HIRcB, a rat fibroblast cell line that overexpresses the human insulin receptor, was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum in a humid atmosphere of 5% CO₂/95% O₂ at 37℃. COS-7 cells were maintained in DMEM supplemented with 10% heat-inactivated bovine calf serum. Cells were planted on a 6- or 10 cm tissue culture dish that was precoated with poly-L-lysine (10 g/mL) and were grown for 1 to 2 days until they reached 50% to 80% confluency. Cells then were placed in serum-free DMEM for 12 to 18 hours before treatment with 10 ng/mL of EGFR.

A bright green fluorescent protein (GFP) was attached to the carboxyl-terminus of tubby by standard recombinant techniques using the pEGFP-C1 vector (Invitrogen, Carlsbad, CA, USA).

The cells were grown in 6-well plates and, once at 60% to 70% confluency, were serum-starved for 24 hours before treatment at 37℃ with selected agents. The media were then aspirated, and the cells were washed twice with ice-cold phosphape buffer saline (PBS) and lysed in 100 L of lysis buffer. The samples were briefly sonicated, heated at 95℃ for 5 minutes, and centrifuged for 5 minutes. The supernatant was electrophoresed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 8% to 16%) gradient gels and transferred to a polyvinylidene difluoride membrane. The blots were incubated overnight at 4℃ with primary antibodies and washed six times with Tris-buffered saline with 0.1% tween 20 before being probed with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The blots were then visualized with ECL (Amersham Biosciences, Piscataway, NJ, USA). In some cases, the blots were stripped and reprobed with other antibodies.

After treatment with inhibitors and drugs, the cells were placed on ice, washed twice with ice-cold PBS, lysed in lysis buffer containing 50 mM Tris-HCl at pH 8.0, 100 mM NaCl, 20 mM NaF, 10 mM sodium pyrophosphate, 5 mM ethylenediaminetetraacetic acid, 1% Nonidet P-40, 10 g/mL aprotinin, 10 g/mL leupeptin, 10 g/mL soybean trypsin inhibitor, 10 g/mL pepstatin, and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride. The cells then were probe-sonicated (Sonifer Cell Disruptor, Heat Systems Co., Melville, NY, USA). The solubilized lysates were clarified by centrifugation at 8,000 ×g for 10 minutes, precleared with agarose, and then incubated with specific antibodies and protein-A agarose. The immunoprecipitates were collected and washed four times with lysis buffer. After heating at 95℃ for 5 minutes, the samples were centrifuged briefly, and the supernatants were analyzed by SDS-PAGE on 8% to 16% gradient gels.

Transiently transfected HIRcB cells were grown on glass-bottom microwell dishes (Mat Tek Corp., Ashland, MA, USA) for 24 hours. The cells were then placed in phenol red-free DMEM supplemented with 0.5% fetal bovine serum for 3 to 4 hours prior to experiments. The microwells were placed on a Zeiss Axiovert 135 confocal microscope (Zeiss Inc., Thornwood, NY, USA), and the cells were treated with insulin (100 ng/mL) at room temperature. Detection of GFP was carried out by recording at a wavelength of 488 or 568 nm, respectively.

Phosphotyrosine-mediated signal pathways have been shown to play an important role in insulin signaling. Tubby does not share any regions or domains of homology with other proteins, but Tub does have several potential tyrosine phosphorylation sites on its carboxyl-terminus. To investigate whether Tub becomes phosphorylated on tyrosine residue in response to insulin, we transfected Myc-tubby into HIRcB cells. Following treatment with insulin, immunoprecipitation with d-myc was conducted. Analysis of the whole cell lysate (WCL) by tyrosine-specific phosphorylation antibody (a-Ty[p]) immunoblot demonstrated that Tub was phosphorylated in a time/dose-dependent manner following insulin introduction (Fig. 1). Anti-Tub immunoblotting of the WCL demonstrated equivalent amounts of Tub expressed under the different conditions. These results indicate that insulin induces rapid and transient tyrosine phosphorylation of Tub at physiological doses.

Phosphotyrosine-mediated interactions with SH2-containing signaling proteins have been shown to play an important role in the insulin pathway. Strikingly, several tyrosine residues are conserved in all tubby family members. Among these, a sequence surrounding tyrosine 464 (YIVM) fits the consensus motif YXXM for phosphorylation by insulin. To determine the possibility of whether this sequence may serve as potential site of phosphorylation by insulin, we constructed a point mt by replacing tyrosine 464 residue with alanine. To investigate whether Tub becomes phosphorylated on tyrosine residue 464 in response to insulin, we transfected wild-type (wt) flag-Tub and mt flag-Tub into HIRcB cells. Following treatment with insulin, immunoprecipitation with a-flag was performed. Analysis of the WCL by tyrosine-specific phosphorylation antibody immunoblot demonstrated that wt Tub was phosphorylated following insulin, but mt Tub was not (Fig. 2). Anti-tubby immunoblotting of the WCL demonstrated equivalent amounts of tubby expressed under the different conditions. These results demonstrated that tyrosine residue 464 of Tub was specifically phosphorylated by insulin.

To analyze the insulin-dependent changes in an intracellular Tub location, GFP-tagged tubby proteins were constructed. HIRcB cells then were transiently transfected with the GFP-tubby. In the absence of insulin, GFP-tubby was uniformly distributed throughout the cytoplasm or plasma membrane with no fluorescence detectable in the nucleus. Analysis of the transfected cells showed that cells expressing GFP were cytoplasmic. Following the addition of insulin, however, substantial amounts of fluorescence appeared in the nucleus (Fig. 3). This result demonstrated that tubby translocated into the nucleus when activated by insulin and suggests that phosphorylation of tyrosine residue may be important in this process.

Since insulin activates the tyrosine phosphorylation of Tub at residue 464, we used insulin to test whether phosphorylation of this residue is important for nuclear translocation. The mt and wt constructs were then transiently expressed in HIRcB cells, and their translocation capacity in response to insulin was evaluated. Following the addition of insulin, cells transfected with the mt GFP-tubby showed substantial amounts of fluorescence in the nucleus (Fig. 4). Analysis of cells transfected with the mt GFP-tubby showed cytoplasmic expressed GFP. This result suggests that tyrosine residue 464 is important for nuclear translocation and tyrosine phosphorylation by insulin.