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.

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.

Selection of inhibitory peptides for Aurora-A kinase from a phage-displayed library of helix-loop-helix peptides.

Conformationally constrained peptide libraries have been made by grafting randomized amino acid sequences onto a rigid scaffold derived from natural proteins. Here, as a library scaffold, we propose a de novo designed helix-loop-helix motif. We constructed a peptide library of the loop region and screened against Aurora-A, which is a member of the Aurora family of serine/threonine protein kinases, to successfully isolate the inhibitory peptides.

A silent mutation made possible efficient production of active human Frk tyrosine kinase in Escherichia coli.

Fyn-related kinase (Frk) was first identified using human breast cancer cells. It shares 51% identity with c-Src. Like all members of the Src family, Frk is thought to cause several cancers via dysregulations in signal transduction from cell-surface receptors.

Crystal structure of human mono-phosphorylated ERK1 at Tyr204.

Extracellular signal-regulated kinase (ERK) is a member of the MAP kinase family, and can regulate several cellular responses. The isoforms ERK1 and ERK2 have markedly similar amino acid sequences, but exhibit distinctive physiological functions. As well as ERK2, ERK1 was auto- and mono-phosphorylated at Tyr204 in the activation loop during Escherichia coli production, resulting in basal level activity, approximately 500-fold less compared with fully-active ERK1 dual-phosphorylated at Thr202 and Tyr204.

Use of RNase P for efficient preparation of yeast tRNATyr transcript and its mutants.

Because T7 RNA polymerase has a strong preference for particular sequences to initiate transcription, some RNAs having pyrimidine-rich sequences at their 5'-end (yeast tRNA(Tyr), for example) are hardly transcribed by this enzyme. To circumvent this inconvenience, we have developed an efficient method for in vitro preparation of such tRNAs. The RNA of interest is first transcribed as a precursor form that has purine-rich extra sequences at its 5'-end, then processed with RNase P to generate the objective tRNAs.

MYRbase: analysis of genome-wide glycine myristoylation enlarges the functional spectrum of eukaryotic myristoylated proteins.

We evaluated the evolutionary conservation of glycine myristoylation within eukaryotic sequences. Our large-scale cross-genome analyses, available as MYRbase, show that the functional spectrum of myristoylated proteins is currently largely underestimated. We give experimental evidence for in vitro myristoylation of selected predictions.

The beta subunit of Aquifex aeolicus leucyl-tRNA synthetase is responsible for cognate tRNA recognition.

The active form of the leucyl-tRNA synthetase from an extreme thermophile Aquifex aeolicus has a heterodimeric (alpha/beta type) quaternary structure that is unique among class I aminoacyl-tRNA synthetases. In an attempt to clarify the individual roles of each subunit in the function of leucyl-tRNA synthetase, several elementary activities were separately measured using each of the subunits alone or the reconstructed alpha/beta complex. It was found that the beta subunit alone is capable of recognizing its cognate tRNA, while the leucyl-adenylate formation and the overall leucyl-tRNA formation are detected only when both of the subunit proteins coexisted.

Leucyl-tRNA synthetase from the extreme thermophile Aquifex aeolicus has a heterodimeric quaternary structure.

Class I aminoacyl-tRNA synthetases have been thought to be single polypeptide enzymes. However, the complete genome sequence of a hyper thermophile Aquifex aeolicus suggests that the gene for leucyl-tRNA synthetases (LeuRS) is probably split into two pieces (leuS and leuS'). In this research, each gene was separately cloned and overexpressed in Escherichia coli and the protein products were examined for LeuRS activity.