Oncohematological diseases are among the most common types of cancer characterized by clonal proliferation of transformed cells, which due to mutations are able to avoid apoptosis and have a selective advantage over differentiated hematopoietic cells. Development of oncohematological disorder due to chromosomal translocation is quite common case. Translocation results in fusion of two genes, leading to deregulation of signaling pathways due to the acquisition of atypical functions: constitutive expression, tyrosine kinase activity and abnormal protein localization within the cell. The t(9; 22) (q34; q11) reciprocal translocation between 9 and 22 chromosomes results in fusion of Bcr gene on chromosome 22 to the Abl gene on chromosome 9. BCR-ABL hybrid protein resulting from such fusion is characterized by the constitutive tyrosine kinase activity provided by its ABL portion. This leads to uncontrolled phosphorylation of proteins inside the cell and as a result, the cell becomes malignant and loses the ability to differentiate.
Because of different breakpoints in the Bcr gene, translocation can give rise to different types of chimeric protein – BCR-ABLp190, BCR-ABLp210, BCR-ABLp230. They differ in the presence or absence of a BCR region containing the PH and DH domains. These three forms are associated with different types of leukemia, the short type p190 – acute lymphoblastic leukemia, type p210 – chronic myeloid leukemia, type p230 – chronic neutrophilic leukemia. Thus, the study of the function of these domains will help to understand which molecular pathways determine the specific phenotype of the disease. In previous studies, mass spectrometry identified 23 potential candidates for interaction with the PH domain of the BCR among the proteins of the K562 cell line, which originates from a patient with chronic myeloid leukemia and contains t(9; 22) (q34; q11) rearrangement, expressing BCR-ABLp210. Among the candidates for the interaction are cortactin, which belongs to protein family of actin regulators, and β-tubulin, which is one of the two heterodimers that form microtubules.
We determined that PH domain of BCR protein and full-length BCR are able to colocalize with cortactin in the perimembrane region at the points of actin branching and in the sites of cell adhesion to surface. We obtained a subdiffraction image of PH domain of BCR, as well as an image of distribution of clathrin, BCR and cortactin using STED super-resolution microscopy. We found that PH domain and full-length BCR are present on the centrosome along with cortactin. Since the centrosome is the nucleation center of microtubules, we suggest that it is in this region that the interaction between the PH domain and β-tubulin may take place. According to recent studies, centrosome is also the actin-organizing center, so the simultaneous presence of BCR / ABL and cortactin may be an indication that cortactin is activated by tyrosine kinase activity of ABL moiety. To test this hypothesis, we performed a bioinformatic analysis of phosphorylation sites and found that ABL and cortactin activator - SRC kinase share seven predicted sites. For the first time it is shown that PH domain provides anchoring of BCR to the centrosome. We believe that uncontrolled phosphorylation of cortactin can lead to disruption of centrosome integrity, which is one of the distinct features of cancer cells. We have demonstrated colocalization between BCR and cortactin in the nucleus at the terminal stage of cell division, characterized by the presence of contractile ring between the nuclei. We hypothesize that BCR and cotractin may thus provide the branching of nuclear actin that is required for a coordinated change in the nuclear architecture during cell division.
We hypothesize that data of colocalization with clathrin and cortactin in different cell compartments demonstrate involvement of PH domain of BCR in endosomal trafficking and sorting system, comprising clathrin-mediated endocytosis, early endosome formation, trans-Golgi network, and centrosome. These data may aid in better understanding of pathogenesis mechanisms of chronic myeloid leukemia and provide background for new therapeutic targets that may be an alternative to the existing approach of using tyrosine kinase inhibitors of ABL, which often lead to resistance and requirement to develop next generation of drugs.
