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

Abstract

The paired box domain of PAX5 was reported to fuse with the sequence of promyelocytic leukemia (PML) to produce PAX5-PML chimeric protein in two patients with B-cell acute lymphoblastic leukemia. In the present studies, we found, by gel shift assays, that PAX5-PML 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-PML inhibited wild-type PAX5 target gene transcriptional activity. Studies comparing PAX5-PML with PAX5-PML(DCC) demonstrated that the coiled-coil (CC) protein interaction domain located within the PML moiety was required for PAX5-PML 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-PAX5-PML and YFP-PAX5-PML(DCC) 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-PML 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-PML. These results indicate that the CC domain of PAX5-PML is important for each of the known activities of PAX5-PML fusion proteins.

Anopheles Midgut FREP1 Mediates Plasmodium Invasion

“…Moreover, non-ionic detergent disassociated these oligomers. Consistent with this, coiled-coil motifs in proteins have been reported to mediate homodimer complexes (36) and tetramer complex formation (37). Therefore, interactions between the coiled-coil domains of FREP1 likely mediate FREP1 tetramer formation.…”

The Role of PAX5 in ALL

“…By sub-cellular fractionation and western blot analysis in transfected 293T cells, it has been shown that PAX5/ETV6, PAX5/FOXP1 and PAX5/C20S were present mainly in the nucleus and less abundant in the cytoplasm, while PAX5/C20L was exclusively nuclear (Kawamata et al, 2008). In agreement with these data, fluorescent microscopic examination of transiently expressed proteins in HeLa and 293T cells demonstrated that PAX5/PML exhibited a diffuse granular pattern within the nucleus, similar to PAX5 but not to PML (Qiu et al, 2011).…”

“…Kawamata demonstrated the specific binding for PAX5/ETV6, PAX5/FOXP1 and PAX5/C20orf112 in parallel to wild type PAX5 (Kawamata et al, 2008). In an analogous setting, it has been demonstrated that PAX5/PML is able to bind to PAX5-responsive elements (Qiu et al, 2011). A formal demonstration of DNA binding by PAX5/ELN is missing in literature, although the transcriptional activity of PAX5 fusion genes, including the PAX5/ELN, has been proven by reporter gene assays (Bousquet et al, 2007;Mullighan et al, 2007;Kawamata et al, 2008;Kurahashi et al, 2011;Qiu et al, 2011).…”

The role of the Janus-faced transcription factor PAX5-JAK2 in acute lymphoblastic leukemia

“…14 Generally, it is hypothesized that PAX5 fusions act as aberrant transcription factors antagonizing wild-type PAX5 function in a dominant negative mode. 1,8,9,11,[15][16][17][18] However, in a recent study, we have shown that a subset of the PAX5 fusion proteins may have a cellular context-dependent activation potential, indicating that some PAX5 fusions may also activate target genes, thus arguing against their simplified trans-dominant negative function. 14 Janus kinase 2 (JAK2) belongs to a family of nonreceptor tyrosine kinases and is involved in signal transduction from many cytokine and growth hormone receptors regulating hematopoiesis, immunity, growth, and development.…”