Phosphate Transfer Research Articles

Hydroxyurea Induces Bystander Cytotoxicity in Cocultures of Herpes Simplex Virus Thymidine Kinase–Expressing and Nonexpressing HeLa Cells Incubated with Ganciclovir

Suicide gene therapy with the herpes simplex virus thymidine kinase (HSV-TK) cDNA and ganciclovir can elicit cytotoxicity to transgene-expressing and nonexpressing bystander cells via transfer of ganciclovir phosphates through gap junctions. HeLa cells do not exhibit bystander cytotoxicity, although we showed recently that they transfer low levels of ganciclovir phosphates to bystander cells. Here, we attempted to induce bystander cytotoxicity using hydroxyurea, an inhibitor of ribonucleotide reductase, to decrease the endogenous dGTP pool, which should lessen competition with ganciclovir triphosphate for DNA incorporation. Addition of hydroxyurea to cocultures of HSV-TK-expressing and bystander cells synergistically increased ganciclovir-mediated cytotoxicity to both cell populations while producing primarily an additive effect in cultures of 100% HSV-TK-expressing cells. Whereas HSV-TK-expressing cells in coculture were approximately 50-fold less sensitive to ganciclovir compared with cultures of 100% HSV-TK-expressing cells, addition of hydroxyurea restored ganciclovir sensitivity. Quantification of deoxynucleoside triphosphate pools showed that hydroxyurea decreased dGTP pools without significantly affecting ganciclovir triphosphate levels. Although hydroxyurea significantly increased the ganciclovir triphosphate:dGTP value for 12 to 24 hours in HSV-TK-expressing and bystander cells from coculture (1.4- to 4.9-fold), this value was increased for <12 hours (2.5-fold) in 100% HSV-TK-expressing cells. These data suggest that the prolonged increase in the ganciclovir triphosphate:dGTP value in cells in coculture resulted in synergistic cytotoxicity. Compared with enhancement of bystander cytotoxicity through modulation of gap junction intercellular communication, this strategy is superior because it increased cytotoxicity to both HSV-TK-expressing and bystander cells in coculture. This approach may improve clinical efficacy.

1954‐2006: the long march of protein kinase CK2

The term “Protein kinase” was coined 52 years ago in a JBC paper (G. Burnett & E. P. Kennedy, JBC 211, 969–980, 1954), where an enzymatic activity catalysing the transfer of phosphate from ATP to a protein (namely casein) was described for the first time. Now-a-days protein kinases make up one of the largest families of enzymes, with more than 500 members encoded by the human genome, responsible for the phosphorylation of 30–40% of the whole eukaryotic proteome and controlling nearly all aspects of life. The aim of this presentation is to track out in the eukaryotic “kinome” the enzyme(s) firstly identified as “protein kinases” and to examine what is currently known about their biological role. In hindsight we can say that the activity described by Burnett and Kennedy was due to two distinct protein kinases, presently denoted by the acronyms CK1 and CK2, reminiscent of the old misnomers “casein kinase 1” and “casein kinase 2”. Attention will be focused on CK2 which appears to be the most pleiotropic of all protein kinases, with more than 300 protein substrates already identified and possibly responsible for the generation of a substantial proportion of the eukaryotic phosphoproteome (F. Meggio & L.A. Pinna, FASEB J. 17, 349–368, 2003). The predominance among its targets of signalling proteins and transcription factors sounds paradoxical since CK2, unlike the majority of protein kinases, is constitutively active being not turned on in response to specific stimuli. A number of coincidental arguments and experimental models support the view that CK2 is responsible for unscheduled cell survival and enhances the cancer cell phenotype of tumours whose key feature is dysregulated apoptosis. This makes CK2 a potential target for anti tumour drugs.

