Endocrinol Metab.  2014 Jun;29(2):163-168. 10.3803/EnM.2014.29.2.163.

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

• Kim JW
• Kim HS
• Kim SD
• Park JY

• KMID: 2169423
• DOI: http://doi.org/10.3803/EnM.2014.29.2.163

Abstract

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.

Keyword

Tubby; Insulin; Phosphorylation; Translocation

MeSH Terms

Blotting, Western
Cell Membrane
Immunoprecipitation
Insulin*
Microscopy, Confocal
Phosphorylation
Protein-Tyrosine Kinases
Tyrosine*
Insulin
Protein-Tyrosine Kinases
Tyrosine

Figure

• 

  Fig. 1 Tubby gene (Tub) is phosphorylated by insulin. Phosphorylation of Tub by insulin. (A, B) HIRcB cells were transiently transfected with c-Myc-tubby. Following starvation, insulin was added. Immunoprecipitation (IP) reaction with anti-Myc antibody (Ab) and Western blot with phosphotyrosine Ab were performed to determine the phosphorylation of Tub. The lower panel is a Western blot demonstrating equivalent loading of total tubby in whole cell lysates used for the IP, using the tubby Ab. IB, immunoblotting.

• 

  Fig. 2 Tyrosine residue 464 of tubby gene (Tub) is an important phosphorylation site activated by insulin. Phosphorylation of Tub by insulin. HIRcB cells were transiently transfected with wild-type-flag-tubby and mutant-flag-tubby. Following starvation, insulin was added. Immunoprecipitation (IP) reaction with antiflag antibody (Ab) and Western blot with phosphotyrosine Ab were performed to determine the phosphorylation of Tub. The lower panel is a Western blot demonstrating equivalent loading of total tubby in whole cell lysates used for the IP, using the tubby Ab.

• 

  Fig. 3 Translocation of green fluorescent protein (GFP)-tubby in response to insulin in HIRcB cells. Serum-starved, transiently transfected HIRcB cells were used. Following starvation, insulin was added. After 30 minutes, the cellular location of GFP-tubby was observed using a confocal microscope with objective ×20. mt, mutant; IP, immunoprecipitation; IB, immunoblotting.

• 

  Fig. 4 Phosphorylation of tyrosine residue 464 of tubby gene (Tub) is important for nuclear translocation. Serum-starved HIRcB cells were used after transfection with wild-type green fluorescent protein (GFP)-tubby and mutant-GFP-tubby. Following starvation, insulin was added. After 30 minutes, the cellular locations of wild-type and mutant GFP-tubby were observed using a confocal microscope with objective ×20.

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