Mediation of Af4 protein function in the cerebellum by Siah proteins

We have established that the gene AF4, which had long been recognized as disrupted in childhood leukemia, also plays a role in the CNS. AN is mutated in the robotic mouse that is characterized by ataxia and Purkinje cell loss. To determine the molecular basis of this mutation, we carried out a yeast two-hybrid screen and show that AM binds the E3 ubiquitin ligases Drosophila seven in absentia (sina) homologues (Siah)-1a and Siah-2 in the brain. Siah-1a and AN are expressed in Purkinje cells and colocalize in the nucleus of human embryonic kidney 293T and P19 cells. In vitro binding assays and coimmunoprecipitation reveal a significant reduction in affinity between Siah-1a and robotic mutant AM compared with wild-type, which correlates with the almost complete abolition of mutant AM degradation by Siah-la. These data strongly suggest that an accumulation of mutant AM occurs in the robotic mouse due to a reduction in its normal turnover by the proteasome. A significant increase in the transcriptional activity of mutant AM relative to wild-type was obtained in mammalian cells, suggesting that the activity of AM is controlled through Siahmediated degradation. Another member of the AM family, Fmr2, which is involved in mental handicap in humans, binds Siah proteins in a similar manner. These results provide evidence that a common regulatory mechanism exists that controls levels of the Af4/Fmr2 protein family. The robotic mouse thus provides a unique opportunity to understand how these proteins play a role in disorders as diverse as leukemia, mental retardation, and neurodegenerative disease.