Structural Basis of TPR-Mediated Oligomerization and Activation of Oncogenic Fusion Kinases.

The nuclear pore complex subunit TPR is found in at least five different oncogenic fusion kinases, including TPR-MET, yet how TPR fusions promote activation of kinases and their oncogenic activities remains poorly understood. Here we report the crystal structure of TPR(2-142), the MET fusion partner of oncogenic TPR-MET. TPR(2-142) contains a continuous 124-residue α helix that forms an antiparallel tetramer from two leucine zipper-containing parallel coiled coils. Remarkably, single mutations cause strikingly different conformations of the coiled coil, indicating its highly dynamic nature. We further show that fusion of TPR(2-142) to the MET intracellular domain strongly and selectively stabilizes the αG helix of the MET kinase domain, and mutations of only the TPR leucine zipper residues at the junction to MET, but not other leucine zipper residues, abolish kinase activation. Together, these results provide critical insight into the TPR structure and its ability to induce dimerization and activation of fusion kinases.

A New Transgenic Mouse Model of Heart Failure and Cardiac Cachexia Raised by Sustained Activation of Met Tyrosine Kinase in the Heart.

Among other diseases characterized by the onset of cachexia, congestive heart failure takes a place of relevance, considering the high prevalence of this pathology in most European countries and in the United States, and is undergoing a rapid increase in developing countries. Actually, only few models of cardiac cachexia exist. Difficulties in the recruitment and follow-up of clinical trials implicate that new reproducible and well-characterized animal models are pivotal in developing therapeutic strategies for cachexia. We generated a new model of cardiac cachexia: a transgenic mouse expressing Tpr-Met receptor, the activated form of c-Met receptor of hepatocyte growth factor, specifically in the heart. We showed that the cardiac-specific induction of Tpr-Met raises a cardiac hypertrophic remodelling, which progresses into concentric hypertrophy with concomitant increase in Gdf15 mRNA levels. Hypertrophy progresses to congestive heart failure with preserved ejection fraction, characterized by reduced body weight gain and food intake and skeletal muscle wasting. Prevention trial by suppressing Tpr-Met showed that loss of body weight could be prevented. Skeletal muscle wasting was also associated with altered gene expression profiling. We propose transgenic Tpr-Met mice as a new model of cardiac cachexia, which will constitute a powerful tool to understand such complex pathology and test new drugs/approaches at the preclinical level.

[Construction of a recombinant stable Ba/F3 cell strain containing Tpr-Met].

AIM: To construct a stable cell strain encoding tumor-associated fused gene which expresses oncoprotein Tpr-Met. METHODS: We transfected Tpr-Met vector into Ba/F3 cells and screened the cell strain stably expressing Tpr-Met. The interleukin 3 (IL-3) independent proliferation of the cells was measured using the MTS assay. The expression of Tpr-Met, the activity of downstream signal transduction pathway and SU11274-induced inhibition of the signal pathway were investigated by Western blotting. RESULTS: We obtained a Ba/F3 cell strain stably expressing Tpr-Met. The cells presented IL-3 independent proliferation, suggesting a malignant transformation of the cell line. In Tpr-Met transformed Ba/F3 cells, the phosphorylation of Met and ERK were enhanced; however, specific c-Met inhibitor SU11274 suppressed the cell proliferation and c-Met phosphorylation. CONCLUSION: Tpr-Met transformed Ba/F3 strain has been successfully constructed.