outside the SH3-SH2-tyrosine kinase domain core. We further demonstrate that a catalytic site mutation associated with broad drug resistance, ABL1T315I, remains responsive to stimulation by RIN1. These findings are consistent with an allosteric kinase activation mechanism by which RIN1 binding promotes a more accessible ABL catalytic site through relief of autoinhibition. Direct disruption of RIN1 binding may therefore be a useful strategy to suppress the activity of normal and oncogenic ABL, including inhibitor-resistant mutants that confound current therapeutic strategies. Stimulation through derepression may be applicable to many other tyrosine kinases autoinhibited by coupled SH3 and SH2 domains.     

Domain movements in protein kinases

Structural studies of the catalytic subunit of the cAMP-dependent protein kinase, both by crystallographic methods and in solution, reveal two conformations. Crystal structures of several other protein kinases have also been solved in the past year. With this combined information we can begin to define mobile domains and subdomains within the conserved catalytic core.     

Crystal structures of the N-terminal kinase domain of human RSK1 bound to three different ligands: Implications for the design of RSK1 specific inhibitors

The p90 ribosomal S6 kinases (RSKs) also known as MAPKAP-Ks are serine/threonine protein kinases that are activated by ERK or PDK1 and act as downstream effectors of mitogen-activated protein kinase (MAPK). RSK1, a member of the RSK family, contains two distinct kinase domains in a single polypeptide chain, the regulatory C-terminal kinase domain (CTKD) and the catalytic N-terminal kinase domain (NTKD). Autophosphorylation of the CTKD leads to activation of the NTKD that subsequently phosphorylates downstream substrates. Here we report the crystal structures of the unactivated RSK1 NTKD bound to different ligands at 2.0 A resolution. The activation loop and helix alphaC, key regulatory elements of kinase function, are disordered. The DFG motif of the inactive RSK1 adopts an "active-like" conformation. The beta-PO(4) group in the AMP-PCP complex adopts a unique conformation that may contribute to inactivity of the enzyme. Structures of RSK1 ligand complexes offer insights into the design of novel anticancer agents and into the regulation of the catalytic activity of RSKs.     

Structure and catalytic mechanism of human protein tyrosine phosphatome

Together with protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs) serve as hallmarks in cellular signal transduction by controlling the reversible phosphorylation of their substrates. The human genome is estimated to encode more than 100 PTPs, which can be divided into eleven sub-groups according to their structural and functional characteristics. All the crystal structures of catalytic domains of sub-groups have been elucidated, enabling us to understand their precise catalytic mechanism and to compare their structures across all sub-groups. In this review, I describe the structure and mechanism of catalytic domains of PTPs in the structural context. [BMB Reports 2012; 45(12): 693-699]     

Histidine kinases: diversity of domain organization

Histidine kinases play a major role in signal transduction in prokaryotes for the cellular adaptation to environmental conditions and stresses. Recent progress in the three-dimensional structure determination of two representative members of histidine kinases, EnvZ (class I) and CheA (class II), has revealed common structural features, as well as a kinase catalytic motif topologically similar to those of the ATP-binding domains of a few ATPases. They have also disclosed that there are significant differences in domain organization between class I and II histidine kinases, possibly reflecting their distinct locations, functions and regulatory mechanisms. In spite of this diversity, both class I and II histidine kinases use similar four-helix bundle motifs to relay phosphoryl groups from ATP to regulatory domains of response regulators. The previously known so-called transmitter domain of histidine kinase is further dissected into two domains: a CA (Catalytic ATP-binding) domain and a DHp (Dimerization Histidine phosphotransfer) domain for class I, or a CA domain and an HPt (Histidine-containing Phosphotransfer) domain for class II histidine kinases. From a comparative analysis of the CA domains of EnvZ, CheA and their ATPase homologues, the core elements of the CA domain have been derived. The apparent resemblance between DHp and HPt domains is only superficial, and significant differences between them are discussed.     

CaMKII structure--an elegant design

Crystal structures of protein kinases continue to reveal new mechanisms for the regulation of catalytic activity of these enzymes. In this issue of Cell, Rosenberg et al. (2005) report the structure of the catalytic and regulatory domains of CaMKII, a protein kinase important in the cellular response to changes in intracellular calcium ion concentration. This study provides new mechanistic insights into the workings of this finely tuned molecular machine.     

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.     

Discovery of a novel mode of protein kinase inhibition characterized by the mechanism of inhibition of human mesenchymal-epithelial transition factor (c-Met) protein autophosphorylation by ARQ 197

A number of human malignancies exhibit sustained stimulation, mutation, or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced, selective, orally bioavailable, and well tolerated c-Met inhibitor, currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein, we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed, novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met, and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases, we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest.     

