The cyclin-dependent kinases cdk2 and cdk5 act by a random, anticooperative kinetic mechanism

cdk2.cyclin E and cdk5.p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2.GST-cyclin E and cdk5.GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K(i) value of 0.6 mm. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K(i) value of 2 microm. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2.GST-cyclin E the kinetic parameters were determined to be K(m, ATP) = 3.6 +/- 1.0 microm, K(m, peptide) = 4.6 +/- 1.4 microm, and the anticooperativity factor, alpha = 130 +/- 44. For cdk5.GST-p25, the K(m, ATP) = 3.2 +/- 0.7 microm, K(m, peptide) = 1.6 +/- 0.3 microm, and alpha = 7.2 +/- 1.8.     

Substrate specificity and kinetic mechanism of human placental insulin receptor/kinase

The insulin receptor has been shown to be a protein kinase which phosphorylates its substrates on tyrosine residues. To examine the acceptor specificity of affinity-purified insulin receptor/kinase, hydroxyamino acid containing analogues of the synthetic peptide substrate Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly were prepared. Substitution of serine, threonine, or D-tyrosine for L-tyrosine completely ablated the acceptor activity of the synthetic peptides. These peptides, along with a phenylalanine-containing analogue, did serve as competitive inhibitors of the insulin receptor/kinase with apparent Ki values in the range of 2-4 mM. These data suggest that the insulin receptor/kinase is specific for tyrosine residues in its acceptor substrate and imply that serine phosphate or threonine phosphate present in receptor is due to phosphorylation by other protein kinases. The kinetics of the phosphorylation of the L-tyrosine-containing peptide were examined by using prephosphorylated insulin receptor/kinase. Prephosphorylation of the receptor was necessary to maximally activate the kinase and to linearize the initial velocity of the peptide phosphorylation reaction. The data obtained rule out a ping-pong mechanism and are consistent with a random-order rapid-equilibrium mechanism for the phosphorylation of this peptide substrate. Additional experiments demonstrated that the autophosphorylated insulin receptor was not able to transfer the preincorporated phosphate to the synthetic peptide substrate. Thus, the insulin receptor/kinase catalyzes the reaction via a mechanism that does not involve transfer of phosphate from a phosphotyrosine-containing enzyme intermediate.     

Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation

The identification of relevant protein kinase–protein substrate partners remains a serious challenge on a genome-wide scale. The design and synthesis of a photo-activatable nucleotide reagent to crosslink protein kinases with their substrates is described in which an azido group is appended to the γ-phosphoryl and purine moieties of ATP. In the absence of UV, compounds of this class were shown to act as competitive inhibitors versus ATP and non-competitive inhibitors versus peptide substrate for the protein tyrosine kinase Csk, suggesting that they can form a ternary complex with kinase and protein substrate. In vitro experiments with protein kinases indicate the bifunctional reagent can induce covalent protein–protein crosslinking that is dependent on UV irradiation. That significant kinase–substrate crosslinking occurs is suggested by the fact that this crosslinking is competitively inhibited by ATP. The crosslinked adducts can be readily cleaved by phosphodiesterase which supports the model for crosslinking and provides a simple method to deconvolute the linked protein partners.     

Microarrayed compound screening (microARCS) to identify activators and inhibitors of AMP-activated protein kinase

A novel and innovative high-throughput screening assay was developed to identify both activators and inhibitors of AMP-activated protein kinase (AMPK) using microarrayed compound screening (microARCS) technology. Test compounds were arrayed at a density of 8640 on a polystyrene sheet, and the enzyme and peptide substrate were introduced into the assay by incorporating them into an agarose gel followed by placement of the gels onto the compound sheet. Adenosine triphosphate (ATP) was delivered via a membrane, and the phosphorylated biotinylated substrate was captured onto a streptavidin affinity membrane (SAM trade mark ). For detection, the SAM trade mark was removed, washed, and imaged on a phosphor screen overnight. A library of more than 700,000 compounds was screened using this format to identify novel activators and inhibitors of AMPK.     

