The double-stranded RNA-dependent protein kinase differentially regulates insulin receptor substrates 1 and 2 in HepG2 cells

Abstract

Initially identified to be activated upon virus infection, the double-stranded RNA-dependent protein kinase (PKR) is best known for triggering cell defense responses by phosphorylating eIF-2α, thus suppressing RNA translation. We as well as others showed that the phosphorylation of PKR is down-regulated by insulin. In the present study, we further uncovered a novel function of PKR in regulating the IRS proteins. We found that PKR up-regulates the inhibitory phosphorylation of IRS1 at Ser312, which suppresses the tyrosine phosphorylation of IRS1. This effect of PKR on the phosphorylation of IRS1 is mediated by two other protein kinases, JNK and IKK. In contrast, PKR regulates IRS2, another major IRS family protein in the liver, at the transcriptional rather than the posttranslational level, and this effect is mediated by the transcription factor, FoxO1, which has been previously shown to be regulated by insulin and plays a significant role in glucose homeostasis and energy metabolism. In summary, we found for the first time that initially known as a virus infection response gene, PKR regulates the upstream central transmitters of insulin signaling, IRS1 and IRS2, through different mechanisms.

Figure 1.

Involvement of PKR in regulating the phosphorylation of IRS1. Reverse transfection of suspended HepG2 cells was performed with scrambled siRNA (negative control) or siRNA of PKR for 24 h, and the transfected cells were cultured in regular media for another 24 h (A). Cells were then treated with different concentrations of insulin for 15 min and harvested after the treatment (A). HepG2 cells were exposed to 5 μM PKR inhibitor (PI) or its analogue as a negative control (NC) or 10 mM 2-AP dissolved in PBS:glacial acetic acid (200:1; GA, control) for 12 h followed by the treatment of 0.2 nM insulin for 15 min (B). Western blot analysis was performed to detect the levels of β-actin and PKR and the total and phosphorylated levels of PKR and IRS1. The phosphorylation levels of IRS1 at Ser312 and Tyr941 were quantified by normalizing to total IRS1 levels and are expressed as the average of three samples ± SD from three independent experiments (A, middle and bottom). Student's t test was performed for analyzing the differences between samples transfected with siPKR and scrambled siRNA (negative control; A). Significantly higher (Tyr941) or lower (Ser312) than negative control; i.e., scrambled siRNA; *p < 0.01.

Figure 2.

PKR mediates the effects of ceramide on the phosphorylation of IRS1. HepG2 cells were exposed to different levels of ceramide for 12 h (A). Reverse transfection of suspended HepG2 cells was performed with scrambled siRNA (negative control) or siRNA of PKR for 24 h, and the transfected cells were cultured in regular media for another 12 h (B). Cells were then treated with ceramide (10 μM) for 12 h followed by insulin (0.5 nM) treatment for 15 min (B). Pretreated with 10 μM ceramide for 12 h, HepG2 cells were exposed to different levels of PKR inhibitor dissolved in DMSO (control; C) or 10 mM 2-AP dissolved in PBS:glacial acetic acid (200:1; GA, control; D) for another 12 h. After treatment, the cells were harvested, and Western blot analysis was performed to detect the level of β-actin and the total and phosphorylated levels of PKR and IRS1.

Figure 3.

Involvement of JNK and IKK in regulating the phosphorylation of IRS1. Reverse transfection of suspended HepG2 cells was performed with scramble siRNA (negative control) or siRNA of PKR (siPKR) for 24 h, and the transfected cells were cultured in regular medium for another 24 h (A). Cells were harvested, and Western blot analysis was performed to detect the total and phosphorylated levels of JNK and IKK (A). Reverse transfection of suspended HepG2 cells was performed with scrambled siRNA (control) or siRNAs of JNK1 and JNK2 together or siRNA of IKK for 24 h, and the transfected cells were cultured in regular media for another 24 h. Next, the forward transfection of empty vector pCMV6-XL5 (pCMV6) or plasmid containing PKR cDNA sequence (pCMV6-hPKR) was performed, followed by insulin treatment (0.5 nM) for 15 min (B). After treatment, cells were then harvested, and Western blot analysis was performed to detect the protein level of IKK and JNK and total and phosphorylated levels of PKR and IRS1 (B).