Subdomain switching reveals regions that harbor substrate specificity and regulatory properties of protein tyrosine kinases

Csk and Src are two protein tyrosine kinases that share a similar overall multidomain structural organization and a high degree of sequence homology but have different substrate specificities and regulatory properties. In this study, we generated chimeric kinases of Csk and Src by switching the C-terminal lobes of their catalytic domains, and we characterized their substrate specificity and regulatory properties. First, both Csk and Src phosphorylate Src as a common substrate, but on different Tyr residues. The C-terminal lobes of the kinase catalytic domain determined the site of phosphorylation on Src. Furthermore, toward several physiological substrates of Src, the substrate specificity was also determined by the C-terminal lobe of the catalytic domain regardless of the regulatory domains and the N-terminal lobe of the catalytic domain. Second, Csk and Src represent two general regulatory strategies for protein tyrosine kinases. Csk catalytic domain is inactive and is positively regulated by the regulatory domains, while Src catalytic domain is active and suppressed by its interactions with the regulatory domains. The regulatory properties of the chimeric kinases were more complicated. The regulatory domains and the N-lobe did not fully determine the response to a regulatory ligand, suggesting that the C-lobe also contributes to such responses. On the other hand, the intrinsic kinase activity of the catalytic domain correlates with the identity of the N-lobe. These results demonstrate that the chimeric strategy is useful for detailed dissection of the mechanistic basis of substrate specificity and regulation of protein tyrosine kinases.     

Binding, domain orientation, and dynamics of the Lck SH3-SH2 domain pair and comparison with other Src-family kinases

The catalytic activity of Src-family kinases is regulated by association with its SH3 and SH2 domains. Activation requires displacement of intermolecular contacts by SH3/SH2 binding ligands resulting in dissociation of the SH3 and SH2 domains from the kinase domain. To understand the contribution of the SH3-SH2 domain pair to this regulatory process, the binding of peptides derived from physiologically relevant SH2 and SH3 interaction partners was studied for Lck and its relative Fyn by NMR spectroscopy. In contrast to Fyn, activating ligands do not induce communication between SH2 and SH3 domains in Lck. This can be attributed to the particular properties of the Lck SH3-SH2 linker which is shown to be extremely flexible thus effectively decoupling the behavior of the SH3 and SH2 domains. Measurements on the SH32 tandem from Lck further revealed a relative domain orientation that is distinctly different from that found in the Lck SH32 crystal structure and in other Src kinases. These data suggest that flexibility between SH2 and SH3 domains contributes to the adaptation of Src-family kinases to specific environments and distinct functions.     

Crystal structures of c-Src reveal features of its autoinhibitory mechanism.

Src family kinases are maintained in an assembled, inactive conformation by intramolecular interactions of their SH2 and SH3 domains. Full catalytic activity requires release of these restraints as well as phosphorylation of Tyr-416 in the activation loop. In previous structures of inactive Src kinases, Tyr-416 and flanking residues are disordered. We report here four additional c-Src structures in which this segment adopts an ordered but inhibitory conformation. The ordered activation loop forms an alpha helix that stabilizes the inactive conformation of the kinase domain, blocks the peptide substrate-binding site, and prevents Tyr-416 phosphorylation. Disassembly of the regulatory domains, induced by SH2 or SH3 ligands, or by dephosphorylation of Tyr-527, could lead to exposure and phosphorylation of Tyr-416.     

Hybrid and Rogue Kinases Encoded in the Genomes of Model Eukaryotes

The highly modular nature of protein kinases generates diverse functional roles mediated by evolutionary events such as domain recombination, insertion and deletion of domains. Usually domain architecture of a kinase is related to the subfamily to which the kinase catalytic domain belongs. However outlier kinases with unusual domain architectures serve in the expansion of the functional space of the protein kinase family. For example, Src kinases are made-up of SH2 and SH3 domains in addition to the kinase catalytic domain. A kinase which lacks these two domains but retains sequence characteristics within the kinase catalytic domain is an outlier that is likely to have modes of regulation different from classical src kinases. This study defines two types of outlier kinases: hybrids and rogues depending on the nature of domain recombination. Hybrid kinases are those where the catalytic kinase domain belongs to a kinase subfamily but the domain architecture is typical of another kinase subfamily. Rogue kinases are those with kinase catalytic domain characteristic of a kinase subfamily but the domain architecture is typical of neither that subfamily nor any other kinase subfamily. This report provides a consolidated set of such hybrid and rogue kinases gleaned from six eukaryotic genomes–S.cerevisiae, D. melanogaster, C.elegans, M.musculus, T.rubripes and H.sapiens–and discusses their functions. The presence of such kinases necessitates a revisiting of the classification scheme of the protein kinase family using full length sequences apart from classical classification using solely the sequences of kinase catalytic domains. The study of these kinases provides a good insight in engineering signalling pathways for a desired output. Lastly, identification of hybrids and rogues in pathogenic protozoa such as P.falciparum sheds light on possible strategies in host-pathogen interactions.     

