RESUMEN
Fasciculation and elongation zeta-1 (FEZ1) protein is involved in axon outgrowth and is highly expressed in the brain. It has multiple interaction partners, with functions varying from the regulation of neuronal development and intracellular transport mechanisms to transcription regulation. One of its interactors is retinoic acid receptor (RAR), which is activated by retinoic acid and controls many target genes and physiological process. Based on previous evidence suggesting a possible nuclear role for FEZ1, we wanted to deepen our understanding of this function by addressing the FEZ1-RAR interaction. We performed in vitro binding experiments and assessed the interface of interaction between both proteins. We found that FEZ1-RAR interacted with a similar magnitude as RAR to its responsive element DR5 and that the interaction occurred in the coiled-coil region of FEZ1 and in the ligand-binding domain of RAR. Furthermore, cellular experiments were performed in order to confirm the interaction and screen for induced target genes from an 86-gene panel. The analysis of gene expression showed that only in the presence of retinoic acid did FEZ1 induce hoxb4 gene expression. This finding is consistent with data from the literature showing the hoxb4 gene functionally involved in development and acute myeloid leukemia, as is FEZ1.
RESUMEN
BACKGROUND: The FEZ (fasciculation and elongation protein zeta) family designation was purposed by Bloom and Horvitz by genetic analysis of C. elegans unc-76. Similar human sequences were identified in the expressed sequence tag database as FEZ1 and FEZ2. The unc-76 function is necessary for normal axon fasciculation and is required for axon-axon interactions. Indeed, the loss of UNC-76 function results in defects in axonal transport. The human FEZ1 protein has been shown to rescue defects caused by unc-76 mutations in nematodes, indicating that both UNC-76 and FEZ1 are evolutionarily conserved in their function. Until today, little is known about FEZ2 protein function. METHODOLOGY/PRINCIPAL FINDINGS: Using the yeast two-hybrid system we demonstrate here conserved evolutionary features among orthologs and non-conserved features between paralogs of the FEZ family of proteins, by comparing the interactome profiles of the C-terminals of human FEZ1, FEZ2 and UNC-76 from C. elegans. Furthermore, we correlate our data with an analysis of the molecular evolution of the FEZ protein family in the animal kingdom. CONCLUSIONS/SIGNIFICANCE: We found that FEZ2 interacted with 59 proteins and that of these only 40 interacted with FEZ1. Of the 40 FEZ1 interacting proteins, 36 (90%), also interacted with UNC-76 and none of the 19 FEZ2 specific proteins interacted with FEZ1 or UNC-76. This together with the duplication of unc-76 gene in the ancestral line of chordates suggests that FEZ2 is in the process of acquiring new additional functions. The results provide also an explanation for the dramatic difference between C. elegans and D. melanogaster unc-76 mutants on one hand, which cause serious defects in the nervous system, and the mouse FEZ1 -/- knockout mice on the other, which show no morphological and no strong behavioural phenotype. Likely, the ubiquitously expressed FEZ2 can completely compensate the lack of neuronal FEZ1, since it can interact with all FEZ1 interacting proteins and additional 19 proteins.
