Crystal structures of the catalytic domain of human protein kinase associated with apoptosis and tumor suppression

We have determined X-ray crystal structures with up to 1.5 A resolution of the catalytic domain of death-associated protein kinase (DAPK), the first described member of a novel family of pro-apoptotic and tumor-suppressive serine/threonine kinases. The geometry of the active site was studied in the apo form, in a complex with nonhydrolyzable AMPPnP and in a ternary complex consisting of kinase, AMPPnP and either Mg2+ or Mn2+. The structures revealed a previously undescribed water-mediated stabilization of the interaction between the lysine that is conserved in protein kinases and the beta- and gamma-phosphates of ATP, as well as conformational changes at the active site upon ion binding. Comparison between these structures and nucleotide triphosphate complexes of several other kinases disclosed a number of unique features of the DAPK catalytic domain, among which is a highly ordered basic loop in the N-terminal domain that may participate in enzyme regulation.     

Death-associated protein kinase 2: Regulator of apoptosis,autophagy and inflammation

Death-associated protein kinase 2 (DAPK2/DRP-1) belongs to a family of five related serine/threonine kinases that mediate a range of cellular processes, including membrane blebbing, apoptosis, and autophagy, and possess tumour suppressive functions. The three most conserved family members DAPK1/DAPK, DAPK2 and DAPK3/ZIPK share a high degree of homology in their catalytic domain, but differ significantly in their extra-catalytic structures and tissue-expression profiles. Hence, each orthologue binds to various unique interaction partners, localizes to different subcellular regions and controls some dissimilar cellular functions. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms that activate DAPK2 and that execute DAPK2-mediated apoptosis, autophagy and inflammation. In this “molecules in focus” review on DAPK2, the structure, modes of regulation and various cellular functions of DAPK2 will be summarized and discussed.     

Diversity of Bisubstrate Binding Modes of Adenosine Analogue-Oligoarginine Conjugates in Protein Kinase A and Implications for Protein Substrate Interactions

Crystal structures of the catalytic subunit α of cAMP-dependent protein kinase (PKAc) with three adenosine analogue-oligoarginine conjugates (ARCs) are presented. The rationally designed ARCs include moieties that, in combination, target both the ATP- and the peptide-substrate-binding sites of PKAc, thereby taking advantage of high-affinity binding interactions offered by the ATP site while utilizing an additional mechanism for target specificity via binding to the peptide substrate site. The crystal structuresdemonstrate that, in accord with the previously reported bisubstrate character of ARCs, the inhibitors occupy both binding sites of PKAc. Further, they show new binding modes that may also apply to natural protein substrates of PKAc, which have not been revealed by previous crystallographic studies. The crystal structures described here contribute to the understanding of the substrate-binding patterns of PKAc and should also facilitate the design of inhibitors targeting PKAc and related protein kinases.     

p38α MAP kinase dimers with swapped activation segments and a novel catalytic loop conformation

Many protein kinase functions, including autophosphorylation in trans, require dimerization, possibly by activation segment exchange. Such dimers have been reported for a few autophosphorylating protein kinases, but not for mitogen-activated protein kinases (MAPKs). Activation of MAPKs proceeds not only via the well-characterized action of dual T/Y specificity MAPK kinases, phosphorylating both residues of the MAPK TxY activation loop motif, but also via a noncanonical activation pathway triggered by phosphorylation at Tyr323 and homodimerization. Here, we report the 2. 7-Å-resolution structure of p38α MAPK from Salmo salar in a novel domain-swapped homodimeric form. The tyrosines of the conserved sequence YxAPE anchor the swapped activation segments in a configuration suitable for autophosphorylation in trans and provide a model for the noncanonical pathway. In the dimer, a structural unit containing Tyr323 is formed at a dimerization contact region that stabilizes the HRD catalytic loop in a unique inactive geometry. This feature is consistent with the requirement of Tyr323 phosphorylation for the initiation of the noncanonical pathway. Despite the interacting surface of more than 2600 Å², the dimer is not obligate, as gel filtration shows the dimerization to occur only at relatively high concentrations. The transition from monomer to dimer involves a relatively simple hinged displacement of helix EF and adjacent residues. Thus, dimer formation is likely to be transient, compatible with functional requirements for autophosphorylation, allowing further modulation, for example, by scaffolding mechanisms.     