Selection of cyclic-peptide inhibitors targeting Aurora kinase A: problems and solutions

The critical role of Aurora kinase in cell cycle progression and its deregulation in cancer has garnered significant interest. As such, numerous Aurora targeted inhibitors have been developed to date, almost all of which target the ATP cleft at the active site. These current inhibitors display polypharmacology; that is, they target multiple kinases, and some are being actively pursued as therapeutics. Currently, there are no general approaches for targeting Aurora at sites remote from the active site, which in the long term may provide new insights regarding the inhibition of Aurora as well as other protein kinases, and provide pharmacological tools for dissecting Aurora kinase biology. Toward this long term goal, we have recently developed a bivalent selection strategy that allows for the identification of cyclic peptides that target the surface of PKA, while the active site is blocked by an ATP-competitive compound. Herein, we extend this approach to Aurora kinase (Aurora A), which required significant optimization of selection conditions to eliminate background peptides that target the streptavidin matrix upon which the kinases are immobilized. Using our optimized selection conditions, we have successfully selected several cyclic peptide ligands against Aurora A. Two of these inhibitors demonstrated IC(50) values of 10 μM and were further interrogated. The CTRPWWLC peptide was shown to display a noncompetitive mode of inhibition suggesting that alternate sites on Aurora beyond the ATP and peptide substrate binding site may be potentially targeted.     

Development of a fluorescence polarization bead-based coupled assay to target different activity/conformation states of a protein kinase

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP)-based couple d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z' of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC trade mark and validated with the protein kinase C inhibitor staurosporine. The IC(50) value generated was comparable to the value obtained by the radioactive (33)P-gamma-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.     

Peptide inhibitors of protein kinases-discovery, characterisation and use

Protein kinases are now the second largest group of drug targets, and most protein kinase inhibitors in clinical development are directed towards the ATP-binding site. However, these inhibitors must compete with high intracellular ATP concentrations and they must discriminate between the ATP-binding sites of all protein kinases as well the other proteins that also utilise ATP. It would therefore be beneficial to target sites on protein kinases other than the ATP-binding site. This review describes the discovery, characterisation and use of peptide inhibitors of protein kinases. In many cases, the development of these peptides has resulted from an understanding of the specific protein-binding partners for a particular protein kinase. In addition, novel peptide sequences have been discovered in library screening approaches and have provided new leads in the discovery and/or design of peptide inhibitors of protein kinases. These approaches are therefore providing exciting new opportunities in the development of ATP non-competitive inhibitors of protein kinases.     

Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.     

Properties of several protein kinases that copurify with rat spinal cord neurofilaments

Several protein kinases that copurify with neurofilaments (NF) were identified and each kinase was assessed for its ability to phosphorylate NF proteins. NFs were isolated using an axonal flotation procedure and the kinases were extracted from NFs with 0.8 M KCl. NF kinases were incubated with peptide substrates for selected protein kinases, [32P]ATP and protein kinase cofactors and inhibitors to characterize the kinases. Using peptide substrates, three types of kinase were identified, and a fourth was identified using NF protein as substrate. The first three kinases were the catalytic subunit of cAMP-dependent protein kinase, calcium-calmodulin dependent protein kinase II and a cofactor-independent kinase that phosphorylated prepro VIP sequence 156-170 and was inhibited by heparin. Using NF proteins as substrate, a fourth kinase was identified which was cofactor-independent and was not inhibited by heparin. Neither cofactor-independent kinase was casein kinase II. NF proteins were phosphorylated in vitro on serine and threonine, primarily by the two cofactor-independent kinases. Using [alpha-32P]8-N3ATP for affinity labeling, one kinase of 43,800 Da was identified. Thus, in addition to cAMP-dependent protein kinase and calcium-calmodulin dependent protein kinase II, two kinases have been found which are primarily responsible for NF phosphorylation in vitro and are cofactor-independent.     

Unique MAP Kinase binding sites

Map kinases are drug targets for autoimmune disease, cancer, and apoptosis-related diseases. Drug discovery efforts have developed MAP kinase inhibitors directed toward the ATP binding site and neighboring "DFG-out" site, both of which are targets for inhibitors of other protein kinases. On the other hand, MAP kinases have unique substrate and small molecule binding sites that could serve as inhibition sites. The substrate and processing enzyme D-motif binding site is present in all MAP kinases, and has many features of a good small molecule binding site. Further, the MAP kinase p38alpha has a binding site near its C-terminus discovered in crystallographic studies. Finally, the MAP kinases ERK2 and p38alpha have a second substrate binding site, the FXFP binding site that is exposed in active ERK2 and the D-motif peptide induced conformation of MAP kinases. Crystallographic evidence of these latter two binding sites is presented.     

