Abstract
Her4 is a transmembrane receptor tyrosine kinase belonging to the ErbB-EGFR family. It plays a vital role in the cardiovascular and nervous systems, and mutations in Her4 have been found in melanoma and lung cancer. The kinase domain of Her4 forms a dimer complex, called the asymmetric dimer, which results in kinase activation. Although a crystal structure of the Her4 asymmetric dimer is known, the dimer affinity and the effect of the subsequent phosphorylation steps on kinase domain conformation are unknown. We report here the use of carboxyl-group footprinting MS on a recombinant expressed, Her4 kinase-domain construct to address these questions. Carboxyl-group footprinting uses a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, in the presence of glycine ethyl ester, to modify accessible carboxyl groups on glutamate and aspartate residues. Comparisons of Her4 kinase-domain monomers versus dimers and of unphosphorylated versus phosphorylated dimers were made to map the dimerization interface and to determine phosphorylation induced-conformational changes. We detected 37 glutamate and aspartate residues that were modified, and we quantified their extents of modification by liquid chromatography MS. Five residues showed changes in carboxyl-group modification. Three of these residues are at the predicted dimer interface, as shown by the crystal structure, and the remaining two residues are on loops that likely have altered conformation in the kinase dimer. Incubating the Her4 kinase dimers with ATP resulted in dramatic increase in Tyr-850 phosphorylation, located on the activation loop, and this resulted in a conformational change in this loop, as evidenced by reduction in carboxyl-group modification. The kinase monomer-dimer equilibrium was measured using a titration format in which the extent of carboxyl-group footprinting was mathematically modeled to give the dimer association constant (1.5-6.8 × 10(12) dm(2)/mol). This suggests that the kinase-domain makes a significant contribution to the overall dimerization affinity of the full-length Her4 protein.