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.     

Evolved to be active: sulfate ions define substrate recognition sites of CK2alpha and emphasise its exceptional role within the CMGC family of eukaryotic protein kinases

CK2alpha is the catalytic subunit of protein kinase CK2 and a member of the CMGC family of eukaryotic protein kinases like the cyclin-dependent kinases, the MAP kinases and glycogen-synthase kinase 3. We present here a 1.6 A resolution crystal structure of a fully active C-terminal deletion mutant of human CK2alpha liganded by two sulfate ions, and we compare this structure systematically with representative structures of related CMGC kinases. The two sulfate anions occupy binding pockets at the activation segment and provide the structural basis of the acidic consensus sequence S/T-D/E-X-D/E that governs substrate recognition by CK2. The anion binding sites are conserved among those CMGC kinases. In most cases they are neutralized by phosphorylation of a neighbouring threonine or tyrosine side-chain, which triggers conformational changes for regulatory purposes. CK2alpha, however, lacks both phosphorylation sites at the activation segment and structural plasticity. Here the anion binding sites are functionally changed from regulation to substrate recognition. These findings underline the exceptional role of CK2alpha as a constitutively active enzyme within a family of strictly controlled protein kinases.     

Site-directed mutagenesis of the glycine-rich loop of death associated protein kinase (DAPK) identifies it as a key structure for catalytic activity

Death associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase that is a therapeutic target for central nervous system (CNS) disorders. We report here the results of studies that test the hypothesis of McNamara et al. (2009) that conformational selection in DAPK's glycine-rich region is key for catalytic activity. The hypothesis was tested by site-directed mutagenesis of glutamine-23 (Q23) in the middle of this loop. The glycine-rich loop exhibits localized differences in structure among DAPK conformations that correlate with different stages of the catalytic cycle. Changing the Q23 to a Valine (V23), found at the corresponding position in another CaM regulated protein kinase, results in a reduced catalytic efficiency. High resolution X-ray crystal structures of various conformations of the Q23V mutant DAPK and their superimposition with the corresponding conformations from wild type catalytic domain reveal localized changes in the glycine-rich region. The effect of the mutation on DAPK catalytic activity and the finding of only localized changes in the DAPK structure provide experimental evidence implicating conformational selection in this domain with activity. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.     

Crystal structure of the kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP PNP

Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine protein kinase of the AGC family which participates in the control of epithelial ion transport and is implicated in proliferation and apoptosis. We report here the 1.9 A crystal structure of the catalytic domain of inactive human SGK1 in complex with AMP-PNP. SGK1 exists as a dimer formed by two intermolecular disulfide bonds between Cys258 in the activation loop and Cys193. Although most of the SGK1 structure closely resembles the common protein kinase fold, the structure around the active site is unique when compared to most protein kinases. The alphaC helix is not present in this inactive form of SGK1 crystal structure; instead, the segment corresponding to the C helix forms a beta-strand that is stabilized by the N-terminal segment of the activation loop through a short antiparallel beta-sheet. Since the differences from other kinases occur around the ATP binding site, this structure can provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of SGK1 kinase.     

Mechanism and functional significance of Itk autophosphorylation

Tec family non-receptor tyrosine kinases (Itk, Btk, Tec, Rlk and Bmx) are characterized by the presence of an autophosphorylation site within the non-catalytic Src homology 3 (SH3) domain. The full-length Itk mutant containing phenylalanine in place of the autophosphorylated tyrosine has been studied in Itk-deficient primary T cells. These studies revealed that the non-phosphorylated enzyme restores Itk mediated signaling only partially. In spite of these insights, the precise role of the Tec kinase autophosphorylation site is unclear and the mechanism of the autophosphorylation reaction within the Tec kinases is not known. Here, we show both in vitro and in vivo that Itk autophosphorylation on Y180 within the SH3 domain occurs exclusively via an intramolecular, in cis mechanism. Using an in vitro kinase assay, we show that mutation of the Itk autophosphorylation site Y180 to Phe decreases kinase activity of the full-length enzyme by increasing Km for a peptide substrate. Moreover, mutation of Y180 to Glu, a residue chosen to mimic the phosphorylated tyrosine, alters the ligand-binding capability of the Itk SH3 domain in a ligand-dependent fashion. NMR chemical shift mapping gives residue-specific structural insight into the effect of the Y180E mutation on ligand binding. These data provide a molecular level context with which to interpret in vivo functional data and allow development of a structural model for Itk autophosphorylation.     