Site-specific phosphorylation of beta-casein by proetin kinases from rabbit reticulocytes

The B variant of beta-casein was phosphorylated with [gamma-32P]ATP using four different protein kinases isolated from rabbit reticulocytes. Casein was maximally phosphorylated by the individual protein kinase activities and subjected to chymotrptic digestion. The peptides were separated by a two-dimensional peptide fingerprinting technique, and the phosphorylated peptides were identified by autoradiography, The two phosphorylated peptides obtained from the action of casein kinase I were shown to have different migration patterns from those obtained with casein kinase II. The cAMP-regulated protein kinases had the same substrate specificity with beta-casein B, and the two phosphorylated peptides obtained using these enzymes were distinct from those phosphorylated by the cAMP-independent enzymes. Thus, the different protein kinases can be identified by substrate specificity using beta-casein.     

Design, synthesis, and characterization of an ATP-peptide conjugate inhibitor of protein kinase A

An ATP-peptide conjugate was synthesized as a bisubstrate analogue inhibitor of the serine/threonine kinase protein kinase A. The compound was found to be a linear, competitive inhibitor with respect to ATP substrate, exhibiting a Ki of 3.8 microM. The compound was noncompetitive with respect to peptide substrate. The inhibitor was shown to be selective for protein kinase A versus the closely related protein kinase C as well as tyrosine kinase Csk. This analysis provides new evidence for the dissociative transition state of protein serine/threonine kinases and illustrates a simple method to convert a low affinity peptide substrate to a selective and moderately potent inhibitor for these enzymes.     

Use of a semisynthetic epitope to probe histidine kinase activity and regulation

Histidine-aspartic acid phosphotransfer pathways are central components of prokaryotic signal transduction pathways and are also found in many eukaryotes. Tools to study histidine kinases, however, are currently quite limited. In this article, we present a new tool to study histidine-aspartic acid phosphotransfer pathways. We show that many histidine kinases will accept ATPγS as a substrate to form a stable thiophosphohistidine even when they do not form stable phosphohistidines using the natural substrate ATP. An antibody that has previously been used to detect thiophosphorylated serine, threonine, and tyrosine residues is shown to recognize thiophosphohistidine and thiophosphoaspartic acid residues. Histidine kinase autothiophosphorylation is regulated by other protein sensor domains in the same way as autophosphorylation, and thiophosphate is transferred to downstream aspartic acid containing response regulators.     

Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity.

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [gamma-32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purposes, the ELISAs would be an attractive alternative to radioactive assays.     

Profiling of protein thiophosphorylation by Phos‐tag affinity electrophoresis: Evaluation of adenosine 5′‐O‐(3‐thiotriphosphate) as a phosphoryl donor in protein kinase reactions

Adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS) has been widely used as a phosphoryl donor to trace protein kinase activities. However, the question remains whether particular kinases accept ATPγS as readily as they accept natural ATP. We investigated the characteristics of several kinase reactions in the presence of ATPγS by using Phos‐tag affinity electrophoresis. The Phos‐tag gel permitted quantitative analysis of thiophosphorylated proteins produced by kinase reactions in vitro and it identified differences in the efficiencies of utilization of ATPγS and ATP in these reactions. Using the method, we evaluated the utility of ATPγS as a phosphoryl donor in studies on bacterial two‐component systems. Histidine kinases accepted ATPγS as readily as they accepted ATP in autophosphorylation reactions. However, downstream phosphotransfer reactions with ATPγS were markedly slower than the corresponding reactions with ATP. In an analysis of the sluggish thiophosphate transfer, we found that detergent‐denatured thiophosphorylated histidine kinases gradually hydrolyzed at the P–N bond, even at neutral pH, during incubation for 24 h, whereas the native form of the thiophosphorylated enzymes were much more stable. Profiling of protein thiophosphorylation by using Phos‐tag affinity electrophoresis might provide new insights into the characteristics of various types of kinase reactions with ATPγS.     

Selection of peptide inhibitors for double-stranded RNA-dependent protein kinase PKR

Protein kinase inhibitors have been developed and applied as antitumor drugs. The majority of these inhibitors are derived from ATP analogs with limited specificity towards the kinase target. Here we present our proof-of-principle study on peptide inhibitors for kinases. Two peptides were selected by phage display against double-stranded RNA-dependent protein kinase (PKR). In vitro assay revealed that these peptides exhibit an inhibitory effect on PKR-catalyzed phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). The peptides also interrupt PKR activity in cells infected by viruses, as PKR activation is one of the hallmarks of host response to viral infection. Kinetic study revealed that one of the peptides, named P1, is a competitive inhibitor for PKR, while the other, named P2, exhibits a more complicated pattern of inhibition on PKR activity. Fragment-based docking of the PKR-peptide complex suggests that P1 occupies the substrate pocket of PKR and thus inhibits the binding between PKR and eIF2α, whereas P2 sits near the substrate pocket. The computational model of PKR-peptide complex agrees with their kinetic behavior. We surmise that peptide inhibitors for kinases have higher specificity than ATP analogs, and that they provide promising leads for the optimization of kinase inhibitors.     

