Insulin Phosphorylates Tyrosine Residue 464 of Tub and Translocates Tubby into the Nucleus in HIRcB Cells

BACKGROUND: The tubby protein has a motif that might be relevant for its action in the insulin signaling pathway. Previous studies have indicated that tubby undergoes phosphorylation on tyrosine residues in response to several stimuli and is known to localize in the nucleus as well as in the plasma membrane. However, the relationship between phosphorylation and nuclear translocation is not well understood. Here, we report that insulin directly phosphorylates tubby, which translocates into the nucleus. METHODS: The effects of insulin on Tubby were performed with Western blot. The immunoprecipitation and confocal microscopy were performed to prove phosphorylation and nuclear translocation. RESULTS: Mutation study reveals that tyrosine residue 464 of tubby gene (Tub) is a phosphorylation site activated by insulin. In addition, major portions of tubby protein in the plasma membrane are translocated into the nucleus after insulin treatment. Tyrosine kinase inhibitor pretreatment blocked insulin-induced tubby translocation, suggesting that phosphorylation is important for nuclear translocation. Moreover, mutant tyrosine residue 464 did not translocate into the nucleus in respond to insulin. These findings demonstrate that insulin phosphorylates tyrosine residue 464 of Tub, and this event is important for insulin-induced tubby nuclear translocation. CONCLUSION: Insulin phosphorylates tyrosine residue 464 of Tub and translocates tubby into the nuclei of HIRcB cells.

Microarray Analysis of Differentially Expressed Genes in the Brains of Tubby Mice

The tubby mouse is characterized by progressive retinal and cochlear degeneration and late-onset obesity. These phenotypes are caused by a loss-of-function mutation in the tub gene and are shared with several human syndromes, suggesting the importance of tubby protein in central nervous system (CNS) functioning. Although evidence suggests that tubby may act as a transcription factor mediating G-protein coupled receptor (GPCR) signaling, any downstream gene regulated by tubby has yet to be identified. To explore potential target genes of tubby with region-specific transcription patterns in the brain, we performed a microarray analysis using the cerebral cortex and hypothalamus of tubby mice. We also validated the changes of gene expression level observed with the microarray analysis using real-time RT-PCR. We found that expression of erythroid differentiation factor 1 (Erdr1) and caspase 1 (Casp1) increased, while p21-activated kinase 1 (Pak1) and cholecystokinin 2 receptor (Cck2r) expression decreased in the cerebral cortex of tubby mice. In the hypothalamic region, Casp 1 was up-regulated and micro-crystallin (CRYM) was down-regulated. Based on the reported functions of the differentially expressed genes, these individual or grouped genes may account for the phenotype of tubby mice. We discussed how altered expression of genes in tubby mice might be understood as the underlying mechanism behind tubby phenotypes.