Autophosphorylation of gatekeeper tyrosine by symbiosis receptor kinase

Plant receptor-like kinases (RLKs) share their evolutionary origin with animal interleukin-1 receptor-associated kinase (IRAK)/Pelle family of soluble kinases and are distinguished by having tyrosine as ‘gatekeeper’. This position is adjacent to the hinge region and is hidden in a hydrophobic pocket of the catalytic cleft of protein kinases and is therefore least probable to be a target for any modification. This communication illustrates the accessibility of the gatekeeper site (Y670) towards both autophosphorylation and dephosphorylation in the recombinant cytoplasmic domain of symbiosis receptor kinase from Arachis hypogaea (AhSYMRK). Autophosphorylation on gatekeeper tyrosine was detected prior to extraction but never under in vitro conditions. We hypothesize gatekeeper phosphorylation to be associated with synthesis/maturation of AhSYMRK and this phenomenon may be prevalent among RLKs.     

Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site

The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.     

Comparative surface geometry of the protein kinase family

Identifying conserved pockets on the surfaces of a family of proteins can provide insight into conserved geometric features and sites of protein–protein interaction. Here we describe mapping and comparison of the surfaces of aligned crystallographic structures, using the protein kinase family as a model. Pockets are rapidly computed using two computer programs, FADE and Crevasse. FADE uses gradients of atomic density to locate grooves and pockets on the molecular surface. Crevasse, a new piece of software, splits the FADE output into distinct pockets. The computation was run on 10 kinase catalytic cores aligned on the αF‐helix, and the resulting pockets spatially clustered. The active site cleft appears as a large, contiguous site that can be subdivided into nucleotide and substrate docking sites. Substrate specificity determinants in the active site cleft between serine/threonine and tyrosine kinases are visible and distinct. The active site clefts cluster tightly, showing a conserved spatial relationship between the active site and αF‐helix in the C‐lobe. When the αC‐helix is examined, there are multiple mechanisms for anchoring the helix using spatially conserved docking sites. A novel site at the top of the N‐lobe is present in all the kinases, and there is a large conserved pocket over the hinge and the αC‐β4 loop. Other pockets on the kinase core are strongly conserved but have not yet been mapped to a protein–protein interaction. Sites identified by this algorithm have revealed structural and spatially conserved features of the kinase family and potential conserved intermolecular and intramolecular binding sites.     

Mammalian NDR protein kinases: from regulation to a role in centrosome duplication

The NDR (nuclear Dbf2-related) family of kinases is highly conserved from yeast to human, and has been classified as a subgroup of the AGC group of protein kinases based on the sequence of the catalytic domain. Like all other members of the AGC class of protein kinases, NDR kinases require the phosphorylation of conserved Ser/Thr residues for activation. Importantly, NDR family members have two unique stretches of primary sequence: an N-terminal regulatory (NTR) domain and an insert of several residues between subdomains VII and VIII of the kinase domain. The kinase domain insert functions as an auto-inhibitory sequence (AIS), while binding of the co-activator MOB (Mps-one binder) proteins to the NTR domain releases NDR kinases from inhibition of autophosphorylation. However, despite such advances in our understanding of the molecular activation mechanism(s) and physiological functions of NDR kinases in yeast and invertebrates, most biological NDR substrates still remain to be identified. Nevertheless, by showing that the centrosomal subpopulation of human NDR1/2 is required for proper centrosome duplication, the first biological role of human NDR1/2 kinases has been defined recently. How far NDR-driven centrosome overduplication could actually contribute to cellular transformation will also be discussed.     

