Protein network study of human AF4 reveals its central role in the RNA Pol II-mediated transcription and in phosphorylation-dependent regulatory mechanisms.

AF4 belongs to a family of proteins implicated in childhood lymphoblastic leukemia, FRAXE mental retardation and ataxia. AF4 is a transcriptional activator that is involved in transcriptional elongation. Although AF4 has been implicated in MLL-related leukomogenesis, AF4-dependent physiological mechanisms are not clearly defined. Proteins that interact with AF4 may play important roles also in mediating oncogenesis, and are potential targets for novel therapies. Using a functional proteomic approach involving tandem mass spectrometry and bioinformatics, we identified about 50 AF4- interacting proteins of various Gene Ontology Categories. About 60% participate in transcription regulatory mechanisms, including the Mediator complex in eukaryotic cells. Here we report the first extensive proteomic study aimed at elucidating AF4 protein cross-talk. Moreover, we found that the AF4 residues Thr-220 and Ser-212 are phosphorylated, which suggests that AF4 function, depends on phosphorylation mechanisms. We also mapped the AF4 interaction site with CDK9, which is a direct interactor crucial for the protein’s function and regulation. Our findings, significantly expand the number of putative members of the multiprotein complex formed by AF4, which is instrumental in promoting the transcription/elongation of specific genes in human cells.