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.
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(ΔCC) 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(ΔCC) 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.
Qiu
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Department of Medicine and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA. jqiu@bcm.edu
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D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D002884:Chromosomes, Human, Pair 15; D002899:Chromosomes, Human, Pair 9; D016384:Consensus Sequence / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051757:PAX5 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism*; D000071156:Promyelocytic Leukemia Protein; D021381:Protein Transport / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014161:Transduction, Genetic; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; D000071156:Promyelocytic Leukemia Protein; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human