Molecular cloning and characterization of the human protein kinase D2. A novel member of the protein kinase D family of serine threonine kinases

We have isolated the full-length cDNA of a novel human serine threonine protein kinase gene. The deduced protein sequence contains two cysteine-rich motifs at the N terminus, a pleckstrin homology domain, and a catalytic domain containing all the characteristic sequence motifs of serine protein kinases. It exhibits the strongest homology to the serine threonine protein kinases PKD/PKCmicro and PKCnu, particularly in the duplex zinc finger-like cysteine-rich motif, in the pleckstrin homology domain and in the protein kinase domain. In contrast, it shows only a low degree of sequence similarity to other members of the PKC family. Therefore, the new protein has been termed protein kinase D2 (PKD2). The mRNA of PKD2 is widely expressed in human and murine tissues. It encodes a protein with a molecular mass of 105 kDa in SDS-polyacrylamide gel electrophoresis, which is expressed in various human cell lines, including HL60 cells, which do not express PKCmicro. In vivo phorbol ester binding studies demonstrated a concentration-dependent binding of [(3)H]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulated the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorbol esters efficiently phosphorylated the exogenous substrate histone H1. In addition, we could identify the C-terminal Ser(876) residue as an in vivo phosphorylation site within PKD2. Phosphorylation of Ser(876) of PKD2 correlated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfected with the CCK(B)/gastrin receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-stimulated protein kinase.     

Higher-order substrate recognition of eIF2alpha by the RNA-dependent protein kinase PKR

In response to binding viral double-stranded RNA byproducts within a cell, the RNA-dependent protein kinase PKR phosphorylates the alpha subunit of the translation initiation factor eIF2 on a regulatory site, Ser51. This triggers the general shutdown of protein synthesis and inhibition of viral propagation. To understand the basis for substrate recognition by and the regulation of PKR, we determined X-ray crystal structures of the catalytic domain of PKR in complex with eIF2alpha. The structures reveal that eIF2alpha binds to the C-terminal catalytic lobe while catalytic-domain dimerization is mediated by the N-terminal lobe. In addition to inducing a local unfolding of the Ser51 acceptor site in eIF2alpha, its mode of binding to PKR affords the Ser51 site full access to the catalytic cleft of PKR. The generality and implications of the structural mechanisms uncovered for PKR to the larger family of four human eIF2alpha protein kinases are discussed.     

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.     

The extended conformation of the 2.9-Å crystal structure of the three-PASTA domain of a Ser/Thr kinase from the human pathogen Staphylococcus aureus

PASTA (penicillin-binding protein and serine/threonine kinase associated) modules are found in penicillin-binding proteins and bacterial serine/threonine kinases mainly from Gram-positive Firmicutes and Actinobacteria. They may act as extracellular sensors by binding peptidoglycan fragments. We report here the first crystal structure of a multiple-PASTA domain from Ser/Thr kinase, that of the protein serine/threonine kinase 1 (Stk1) from the Firmicute Staphylococcus aureus. The extended conformation of the three PASTA subunits differs strongly from the compact conformation observed in the two-PASTA domain of penicillin-binding protein PBP2x, whereas linear conformations were also reported for two-subunit fragments of the four-PASTA domain of the Actinobacteria Mycobacterium tuberculosis studied by liquid NMR. Thus, a stretched organization appears to be the signature of modular PASTA domains in Ser/Thr kinases. Signal transduction to the kinase domain is supposed to occur via dimerization and ligand binding. A conserved X-shaped crystallographic dimer stabilized by intermolecular interactions between the second PASTA subunits of each monomer is observed in the two crystal forms of Stk1 that we managed to crystallize. Extracellular PASTA domains are composed of at least two subunits, and this molecular assembly is a plausible candidate for the biological dimer. We have also performed docking experiments, which predict that the hinge regions of the PASTA domain can accommodate peptidoglycan. Finally, a three-dimensional homology molecular model of full-length Stk1 was generated, suggesting an interaction between the kinase domain and the cytoplasmic face of the plasma membrane via a eukaryotic-like juxtamembrane domain. A comprehensive activation mechanism for bacterial Ser/Thr kinases is proposed with the support of these structural data.