N-Myristoylated c-Abl Tyrosine Kinase Localizes to the Endoplasmic Reticulum upon Binding to an Allosteric Inhibitor

Yongmun Choi,Markus A Seeliger,Shoghag B Panjarian,Hakjoong Kim,Xianming Deng,Taebo Sim,Brian Couch,Anthony J Koleske,Thomas E Smithgall,Nathanael S Gray

doi:10.1074/jbc.m109.026633

Abstract

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH(2)-terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH(2)-terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.

Highlights

The c-Abl and Arg (Abl-related gene) proteins comprise the Abl family of non-receptor tyrosine kinases
Abl family members localize to multiple subcellular compartments; whereas Arg is mostly found in the cytoplasm, c-Abl shuttles between the nucleus and the cytoplasm, where it localizes to the cytosol, endoplasmic reticulum, and mitochondria [2]
Our results demonstrate that GNF-2 inhibits the Abl family of tyrosine kinases in tissue culture cells and in vitro by binding to a hydrophobic pocket located in the C-lobe of the kinase domain, which is the binding site for an N-terminal myristate group

Summary

In Vitro Tyrosine Kinase Assay

In vitro kinase assays were performed according to three different formats as follows. Aliquots of the diluted proteins were preincubated with either DMSO or compounds for 30 min at room temperature and added to K-LISA PTK EAY reaction plates (EMD Chemicals). The kinase reaction was initiated by adding 0.1 mM ATP and was allowed to proceed for 30 min at room temperature. Labeling with 32P-Labeled Inorganic Phosphate—Recombinant Abl and Arg proteins (100 nM for each protein) were diluted in kinase buffer (20 mM Tris-Cl (pH 7.4), 50 mM KCl, and 1 mM DTT) and were preincubated with either DMSO or compounds for 30 min at room temperature. Diluted proteins were mixed with kinase buffer containing 50 ␮M ATP, 15 mM MgCl2, 1 ␮Ci of [␥-32P]ATP, and 1 ␮g of GST-Abltide (Upstate Biotechnology). The reactions were allowed to proceed for 30 min at room temperature and stopped by adding SDS-sample buffer. The ablϪ/ϪargϪ/Ϫ cells were divided at a 1:5 dilution and further incubated in medium containing 200 ␮g/ml hygromycin

Recombinant Protein Expression and Purification

Indirect Immunofluorescence

RESULTS

DISCUSSION