Structural determinants for potent, selective dual site inhibition of human pp60c-src by 4-anilinoquinazolines

The kinetic mechanisms for the inhibition of pp60(c-src) tyrosine kinase (Src TK) by 4-anilinoquinazolines, an important class of chemicals as protein kinase inhibitors, were investigated. 4-Anilinoquinazolines with a bulky group at the 4'-position of the anilino group were shown to be competitive with both ATP and peptide, whereas molecules lacking such a bulky group only displayed an inhibition pattern typical of those competitive with ATP and noncompetitive with peptide. Modifications of the substituents on the carbocyclic ring did not perturb the inhibition pattern although the affinities of these modified inhibitors for Src TK were affected. Structural modeling of Src TK with inhibitor and peptide substrate bound indicated a direct atomic conflict between the bulky 4-position group and the hydroxy of the peptide tyrosyl to which the gamma-phosphate of ATP is transferred during the kinase reaction. This atomic conflict would likely prevent simultaneous binding of both inhibitor and peptide, consistent with the observed kinetic competitiveness of the inhibitor with peptide. The dual site inhibitors appeared to have both enhanced potency and selectivity for Src TK. One such inhibitor, 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline, had a 15 nM potency against Src TK and was selective over receptor tyrosine kinases VEGFR2 by 88-fold and C-fms by 190-fold.     

N-myristoylation of a peptide substrate for Src converts it into an apparent slow-binding bisubstrate-type inhibitor.

The conversion of a peptide substrate to a potent inhibitor by chemical modification is a promising approach in the development of inhibitors for protein tyrosine kinases. N-acylation of the synthetic peptide substrate NH2-Glu-Phe-Leu-Tyr-Gly-Val-Phe-Asp-CONH2 (EFLYGVFD) resulted in synergistic inhibition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was dependent upon the peptide sequence and the length of the acyl chain. The minimum length of the fatty acyl chain to synergistically inhibit Src was a lauryl (C11H23CO) group. N-myristoylated EFLYGVFD (myr-EFLYGVFD) inhibited the phosphorylation of poly E4Y by Src with an apparent Ki of 3 microm, whereas EFLYGVFD and myristic acid inhibited with Ki values of 260 and 35 microm, respectively. The nonacylated EFLYGVFD was a substrate for Src with Km and Vmax values of 100 microm and 400 nmol/min/mg protein, respectively. However, upon myristoylation, the peptide was no longer a substrate for Src. Both the acylated and non-acylated peptides were competitive inhibitors against the substrate poly E4Y. The non-acylated free peptide showed mixed inhibition against ATP while the myristoylated peptide was competitive against ATP. Myristic acid was uncompetitive against poly E4Y and competitive against ATP. Further analysis indicated that the myristoylated peptide acted as a reversible slow-binding inhibitor with two binding sites on Src. The myristoylated 8-mer peptide was reduced in size to a myristoylated 3-mer without losing the affinity or characteristics of a bisubstrate-type inhibitor. The conversion of a classical reversible inhibitor to a reversible slow-binding multisubstrate analogue has improved the potency of inhibition by the peptide.     

ATP analog specificity of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phosphorylase kinase

The ATP analog specificities of the homogeneous cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase have been compared by the ability of 27 analogs to compete with ATP in the protein kinase reaction. Although the data suggest general similarities between the ATP sites of the two homologous cyclic-nucleotide-dependent protein kinases, specific differences especially in the adenine binding pocket are indicated. These differences in affinity suggest potentially useful ATP analog inhibitors of each kinase. For example, apparent autophosphorylation of the purified regulatory subunit of the cAMP-dependent protein kinase is blocked by nebularin triphosphate, suggesting that the phosphorylation is catalyzed by trace contamination of cGMP-dependent protein kinase. Some of the ATP analogs have also been tested using phosphorylase b kinase in order to compare this enzyme with the cyclic-nucleotide-dependent enzymes. All three protein kinases have high specificity for the purine moiety of ATP, and lower specificity for the ribose or triphosphate. The similarity between the ATP site of phosphorylase b kinase to that of the cyclic-nucleotide-dependent protein kinases suggests that it is related to them. The ATP analog specificities of enzymes examined in this study are different from those reported for several unrelated ATP-utilizing enzymes.