The reduced and altered activities of PAX5 are linked to the protein–protein interaction motif (coiled-coil domain) of the PAX5–PML fusion protein in t(9;15)-associated acute lymphocytic leukemia

摘要

The paired box domain of PAX5 was reported to fuse with the sequence of promyelocytic leukemia (PML) to produce PAX5鈥揚ML chimeric protein in two patients with B-cell acute lymphoblastic leukemia. In the present studies, we found, by gel shift assays, that PAX5鈥揚ML bound to a panel of PAX5-consensus sequence acts as a homodimer with reduction of its DNA-binding affinities in comparison with wild-type PAX5. In transient transfection assays using 293T and HeLa cells, and retrovirus transduction of murine hematopoietic stem/progenitor cells together with quantitative real-time polymerase chain reaction analysis, PAX5鈥揚ML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5鈥揚ML with PAX5鈥揚ML(螖CC) demonstrated that the coiled-coil (CC) protein interaction domain located within the PML moiety was required for PAX5鈥揚ML homodimer complex formation and partial transcriptional repression of genes controlled by PAX5. Fluorescent microscopic examination of transiently expressed YFP-tagged proteins in HeLa and 293T cells demonstrated that YFP鈥揚AX5鈥揚ML and YFP鈥揚AX5鈥揚ML(螖CC) exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but not PML. By fluorescent recovery after photobleach (FRAP), we have shown that PAX5鈥揚ML fusion protein has reduced intranuclear mobility compared with wild-type PAX5. Furthermore, the dimerization domain (CC) of PML was responsible for the reduced intranuclear mobility of PAX5鈥揚ML. These results indicate that the CC domain of PAX5鈥揚ML is important for each of the known activities of PAX5鈥揚ML fusion proteins.