Quantitative microscopic analysis of the regulatory mechanisms of the anaplastic lymphoma kinase in neuroblastoma

The anaplastic lymphoma kinase (ALK) plays an essential role in the pathogenesis of neuroblastoma. While the structure of the ALK-receptor as well as point mutations within the primary amino acid sequence of the receptor are well investigated, the activation mechanism of the ALK-receptor during its autophosphorylation and the resulting malign potential require further research. To gain better insight into the activation mechanism, the Wild-Type ALK, the F1174L-, the I1250T-, the R1275Q- and the Y1604F-mutation of the ALK-receptor were examined with regard to their level of phosphorylation and a possible dimerization during autophosphorylation. To examine these questions the receptors were expressed in the SK-N-AS neuroblastoma cell line. The comparative description of the level of phosphorylation of the Wild-Type ALK and its four point mutations was performed using quantitative wide field fluorescence microscopy. By investigating seven phosphotyrosin epitopes of each subtype of the ALK-receptor, characteristic differences between their levels of phosphorylation during autophosphorylation were subject of this study. Furthermore, a correlation between the morphology of the SK-N-AS neuroblastoma cells and their levels of phosphorylation was examined. A possible dimerization of the ALK-receptor during its autophosphorylation was investigated using acceptor photobleaching. Analyzing the FRET-efficiency of the Wild-Type ALK and its four point mutations permits conclusions regarding their behavior during autophosphorylation. It allows for a comparison between the degrees of dimerization of the Wild-Type ALK and its subtypes. The identification of important phosphotyrosin epitopes and the exploration of the activation mechanisms during autophosphorylation by wide field and confocal laser scanning microscopy improve the understanding of the ALK-receptor in the context of neuroblastoma. Possible therapeutically relevant structural attributes of the ALK-receptor and its selected point mutations are an interesting subject to future research.2020-01-2