Figure 2. PKR regulates JNK activity resulting in inhibition of insulin signaling.

(A) Primary Pkr^+/+ and Pkr^−/− MEFs were treated with 0.5 mM palmitic acid for 2 hours. JNK activity was assessed by a kinase assay using recombinant c-Jun protein as substrate.

(B) Induction of JNK phosphorylation after 300 nM thapsigargin treatment for 1 hour in primary Pkr^+/+ and Pkr^−/− MEFs. Phosphorylation level of JNK was examined with anti-phospho-JNK (Thr183/Tyr185) antibody.

(C and D) Induction of IRS-1 phosphorylation after 0.5 mM palmitic acid (C) or 300 nM thapsigargin (D) treatment for 2 hours in Pkr^+/+ and Pkr^−/− MEFs. Phosphorylation level of IRS-1 on Ser307 was examined with anti-phospho-IRS-1 (serine 307) antibody.

(E) Induction of IRS-1 phosphorylation in retrovirally PKR-reconstituted Pkr^−/− MEFs. The cells were serum-starved for 14 hours followed by western blot analysis with anti-phospho-IRS-1 (serine 307) antibody.

(F) The PKR-reconstituted Pkr^−/− MEFs were stimulated with 10 nM Insulin for 3 minutes. The cell lysates were immunoprecipitated with anti-IRS-1 antibody followed by western blot analysis with anti-phospho-tyrosine and anti-PI3K (p85 subunit) antibodies. The graph on the right shows the quantification of the results. Data are shown as the mean ± SEM. *P<0.05.

(G and H) Induction of palmitic acid- and thapsigargin-induced PKR activity requires intact RNA binding domain of PKR. Pkr^−/− MEFs were reconstituted with vector, flag-tagged wild type (WT), RNA binding domain mutant (K64E), or kinase dead mutant (K296R) of PKR by retrovirus-mediated gene transfer. These cells were maintained in serum-free DMEM containing 0.5% BSA for 14 hours followed by treatment with 0.5 mM palmitic acid for 90 minutes (G) or 300 nM thapsigardin for 1 hour (H). The cell lysates were immunoprecipitated with anti-Flag antibody followed by PKR kinase assay and western blot analysis with anti-Flag antibody.

See also Supplemental Figure S2.