Hydrolysis of ATP by Aminoglycoside 3′-Phosphotransferases: AN UNEXPECTED COST TO BACTERIA FOR HARBORING AN ANTIBIOTIC RESISTANCE ENZYME

Aminoglycoside 3'-phosphotransferases (APH(3')s) are common bacterial resistance enzymes to aminoglycoside antibiotics. These enzymes transfer the gamma-phosphoryl group of ATP to the 3'-hydroxyl of the antibiotics, whereby the biological activity of the drugs is lost. Pre-steady-state and steady-state kinetics with two of these enzymes from Gram-negative bacteria, APH(3')-Ia and APH(3')-IIa, were performed. It is demonstrated that these enzymes in both ternary and binary complexes facilitate an ATP hydrolase activity (ATPase), which is competitive with the transfer of phosphate to the antibiotics. Because these enzymes are expressed constitutively in resistant bacteria, the turnover of ATP is continuous during the lifetime of the organism both in the absence and the presence of aminoglycosides. Concentrations of the enzyme in vivo were determined, and it was estimated that in a single generation of bacterial growth there exists the potential that this activity would consume as much as severalfold of the total existing ATP. Studies with bacteria harboring the aph(3')-Ia gene revealed that bacteria are able to absorb the cost of this ATP turnover, as ATP is recycled. However, the cost burden of this adventitious activity manifests a selection pressure against maintenance of the plasmids that harbor the aph(3')-Ia gene, such that approximately 50% of the plasmid is lost in 1500 bacterial generations in the absence of antibiotics. The implication is that, in the absence of selection, bacteria harboring an enzyme that catalyzes the consumption of key metabolites could experience the loss of the plasmid that encodes for the given enzyme.

Dusty Protein Kinase in Vertebrates: Tissue‐specific Expression, Gene Evolution and Unique Structural Features

Protein kinases catalyze phosphate transfer from ATP to the Ser/Thr- or Tyr-OH groups of protein substrates and have undergone a productive expansion as a family from prokaryotes to metazoans. Here we characterized Dusty, a putative dual Ser/Thr and Tyr protein kinase with potential catalytic activity and unique structural properties. Dusty is a conserved single-copy gene present in all vertebrates and is expressed in multiple tissues, particularly broadly in the central nervous system of human, rat, mouse and chicken. Confocal imaging of Dusty-GFP fusion proteins localized Dusty to the cytoplasm with a punctate distribution. Dusty proteins show increased sequence conservation from the N-terminal non-kinase part to C-terminal catalytic domain. The non-kinase part has no homology with any known conserved domains but multiple potential kinase-docking motifs and phosphorylation sites. The catalytic domain is composite in nature whose N and C-termini bear features conserved in Ser/Thr and Tyr kinase domains, respectively. Comparative analysis of Dusty kinase domains identified the conserved pattern of catalytically essential residues, and structure modeling suggests a catalytic activity. Based on these results, we conclude that Dusty is not a RIP family member, but a unique hybrid of Ser/Thr and Tyr kinases that survived natural selection and may possess novel functional roles in the brain and other tissues.

Small substrate, big surprise: fold, function and phylogeny of dihydroxyacetone kinases.

Dihydroxyacetone (Dha) kinases are a family of sequence-conserved enzymes which utilize either ATP (in animals, plants and eubacteria) or phosphoenolpyruvate (PEP, in eubacteria) as their source of high-energy phosphate. The kinases consist of two domains/subunits: DhaK, which binds Dha covalently in hemiaminal linkage to the Nepsilon2 of a histidine, and DhaL, an eight-helix barrel that contains the nucleotide-binding site. The PEP-dependent kinases comprise a third subunit, DhaM, which rephosphorylates in situ the firmly bound ADP cofactor. DhaM serves as the shuttle for the transfer of phosphate from the bacterial PEP: carbohydrate phosphotransferase system (PTS) to the Dha kinase. The DhaL and DhaK subunits of the PEP-dependent Escherichia coli kinase act as coactivator and corepressor of DhaR, a transcription factor from the AAA(+) family of enhancerbinding proteins. In Gram-positive bacteria genes for homologs of DhaK and DhaL occur in operons for putative transcription factors of the TetR and DeoR families. Proteins with the Dha kinase fold can be classified into three families according to phylogeny and function: Dha kinases, DhaK and DhaL homologs (paralogs) associated with putative transcription regulators of the TetR and DeoR families, and proteins with a circularly permuted domain order that belong to the DegV family.

