14-3-3 θ over-expression increases both the cytosolic amount of AF4 and the expression levels of its target genes.

The human AF4 gene is involved in the t(4;11)(q21;q23) translocation that causes acute lymphoblastic leukemia. AF4 is a transcriptional activator implicated in early lymphoid development. It is a crucial component of a nuclear protein complex that promotes transcription elongation and chromatin remodeling. Noteworthy, AF4 function is regulated by phosphorylation and its cellular levels by proteasomal degradation. In a previous study aimed to analyze the AF4 function and regulation, we found that AF4 over-expression promotes transcription of several genes (like PTPRA, GRB2, BRD7, etc.) involved in different pathways, in HEK-293 cells. Moreover, we demonstrated that AF4 directly interacts with the scaffold protein 14-3-3 θ, which belongs to a family of proteins that bind phosphorylated target proteins and regulate variously their function. To identify the AF4-responding genes in hematopoietic cell lines, we ectopically expressed AF4 in the K562 mielogenous leukemia cells. Real-time PCR analysis showed increased expression of specific transcripts in K562 as well as in HEK-293 cells. We also evaluated the potential role of 14-3-3 θ in regulating the AF4 function. To this aim, we ectopically expressed 14-3-3 θ in K562 cells and measured transcription levels of the AF4-responding genes previously identified. We observed that 14-3-3 θ over-expression greatly stimulates transcription of these genes. Moreover, we found that the cytosolic amount of AF4 significantly increased, while nuclear levels slightly decreased. These data suggest that 14-3-3 θ binds and stabilizes AF4 in cytosol, probably by impairing its proteasomal degradation. Additional analy