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

AF4 belongs to a family of proteins implicated in childhood lymphoblastic leukaemia, FRAXE (Fragile X E site) mental retardation and ataxia. AF4 is a transcriptional activator that is involved in transcriptional elongation. Although AF4 has been implicated in MLL (mixed-lineage leukaemia)-related leukaemogenesis, AF4-dependent physiological mechanisms have not been clearly defined. Proteins that interact with AF4 may also play important roles in mediating oncogenesis, and are potential targets for novel therapies. Using a functional proteomic approach involving tandem MS and bioinformatics, we identified 51 AF4-interacting proteins of various Gene Ontology categories. Approximately 60% participate in transcription regulatory mechanisms, including the Mediator complex in eukaryotic cells. In the present paper we report one of the first extensive proteomic studies 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 (cyclin-dependent kinase 9), which is a direct interactor crucial for the function and regulation of the protein. The findings of the present study 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.