An 18 kDa Acid Phosphatase from Chicken Heart Possesses Phosphotransferase Activity

A low molecular weight acid phosphatase was purified to homogeneity from chicken heart with a specific activity of 42 U/mg and a recovery of about 1%. Nearly 800 fold purification was achieved. The molecular weight was estimated to be 18 kDa by SDS-polyacrylamide gel electrophoresis. Para-nitrophenyl phosphate, phenyl phosphate and flavin mononucleotide were efficiently hydrolysed by the enzyme and found to be good substrates. Fluoride and tartrate had no inhibitory effect while phosphate, vanadate and molybdate strongly inhibited the enzyme. The acid phosphatase was stimulated in the presence of glycerol, ethylene glycol, methanol, ethanol and acetone, which reflected the phosphotransferase activity. When phosphate acceptors such as ethylene glycol concentrations were increased, the ratio of phosphate transfer to hydrolysis was also increased, demonstrating the presence of a transphosphorylation reaction where an acceptor can compete with water in the rate limiting step involving hydrolysis of a covalent phospho enzyme intermediate. Partition experiments carried out with two substrates, para-nitrophenyl phosphate and phenyl phosphate, revealed a constant product ratio of 1.7 for phosphotransfer to ethylene glycol versus hydrolysis, strongly supporting the existence of common covalent phospho enzyme intermediate. A constant ratio of K (cat)/K (m), 4.3 x 10(4), found at different ethylene glycol concentrations, also supported the idea that the rate limiting step was the hydrolysis of the phospho enzyme intermediate.

Large-Scale Transfection of Mammalian Cells for the Fast Production of Recombinant Protein

Recombinant proteins (r-proteins) are increasingly important in fundamental research and for clinical applications. As many of these r-proteins are of human or animal origin, cultivated mammalian cells are the host of choice to ensure their functional folding and proper posttranslational modifications. Large-scale transfection of human embryonic kidney 293 or Chinese hamster ovary cells is now an established technology that can be used in the production of hundreds of milligram to gram quantities of a r-protein in less than 1 mo from cloning of its cDNA. This chapter aims to provide an overview of large-scale transfection technology with a particular emphasis on calcium phosphate and polyethylenimine-mediated gene transfer.

The evolution from asparagine or threonine to cysteine in position 146 contributes to generation of a more efficient and stable form of muscle creatine kinase in higher vertebrates

Creatine kinase, a key enzyme in vertebrate excitable tissues that require large energy fluxes, catalyzes the reversible transfer of phosphate between adenosine triphosphate and creatine. Sequence alignment indicated that the 146th amino acid is cysteine in the muscle creatine kinase of higher vertebrates including Amphibia, Reptilia, Aves and Mammalia. In fishes, it is cysteine in Agnatha and Chondrichthyes, and asparagine or threonine in Osteichthyes, which is the ancestor of Amphibia, Reptilia, Aves and Mammalia. To explore the structural and functional role of this special residue, a series of site-directed mutants of rabbit muscle creatine kinase were constructed, including C146S, C146N, C146T, C146G, C146A, C146D and C146R. A detailed comparison was made between wild-type creatine kinase and the mutants in catalytic activity, physico-chemical properties and structural stability against thermal inactivation and guanidine hydrochloride denaturation. It was found that except for C146S, the mutants had relatively lower catalytic activity and structural stability than Wt-CK. Wt-CK and C146S were the most stable ones, followed by C146N and C146T, and then C146G and C146A, and C146D and C146R were the least stable mutants. These results suggested that the 146th residue plays a crucial role in maintaining the structural stability of creatine kinase, and that the evolution in this amino acid from asparagine or threonine to cysteine contributes to the generation of a more efficient and more stable form of creatine kinase in higher vertebrates.

Histidine 89 is an essential residue for Hsp70 in the phosphate transfer reaction

Autophosphorylation of Hsp70 is detected in the process of substrate refolding in the presence of adenosine triphosphate (ATP) in the reaction mixture. But to date, the role and mechanism of Hsp70 autophosphorylation have not been elucidated. In this study we determined the site of histidine phosphorylation of Hsp70 as an intermediate in the process of phosphate transfer reaction by site-directed mutagenesis. We selected two possible sites (ie, His89 and His227) of intermediate histidine phosphorylation based on our hypothesis of the transfer of gamma-phosphoryl groups and replacement by glycine and serine. Although an acid labile autophosphorylation intermediate of Hsp70 and its cytidine diphosphate-dependent dephosphorylation were detected in wild-type Hsp70, they were markedly suppressed in the H89S mutation of Hsp70, but not on the H227S mutation. The ATPase activity and ATP synthesis activity of Hsp70 were almost completely suppressed in the H89S and H89G mutations. The role of His89 in the phosphate transfer reaction of Hsp70 is discussed.