Bioinformatic search for plant homologs of protein kinase BUB1--the keypoint of mitotic spindle assembly

Fourteen plant homologs of animal, yeast and myxomycetes spindle assembly checkpoint protein kinases were identified bioinformatically. It was shown that the closest plant homologues of the BUB1 protein kinases are unknown proteins XP_002274770.1 (CBI21878.1) from Vitis vinifera, EEC82122.1 from Oryza sativa Indica, EEE67244.1 from O. sativa Japonica, EEF44403.1 from Ricinus communis and CAL57156.1 from Ostreococcus tauri. The reconstruction and analysis of spatial structures of the EEC82122.1, EEE67244.1 and XP_002274770.1 (CBI21878.1), catalytic domains confirmed their conformity to spindle assembly checkpoint protein kinases BUB1.     

Crystal structure of dephospho-coenzyme A kinase from Haemophilus influenzae

Dephospho-coenzyme A kinase catalyzes the final step in CoA biosynthesis, the phosphorylation of the 3'-hydroxyl group of ribose using ATP as a phosphate donor. The protein from Haemophilus influenzae was cloned and expressed, and its crystal structure was determined at 2.0-A resolution in complex with ATP. The protein molecule consists of three domains: the canonical nucleotide-binding domain with a five-stranded parallel beta-sheet, the substrate-binding alpha-helical domain, and the lid domain formed by a pair of alpha-helices. The overall topology of the protein resembles the structures of nucleotide kinases. ATP binds in the P-loop in a manner observed in other kinases. The CoA-binding site is located at the interface of all three domains. The double-pocket structure of the substrate-binding site is unusual for nucleotide kinases. Amino acid residues implicated in substrate binding and catalysis have been identified. The structure analysis suggests large domain movements during the catalytic cycle.     

Detecting protein kinase recognition modes of calmodulin by residual dipolar couplings in solution NMR

Calmodulin-regulated serine/threonine kinases (CaM kinases) play crucial roles in Ca2+-dependent signaling transduction pathways in eukaryotes. Despite having a similar overall molecular architecture of catalytic and regulatory domains, CaM kinases employ different binding modes for Ca2+/CaM recruitment which is required for their activation. Here we present a residual dipolar coupling (RDC)-based NMR approach to characterizing the molecular recognition of CaM with five different CaM kinases. Our analyses indicate that CaM kinase I and likely IV use the same CaM binding mode as myosin light chain kinase (1-14 motif), distinct from those of CaM kinase II (1-10 motif) and CaM kinase kinase (1-16- motif). This NMR approach provides an efficient experimental guide for homology modeling and structural characterization of CaM-target complexes.     

Domain characterization of rabbit skeletal muscle myosin light chain kinase.

Myosin light chain kinase can be divided into three distinct structural domains, an amino-terminal "tail," of unknown function, a central catalytic core and a carboxy-terminal calmodulin-binding regulatory region. We have used a combination of deletion mutagenesis and monoclonal antibody epitope mapping to define these domains more closely. A 2.95-kilobase cDNA has been isolated that includes the entire coding sequence of rabbit skeletal muscle myosin light chain kinase (607 amino acids). This cDNA, expressed in COS cells encoded a Ca2+/calmodulin-dependent myosin light chain kinase with a specific activity similar to that of the enzyme purified from rabbit skeletal muscle. Serial carboxy-terminal deletions of the regulatory and catalytic domains were constructed and expressed in COS cells. The truncated kinases had no detectable myosin light chain kinase activity. Monoclonal antibodies which inhibit the activity of the enzyme competitively with respect to myosin light chain were found to bind between residues 235-319 and 165-173, amino-terminal of the previously defined catalytic core. Thus, residues that are either involved in substrate binding or in close proximity to a light chain binding site may be located more amino-terminal than the previously defined catalytic core.     

Structure of the dimeric autoinhibited conformation of DAPK2, a pro-apoptotic protein kinase

The death-associated protein kinase (DAPK) family has been characterized as a group of pro-apoptotic serine/threonine kinases that share specific structural features in their catalytic kinase domain. Two of the DAPK family members, DAPK1 and DAPK2, are calmodulin-dependent protein kinases that are regulated by oligomerization, calmodulin binding, and autophosphorylation. In this study, we have determined the crystal and solution structures of murine DAPK2 in the presence of the autoinhibitory domain, with and without bound nucleotides in the active site. The crystal structure shows dimers of DAPK2 in a conformation that is not permissible for protein substrate binding. Two different conformations were seen in the active site upon the introduction of nucleotide ligands. The monomeric and dimeric forms of DAPK2 were further analyzed for solution structure, and the results indicate that the dimers of DAPK2 are indeed formed through the association of two apposed catalytic domains, as seen in the crystal structure. The structures can be further used to build a model for DAPK2 autophosphorylation and to compare with closely related kinases, of which especially DAPK1 is an actively studied drug target. Our structures also provide a model for both homodimerization and heterodimerization of the catalytic domain between members of the DAPK family. The fingerprint of the DAPK family, the basic loop, plays a central role in the dimerization of the kinase domain.