Discovery and characterization of a cell-permeable, small-molecule c-Abl kinase activator that binds to the myristoyl binding site.

c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site.

Assay development and high-throughput screening of small molecular c-Abl kinase activators.

A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur.

Using 2H2O to study the influence of feeding on protein synthesis: effect of isotope equilibration in vivo vs. in cell culture.

We previously reported that 2H2O can be used to measure rates of protein synthesis during prolonged steady-state conditions (Previs SF, Fatica R, Chandramouli V, Alexander JC, Brunengraber H, and Landau BR. Am J Physiol Endocrinol Metab 286: E665-E672, 2004). The underlying premise of our method is that following the administration of 2H2O, 2H atoms in body water rapidly equilibrate with free alanine before it is incorporated into newly synthesized proteins.

ENU induced mutations causing congenital cardiovascular anomalies.

We used non-invasive high frequency ultrasound to screen N-ethyl-N-nitrosourea mutagenized mouse fetuses for congenital cardiovascular anomalies. We ultrasound scanned 7546 mouse fetuses from 262 mutagenized families, and identified 124 families with cardiovascular defects. Represented were most of the major congenital cardiovascular anomalies seen clinically.

Glucagon represses signaling through the mammalian target of rapamycin in rat liver by activating AMP-activated protein kinase.

The opposing actions of glucagon and insulin on glucose metabolism within the liver are essential mechanisms for maintaining plasma glucose concentrations within narrow limits. Less well studied are the counterregulatory actions of glucagon on protein metabolism. In the present study, the effect of glucagon on amino acid-induced signaling through the mammalian target of rapamycin (mTOR), an important controller of the mRNA binding step in translation initiation, was examined using the perfused rat liver as an experimental model.