Evolution of the arginine kinase gene family

Arginine kinase (AK), catalyzing the reversible transfer of phosphate from MgATP to arginine yielding phosphoarginine and MgADP, is widely distributed throughout the invertebrates and is also present in certain protozoa. Typically, these proteins are found as monomers targeted to the cytoplasm, but true dimeric and contiguous dimeric AKs as well as mitochondrial AK activities have been observed. In the present study, we have obtained the sequences of the genes for AKs from two distantly related molluscs—the cephalopod Nautilus pompilius and the bivalve Crassostrea gigas. These new data were combined with available gene structure data (exon/intron organization) extracted from EST and genome sequencing project databases. These data, comprised of 23 sequences and gene structures from Protozoa, Cnidaria, Platyhelminthes, Mollusca, Arthropoda and Nematoda, provide great insight into the evolution and divergence of the AK family. Sequence and phylogenetic analyses clearly show that the AKs are homologous having arisen from some common ancestor. However, AK gene organization is highly divergent and variable. Molluscan AK genes typically have a highly conserved six-exon/five-intron organization, a structure that is very similar to that of the platyhelminth Schistosoma mansoni Arthropod and nematode AK genes have fewer introns, while the cnidarian and protozoan genes each display unique exon/intron organization when compared to the other AK genes. The non-conservative nature of the AK genes is in sharp contrast to the relatively high degree of conservation of intron positions seen in a homologous enzyme creatine kinase (CK). The present results also show that gene duplication and subsequent fusion events forming unusual two-domain AKs occurred independently at least four times as these contiguous dimers are present in Protozoa, Cnidaria, Platyhelminthes and Mollusca. Detailed analyses of the amino acid sequences indicate that two AKs (one each from Drosophila and Caenorhabditis) have what appear to be N-terminal mitochondrial targeting sequences, providing the first evidence for true mitochondrial AK genes. The AK gene family is ancient and the lineage has undergone considerable divergence as well as multiple duplication and fusion events.

Interactions of Phosphate Groups of ATP and Aspartyl Phosphate with the Sarcoplasmic Reticulum Ca 2+-ATPase: An FTIR Study

Phosphate binding to the sarcoplasmic reticulum Ca 2+-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([ β- 18O 2, βγ- 18O]ATP and [ γ- 18O 3]ATP). Isotopic substitution identified several bands that can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110, and 1085 cm −1 are affected by labeling of the β-phosphate, bands likely near 1154, and 1098–1089 cm −1 are affected by γ-phosphate labeling. The findings indicate that the strength of interactions of β- and γ- phosphate with the protein are similar to those in aqueous solution. Two bands, at 1175 and 1113 cm −1, were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca 2E1P. They indicate terminal and bridging P-O bond strengths that are intermediate between those of ADP-insensitive phosphoenzyme E2P and the model compound acetyl phosphate in water. The bridging bond of Ca 2E1P is weaker than for acetyl phosphate, which will facilitate phosphate transfer to ADP, but is stronger than for E2P, which will make the Ca 2E1P phosphate less susceptible to attack by water.

Beryllofluoride Binding Mimics Phosphorylation of Aspartate in Response Regulators

Beryllofluoride Binding Mimics Phosphorylation of Aspartate in Response Regulators

The capacity for the de novo biosynthesis of creatine is present in the tunicate Ciona intestinalis and is likely widespread in other protochordate and invertebrate groups

