(485ai) The Role of Palmitic Acid in Inhibiting PKR Activity
Palmitic acid, a saturated free fatty acid, has been shown to be a potent inducer of cellular apoptosis in various types of cells, although the mechanism is unclear. Recently, our group suggested that palmitic acid (PA), but not oleic acid (OA), an unsaturated free fatty acid, involved PKR (double-stranded RNA-dependent protein kinase)-mediated apoptosis by reducing autophosphorylation at Thr451 residue. In the present study, we investigated how palmitic acid affects the activity of PKR protein at the molecular and biophysical level. Using a fluorescent palmitic acid, Bodipy-FL-PA, binding assays were performed with three types of recombinant PKR proteins, namely, the double-stranded RNA (dsRNA) binding domain on PKR (PKR-dsRBD), the phosphorylated form of the wild-type PKR protein (p-PKR-wt), and the unphosphorylated form of PKR-wt (PKR-wt). We found that p-PKR-wt has high affinity for Bodipy-FL-PA with KD of 1503.67±79.96 nM, but PKR-dsRBD did not show a significant increase in the fluorescence intensity, suggesting that palmitic acid does not bind to the dsRNA binding domain. Computational docking experiments suggested that PA may dock onto the ATP binding site on PKR, near the K296 residue (within a sphere of 5Å around the side chain of K296) with a higher docking score (34.9) than OA (23.9). We experimentally confirmed through a competition assay with ATP that PA binds to the ATP binding site on p-PKR-wt protein. Furthermore, using western blotting analysis, another competition assay with Bodipy-FL and Bodipy-FL-PA showed only Bodipy-FL-PA reduces the phosphorylation of Thr451 of PKR. These results suggest that PA directly binds to PKR to alter PKR phosphorylation. To determine which PA binding residues on the PKR protein are involved, we first performed computational docking experiments with several mutant PKR proteins and found two specific residues, K296 and F368, are important for PA binding to PKR protein. In-vitro PA binding study with mutant recombinant PKR proteins showed only K296I and K296R significantly decreased Bodipy-FL-PA binding affinity, suggesting the charge state of K296 residue is structurally important for PA binding, similar to the case of ATP binding to PKR. Thus, these findings raise the question of whether palmitic acid may have broader effects, with a potential of modulating kinases with similar ATP binding sites. Thereby with implications on how palmitic acid may be involved in signaling pathways, and possibly in the efficacy of current drug therapies that target kinases.