Figure 4.

Involvement of PKR in regulating IRS2. Reverse transfection of suspended HepG2 cells was performed with scrambled siRNA (negative control) or siRNA of PKR for 24 h, and the transfected cells were cultured in regular media for another 24 h. Cells were then harvested (D) or treated with different concentrations of insulin for 15 min and harvested after the treatment (A). Confluent HepG2 cells were treated with 5 μM PKR inhibitor (PI) or its analogue as a negative control (NC) or 10 mM 2-AP dissolved in PBS:glacial acetic acid (200:1) (GA) for 12 h (B and C). Western blot analysis was performed to detect the total and phosphorylated levels of IRS2 at Ser731 (A and B). RT-PCR was performed to detect the gene expression levels of IRS1 and IRS2 in response to the PKR inhibitors (C) or siRNA of PKR (D). Gene expression data were expressed as the average of nine samples ± SD from three independent experiments. The protein levels of IRS2 were quantified by normalizing to β-actin, and the phosphorylation levels of IRS2 at Ser731 were quantified by normalizing to total IRS2 levels. Both the protein and phosphorylation levels of IRS2 are expressed as the average of three samples ± SD from three independent experiments. Student's t test was performed for analyzing the differences between siPKR and scrambled siRNA (negative control). Significantly lower than negative control; i.e., scrambled siRNA (A and D) or chemical analogue of the PKR inhibitor (B and C); *p < 0.05. Significantly lower than control; GA, solvent of 2-AP; **p < 0.05.

Figure 5.

Involvement of FoxO1 in mediating the effect of PKR on IRS2. Reverse transfection of suspended HepG2 cells was performed with scrambled siRNA (control) or siRNA of PKR (A and B) or siRNA of FoxO1 (D) for 24 h, and the transfected cells were cultured in regular media for another 24 h. The forward transfection of empty vector pCMV6-XL5 (pCMV6) or plasmid containing PKR cDNA sequence (pCMV6-hPKR) was performed, and the cells were then treated with OA (2 nM) or its vehicle, ethanol, as a control, for 1 h (C). Reverse transfection of scramble siRNA (negative control, lanes 1 and 2) or siRNA of FoxO1 (siPKR, lanes 3 and 4) was performed, followed by the forward transfection of empty vector pCMV6-XL5 (pCMV, lanes 1 and 3) or the plasmid containing PKR cDNA sequence (hPKR, lanes 2 and 4; E). After treatment, the cells were harvested, and Western blot analysis was performed to detect the total and phosphorylated levels of FoxO1 (A and C), the total levels of IRS1, IRS2, FoxO1 and β-actin (D and E), and the total and phosphorylated levels of PKR (E). PP2A activity assay was performed to detect the phosphatase activity of PP2A (B). The phosphorylations of FoxO1 at Ser256 normalized to the total FoxO1 protein levels (C) and the protein levels of IRS1 and IRS2 normalized to β-actins (E) are expressed as the average of three samples ± SD from three independent experiments. Student's t test was performed, and p values were calculated for analyzing the differences between the indicated samples.

Figure 6.

Proposed signaling pathways through which PKR is involved in insulin signaling network in HepG2 cells. Insulin activates insulin signaling by IR and IRS, leading to the suppression of PKR phosphorylation at Thr451. PKR induces the phosphorylation of IRS1 at Ser312 through two other kinases, JNK and IKK. In addition, by activating PP2A, PKR dephosphorylates a transcription factor, FoxO1, which up-regulates the gene expression of IRS2.