Creatine kinase (CK) catalyzes the reversible transfer of the γ-terminal phosphate of MgATP to the guanidine creatine (Cr) forming MgADP and phosphocreatine (PCr). The CK reaction plays a central role in both temporal and spatial ATP buffering in cells displaying high and variable rates of ATP turnover. There is a constant non-enzymatic conversion of Cr and PCr to creatinine that must be compensated for by biosynthesis and/or dietary uptake. In all true vertebrate craniates, there is a capacity for de novo biosynthesis of Cr as evidenced by the presence of the two enzymes involved in the biosynthetic pathway—arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Many protochordates and a broad spectrum of invertebrates, including the basal metazoan sponges, express CK and contain significant pools of Cr/PCr, particularly in primitive-type spermatozoa. However, attempts at demonstrating the enzymes of Cr biosynthesis in these organisms have failed and it has been suggested that Cr is derived from the diet and/or by direct uptake from seawater. We show in this communication that the protochordate tunicate Ciona intestinalis expresses GAMT based on the deduced amino acid sequence of a cDNA amplified by reverse transcription PCR. To validate that the transcript coded for GAMT, a full length cDNA was generated by PCR amplification and ligated into an expression vector. The resulting recombinant protein had an N-terminal amino acid sequence and relative molecular mass as predicted by the deduced amino acid sequence from the cDNA. Catalytic studies of this recombinant GAMT showed that it indeed had the capacity to methylate guanidinocetate to Cr with an apparent K m and maximal velocity comparable to GAMTs from vertebrates. Real time PCR showed that this GAMT is primarily expressed in the stomach and gonad, in close proximity to where Cr is packaged into spermatozoa, but also is expressed in two other tissue complexes. Analysis of the C. intestinalis genome and EST sequencing projects showed that the AGAT gene is present and is expressed demonstrating that this species has the complete Cr biosynthetic pathway. Perusal of other EST and genome sequencing projects revealed that true GAMTs are present in the lancelet Branchiostoma, the sea urchin Strongylocentrotus and the hydroid Hydractinia and AGAT genes are present in both Branchiostoma and Strongylocentrotus. Given our present experimental results and the emerging EST/genome sequencing data, it is clear that the capacity for de novo Cr biosynthesis is likely widespread in protochordates and invertebrates expressing CK and that the genes for GAMT/AGAT likely evolved coincident with CK.

Crystal structure of ADP/AMP complex of Escherichia coli adenylate kinase

Crystal structure of ADP/AMP complex of <i>Escherichia coli</i> adenylate kinase

Conformational dependence of a protein kinase phosphate transfer reaction

Atomic motions and energetics for a phosphate transfer reaction catalyzed by the cAMP-dependent protein kinase are calculated by plane-wave density functional theory, starting from structures of proteins crystallized in both the reactant conformation (RC) and the transition-state conformation (TC). In TC, we calculate that the reactants and products are nearly isoenergetic with a 20-kJ/mol barrier, whereas phosphate transfer is unfavorable by 120 kJ/mol in the RC, with an even higher barrier. With the protein in TC, the motions involved in reaction are small, with only P(gamma) and the catalytic proton moving >0.5 A. Examination of the structures reveals that in the RC the active site cleft is not completely closed and there is insufficient space for the phosphorylated serine residue in the product state. Together, these observations imply that the phosphate transfer reaction occurs rapidly and reversibly in a particular conformation of the protein, and that the reaction can be gated by changes of a few tenths of an angstrom in the catalytic site.

Crystal Structures of an NAD Kinase from Archaeoglobus fulgidus in Complex with ATP, NAD, or NADP

NAD kinase is a ubiquitous enzyme that catalyzes the phosphorylation of NAD to NADP using ATP or inorganic polyphosphate (poly(P)) as phosphate donor, and is regarded as the only enzyme responsible for the synthesis of NADP. We present here the crystal structures of an NAD kinase from the archaeal organism Archaeoglobus fulgidus in complex with its phosphate donor ATP at 1.7 A resolution, with its substrate NAD at 3.05 A resolution, and with the product NADP in two different crystal forms at 2.45 A and 2.0 A resolution, respectively. In the ATP bound structure, the AMP portion of the ATP molecule is found to use the same binding site as the nicotinamide ribose portion of NAD/NADP in the NAD/NADP bound structures. A magnesium ion is found to be coordinated to the phosphate tail of ATP as well as to a pyrophosphate group. The conserved GGDG loop forms hydrogen bonds with the pyrophosphate group in the ATP-bound structure and the 2' phosphate group of the NADP in the NADP-bound structures. A possible phosphate transfer mechanism is proposed on the basis of the structures presented.

Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum. Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair

The thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent pyruvate oxidase from Lactobacillus plantarum catalyses the conversion of pyruvate, inorganic phosphate, and oxygen to acetyl-phosphate, carbon dioxide, and hydrogen peroxide. Central to the catalytic sequence, two reducing equivalents are transferred from the resonant carbanion/enamine forms of alpha-hydroxyethyl-ThDP to the adjacent flavin cofactor over a distance of approximately 7 A, followed by the phosphorolysis of the thereby formed acetyl-ThDP. Pre-steady-state and steady-state kinetics using time-resolved spectroscopy and a 1H NMR-based intermediate analysis indicate that both processes are kinetically coupled. In the presence of phosphate, intercofactor electron-transfer (ET) proceeds with an apparent first-order rate constant of 78 s(-1) and is kinetically gated by the preceding formation of the tetrahedral substrate-ThDP adduct 2-lactyl-ThDP and its decarboxylation. No transient flavin radicals are detectable in the reductive half-reaction. In contrast, when phosphate is absent, ET occurs in two discrete steps with apparent rate constants of 81 and 3 s(-1) and transient formation of a flavin semiquinone/hydroxyethyl-ThDP radical pair. Temperature dependence analysis according to the Marcus theory identifies the second step, the slow radical decay to be a true ET reaction. The redox potentials of the FAD(ox)/FAD(sq) (E1 = -37 mV) and FAD(sq)/FAD(red) (E2 = -87 mV) redox couples in the absence and presence of phosphate are identical. Both the Marcus analysis and fluorescence resonance energy-transfer studies using the fluorescent N3'-pyridyl-ThDP indicate the same cofactor distance in the presence or absence of phosphate. We deduce that the exclusive 10(2)-10(3)-fold rate enhancement of the second ET step is rather due to the nucleophilic attack of phosphate on the kinetically stabilized hydroxyethyl-ThDP radical resulting in a low-potential anion radical adduct than phosphate in a docking site being part of a through-bonded ET pathway in a stepwise mechanism of ET and phosphorolysis. Thus, LpPOX would constitute the first example of a radical-based phosphorolysis mechanism in biochemistry.

Strategies for the NMR‐Based Identification and Optimization of Allosteric Protein Kinase Inhibitors

Protein kinases are important drug targets, but kinase inhibitors ought to be selective and specific in order to avoid side effects in the clinic. Kinase inhibitors that do not target the highly conserved ATP-binding site, but that target an allosteric site, are generally expected to be more selective for the target kinase and thus have a better clinical profile. Here we propose an NMR-based strategy to discover and optimize allosteric kinase inhibitors. The approach uses a spin-labeled adenine analogue to detect allosteric kinase ligands by paramagnetic relaxation enhancement. Protein kinases comprise a large family of enzymes that catalyze the transfer of the terminal phosphate from ATP (adenosine triphosphate) to protein substrates, specifically to the hydroxyl group of serine or threonine (Ser/Thr kinases) or tyrosine (Tyr kinases). Protein kinases play a crucial role in signal transduction and thereby regulate central cellular processes such as cell-cycle control, growth control, apoptosis, and transcriptional activation. Kinase activity is generally tightly regulated, but can get out of control with overactive or constitutionally activated kinases. Several pathological states or diseases, such as cancer, can be a consequence of kinase overactivation. Small molecules that can modulate kinase activity in vivo are therefore of high therapeutic interest, and those kinases with a central and specific role in a particular disease are pharmaceutically highly relevant drug targets. 3] A recent example of successful target selection and inhibitor design is the clinical success of Gleevec;, a low-molecular-weight inhibitor of the constitutionally activated tyrosine kinase, Bcr-Abl. Protein kinases generally consist of a catalytic (SH1) domain and one or several regulatory (e.g. SH2 or SH3) domains. The catalytic domains have a conserved three-dimensional fold with a bilobed structure: an N-terminal lobe consisting mainly of b sheets and a C-terminal helical lobe (Figure 1). The catalytic site is located near a hinge region that connects these two domains. Kinases can adopt multiple conformational states that are associated with the degree of catalytic activity : fully active kinases are generally phosphorylated in their activation loop, which adopts a conformation that allows for optimal binding of ATP/Mg and substrate protein, and for efficient transfer of the phosphate group of ATP. There are several regulatory mechanisms by which a kinase becomes down-regulated or “inactive”. The conformational consequence of kinase downregulation can be movement of the activation loop or other components so that the substrate cannot be efficiently bound to the kinase catalytic domain. Besides the ATP-binding site and the substrate binding site, allosteric binding sites occur in kinases, often at sites with regulatory control function. More than 500 kinases are estimated to be encoded in the human genome. All of them bind ATP/Mg , and the ATP-binding site is highly conserved both in amino acid sequence and in three-dimensional structure. Kinase inhibitors that target the ATP site in an active kinase conformation (type I inhibitors) might therefore have a higher risk of clinical liabilities due to lack of selectivity against other kinases. The ATP-binding site changes shape and becomes structurally less conserved when it is in a down-regulated conformation. Kinase inhibitors that target the ATP-binding site in a down-regulated conformation (type II inhibitors) might therefore have better selectivity and specificity, and hopefully a better clinical profile. Glivec/Gleevec is such a type II kinase inhibitor. While not being perfectly selective, it targets the ATP site of Bcr-Abl in its down-regulated conformation. The best selectivity profile might be possible for inhibitors that bind outside the ATP site, at the substrate site or an allosteric binding site. These sites are not generally conserved, and high selectivity against other kinases can hopefully be achieved. Most known kinase inhibitors are type I or II inhibitors. 14] This is probably due to the fact that most kinase inhibitor screens are performed by using biochemical functional assays with purified and activated recombinant kinase. Allosteric kinase inhibitors are not identified by these assay types if they do not inhibit kinase catalysis per se, although they might inhibit kinase activation or signal transduction. Allosteric kinase Figure 1. Principle of the experiment. Spin-labeled adenine analogue 3 is bound to the ATP-binding site. Any ligand that binds simultaneously but at a different binding site feels the paramagnetic relaxation enhancement mediated by 3. The structure of MEK2 in complex with PD334581 and ATP is shown here solely to illustrate the technique.

