Regulation of the wild-type and Y1235D mutant Met kinase activation.

Met receptor tyrosine kinase plays a crucial role in the regulation of a large number of cellular processes and, when deregulated by overexpression or mutations, leads to tumor growth and invasion. The Y1235D mutation identified in metastases was shown to induce constitutive activation and a motile-invasive phenotype on transduced carcinoma cells. Wild-type Met activation requires phosphorylation of both Y1234 and Y1235 in the activation loop.

Searching for biomarkers of Aurora-A kinase activity: identification of in vitro substrates through a modified KESTREL approach.

Aurora-A, -B, and -C are members of a small family of mitotic serine/threonine kinases that regulate centrosome maturation, chromosome segregation, and cytokinesis. They are often overexpressed in different human tumor types and have been identified as attractive targets for anticancer drug development. As specific inhibitors of the Aurora kinases are entering phase I clinical trials, there is a high need for appropriate Aurora-A biomarkers to follow mechanism of action or response.

Kinase selectivity profiling by inhibitor affinity chromatography.

As new drugs rapidly advance into clinical trials, comprehensive identification of their intracellular targets becomes fundamental for the full understanding of the molecular basis of their efficacy and toxicity. This is particularly important when the targets belong to a large family and the inhibitors recognize a conserved site among different members of the class. A typical example is the kinase family, where efforts are aimed at the development of inhibitors of distinct kinases for therapeutic applications in oncology, inflammation and other disease areas.

The oxygen evolving enhancer protein 1 (OEE) of photosystem II in green algae exhibits thioredoxin activity.

A thioredoxin-like chloroplast protein of the fructosebisphosphatase-stimulating f-type, but with an unusually high molecular mass of 28 kDa has previously been identified and purified to homogeneity in a fractionation scheme for resolution of the acid- and heat-stable, regular-size (12kDa) thioredoxins of the unicellular green algae, Scenedesmus obliquus. An apparently analogous protein of 26 kDa was described in a cyanobacterium, Anabaena sp., but no such large thioredoxin species f exists in the thioredoxin profiles of higher plants.

Inhibitor affinity chromatography: profiling the specific reactivity of the proteome with immobilized molecules.

An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor-protein interactions as a complementary approach to two-dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin-dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme.

A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: chemical modification, enrichment via solid-phase reversible binding, and analysis by mass spectrometry.

beta-Elimination of the phosphate group on phosphoserine and phosphothreonine residues and addition of an alkyldithiol is a useful tool for analysis of the phosphorylation states of proteins and peptides. We have explored the influence of several conditions on the efficiency of this PO(4)(3-) elimination reaction upon addition of propanedithiol. In addition to the described influence of different bases, the solvent composition was also found to have a major effect on the yield of the reaction.

Involvement of conserved asparagine and arginine residues from the N-terminal region in the catalytic mechanism of rat liver and Trypanosoma cruzi tyrosine aminotransferases.

Rat liver and Trypanosoma cruzi tyrosine aminotransferases (TATs) share over 40% sequence identity, but differ in their substrate specificities. To explore the molecular features related to these differences, comparative mutagenesis studies were conducted on full length T. cruzi TAT and N-terminally truncated rat TAT recombinant enzymes.