Inhibitors Screening Of Protein Tyrosine Kinases ALK And C-MET And Characterization Of Protein Tyrosine Phosphatase PTPRU

Protein tyrosine kinases play an important role in regulating cell proliferation, differentiation, and transformation. Mutations of these enzymes often cause neoplastic transformation, which makes them unique markers for cancer diagnosis and specific targets for anti-cancer drug development. Alk and c-MET are two typical tyrosine kinases that are frequently mutated in cancer cells. Alk was originally identified as a fusion partner of NPM-ALK in anaplastic large cell lymphoma (ALCL). Recent studies revealed its fusion with EML4 in non-small cell lung cancer (NSCLC) and activation mutations in neuroblastoma as well. c-MET is the receptor of hepatocyte growth factor. It is mutated or over-expressed in various types of tumors including breast, liver, lung, ovary, kidney, and thyroid cancers. ALK and c-MET are hot targets for therapeutic drug development.In this study, we cloned the catalytic domains of ALK and MET and expressed them as recombinant proteins by using the baculovirus expression system. We further established an activity assay method and used it to identify inhibitors of ALK and MET. By screening a chemical compound library containing 92 protein kinsase inhibitors, we identified GO6976 as a potent inhibitor of ALK with an IC50 value of 30 nM and staurosporine as a MET inhibitor with an IC50 value of 20 nM. We further characterized the ALK inhibitor GO6976 with NPM-ALK-positive Karpas 299 ALCL cells and EML4-ALK- positive H2228 NSCLC cells. Our data demonstrated that Go6976, at sub-micromolar concentrations, effectively blocked the growth of Karpas 299 and H2228 cells and inhibited activations of major signal transduction pathways within the cells. Together, we not only identified potent inhibitors of Alk and c-MET but also established a good system for further endeavors to identify more selective and potent protein tyrosine kinases inhibitors with therapeutic potentials.PTPRU is a MAM domain-containing receptor-like protein tyrosine phosphatase. Previous studies demonstrated an important role of the enzyme in the maintenance of epithelial integrity and in regulation of the Wnt/β-catenin signaling pathway. To better understand the function of PTPRU, we cloned and expressed the intracellular portion of PTPRU as a GST fusion protein in E. coli cells. We purified the protein to homogeneity and used it to immunize mice for antibody production. The resultant antibodies specifically recognized PTPRU over-expressed in cell lines. Western blotting analyses demonstrated the partition of truncated forms of PTPRU containing the cadherin-like domain in the Triton X-100-insoluble fraction, and immunofluorescent cell staining revealed localization of these proteins in the punctate intracellular structures. Our data suggest that the cadherin-like domain of PTPRU has a major role in determining its intracellular localization.