Kinetics of β2‐Microglobulin and Phosphate during Hemodialysis: Effects of Treatment Frequency and Duration

Current understanding of beta2-microglobulin (beta2M) and phosphate (or inorganic phosphorus) kinetics during hemodialysis is reviewed. The postdialysis:predialysis concentration ratio for beta2M is determined by dialyzer clearance for beta2M, treatment time, patient body size (specifically, extracellular fluid volume), and total ultrafiltration volume during the treatment. Evaluation of these treatment parameters can be used to calculate dialyzer clearance for beta2M; however, such calculated values are only approximations, since they neglect intradialytic generation, nonrenal (nondialyzer) clearance, and postdialysis rebound of beta2M. The detailed kinetics of beta2M during hemodialysis are best described using a two-compartment model. Theoretical predictions from such two-compartment models suggest that the product of dialyzer clearance for beta2M and weekly treatment duration, independent of treatment frequency, is the main determinant of plasma beta2M concentrations. The kinetics of phosphate removal during hemodialysis are incompletely understood. Phosphate is removed from both extracellular and intracellular compartments during hemodialysis; the plasma phosphate concentration levels off after the first 1 or 2 hours of treatment and plasma concentrations can rebound even before therapy is complete. Increases in dialyzer clearance of phosphate have been previously achieved only by increasing dialysis membrane surface area or by the use of hemodiafiltration. A four-compartment model of phosphate kinetics proposed recently by Spalding et al. suggests that the major barrier to phosphate removal is limited transfer of phosphate between the intracellular and extracellular compartments, although other complex factors also play important roles. Theoretical predictions using the model of Spalding et al. suggest that increasing either treatment frequency or treatment duration can increase phosphate removal. The kinetics of beta2M are representative of middle molecules whose removal during hemodialysis is governed predominantly by clearance at the dialyzer. In contrast, phosphate removal is limited primarily by its sequestration in the intracellular compartment (and possibly other compartments), not by its clearance at the dialyzer. The kinetics of phosphate may therefore be representative of uremic toxins whose removal is limited by sequestration into compartments or by protein binding. Enhanced removal of both of these uremic toxins using a given therapy will require treatments of increased frequency and longer duration.

Tyrosine Kinases as Targets for Cancer Therapy

Tyrosine kinases, enzymes that catalyze the transfer of phosphate from ATP to tyrosine residues in polypeptides, are ubiquitous, numerous, and of considerable clinical interest because they participate in the development of cancer and have become choice targets for therapeutic intervention. This comprehensive review discusses the molecular and clinical aspects of tyrosine kinases.