A crucial role of Cys218 in configuring an unprecedented auto-inhibition form of MAP2K7.

Mitogen-activated protein kinase kinase 7 (MAP2K7) is an indispensable kinase of the c-Jun N-terminal kinase signal cascade and is rigorously regulated via phosphorylation. To investigate the regulatory mechanism of the inactive non-phosphorylated state of MAP2K7, the crystal structures of the wild-type and C218S mutant were solved. The wild-type apo-structure revealed an unprecedented auto-inhibition form that occluded the ATP site.

5Z-7-Oxozeaenol covalently binds to MAP2K7 at Cys218 in an unprecedented manner.

5Z-7-Oxozeaenol (5Z7O) is a covalent bonding inhibitor against the several protein kinases (e.g., ERK2 and TAK1) that possess a free cysteine at the gatekeeper-2 position.

Quick evaluation of kinase inhibitors by surface plasmon resonance using single-site specifically biotinylated kinases.

In evaluating kinase inhibitors, kinetic parameters such as association/dissociation rate constants are valuable information, as are equilibrium parameters KD and IC50 values. Surface plasmon resonance (SPR) is a powerful technique to investigate these parameters. However, results are often complicated because of impaired conformations by inappropriate conditions required for protein immobilization and/or heterogeneity of the orientation of immobilization.

Activity-based kinase profiling of approved tyrosine kinase inhibitors.

The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP.

Seven cysteine-deficient mutants depict the interplay between thermal and chemical stabilities of individual cysteine residues in mitogen-activated protein kinase c-Jun N-terminal kinase 1.

Intracellular proteins can have free cysteines that may contribute to their structure, function, and stability; however, free cysteines can lead to chemical instabilities in solution because of oxidation-driven aggregation. The MAP kinase, c-Jun N-terminal kinase 1 (JNK1), possesses seven free cysteines and is an important drug target for autoimmune diseases, cancers, and apoptosis-related diseases. To characterize the role of cysteine residues in the structure, function, and stability of JNK1, we prepared and evaluated wild-type JNK1 and seven cysteine-deficient JNK1 proteins.

Crystal and solution structures disclose a putative transient state of mitogen-activated protein kinase kinase 4.

Mitogen-activated protein kinase kinase 4 (MAP2K4) plays a crucial role in the stress-activated signal cascade and is enzymatically regulated by ligand or substrate binding, and/or post-translational modification. Crystal structures combined with small-angle X-ray scattering experiments revealed that the apo form of non-phosphorylated MAP2K4 (npMAP2K4) exists in a transient state which has a longer conformation compared with the typical kinase folding. Upon ATP-binding, the transient conformation adopted the configuration of typical kinase folding.

Crystal structure of non-phosphorylated MAP2K6 in a putative auto-inhibition state.

Mitogen-activated protein kinase kinase 6 (MAP2K6) plays a crucial role in the p38 MAP kinase signal cascade that regulates various stress-induced responses and is associated with pathological conditions. The crystal structure of human non-phosphorylated MAP2K6 (npMAP2K6) complexed with an ATP analogue was determined at 2.6 Å resolution and represents an auto-inhibition state of MAP2K6.

Characterization of kinase inhibitors using different phosphorylation states of colony stimulating factor-1 receptor tyrosine kinase.

It is known that some kinase inhibitors are sensitive to the phosphorylation state of the kinase, and therefore those compounds can discriminate between a phosphorylated and unphosphorylated protein. In this study, we prepared two colony stimulating factor-1 receptor (CSF-1R) tyrosine kinase proteins: one highly phosphorylated by autophosphorylation and the other dephosphorylated by phosphatase treatment. These kinases were subjected to an activity-based assay to investigate the effect of their phosphorylation state on the potency of several kinase inhibitors.

Crystal structures of MKK4 kinase domain reveal that substrate peptide binds to an allosteric site and induces an auto-inhibition state.

MKK4 activates both JNKs and p38s. We determined the crystal structures of human non-phosphorylated MKK4 kinase domain (npMKK4) complexed with AMP-PNP (npMKK4/AMP) and a ternary complex of npMKK4, AMP-PNP and p38α peptide (npMKK4/AMP/p38). These crystal structures revealed that the p38α peptide-bound npMKK4 at the allosteric site rather than at the putative substrate binding site and induced an auto-inhibition state.

Structural basis for the inhibitor recognition of human Lyn kinase domain.

Human Lyn tyrosine kinase is expressed in hematopoietic tissues and plays crucial roles in the signal transduction of hematopoietic immune system. Its excess activity is involved in several tumors. The crystal structure has revealed that the potent inhibitor staurosporine binds to human Lyn kinase domain at the ATP-binding site.

Molecular cloning, functional characterization, and enzyme-linked immunosorbent assay of cynomolgus monkey Fas ligand.

Fas ligand (FasL) cDNAs were cloned and sequenced from cynomolgus, rhesus, and pig-tailed monkeys. The 840-bp cDNAs were identical among these three species of monkeys except for one nucleotide. The deduced 280 amino acids were completely identical and displayed 97% homology with human FasL (hFasL).

Exacerbated and prolonged allergic and non-allergic inflammatory cutaneous reaction in mice with targeted interleukin-18 expression in the skin.

Interleukin 18 induces both T helper 1 and T helper 2 cytokines, proinflammatory cytokines, chemokines, and IgE and IgG1 production. A role of interleukin 18 in inflammatory cutaneous reactions is still unclear, however. Here we generated keratin 5/interleukin 18 transgenic mice overexpressing mature murine interleukin 18 in the skin using a human keratin 5 promoter.

New type of metalloproteinase inhibitor: design and synthesis of new phosphonamide-based hydroxamic acids.

A series of phosphonamide-based hydroxamate derivatives were synthesized, and the inhibitory activities were evaluated against various metalloproteinases in order to clarify its selectivity profile. Among the four diastereomeric isomers resulting from the chirality at the C-3 and P atoms, the compound with a (R,R)-configuration both at the C-3 position and the phosphorus atom was found to be potently active, while the other diastereomeric isomers were almost inactive. A number of (R,R)-compounds synthesized here exhibited broad spectrum activities with nanomolar K(i) values against MMP-1, -3, -9, and TACE and also showed nanomolar IC(50) values against HB-EGF shedding in a cell-based inhibition assay.