Molecular Mechanism by Which Palmitate Inhibits PKR Autophosphorylation

Abstract

PKR (double-stranded RNA-activated protein kinase) is an important component of the innate immunity, antiviral and apoptotic pathways. Recently, our group found that palmitate, a saturated fatty acid, is involved in apoptosis by reducing the autophosphorylation of PKR at the Thr451 residue, however, the molecular mechanism by which palmitate reduces PKR autophosphorylation is not known. Thus, we investigated how palmitate affects the phosphorylation of the PKR protein at the molecular and biophysical levels. Biochemical and computational studies show that palmitate binds to PKR, near the ATP-binding site, thereby inhibiting its autophosphorylation at Thr451 and Thr446. Mutation studies suggests that Lys296 and Asp432 in the ATP binding site on the PKR protein are important for palmitate binding. We further confirmed that palmitate also interacts with other kinases, due to the conserved ATP-binding site. A better understanding of how palmitate interacts with the PKR protein, as well as other kinases, could shed light onto possible mechanisms by which palmitate mediates kinase signaling pathways, that could have implications on the efficacy of current drug therapies that target kinases.

APOL1 risk allele RNA contributes to renal toxicity by activating protein kinase R

“…Stable HEK293 cells bearing the APOL1-G0, G1, or G2 variant were mock-treated with the following: (a) 10 3 U per ml IFNα for 24–72 h and 100 μM palmitic acid (Sigma) for 2 h, or (b) 100 mM 103 U per ml IFNα for 24–72 h and calyculin A for 30 min. Palmitic acid binds PKR directly and inhibits the dsRNA binding to PKR 44 and therefore palmitic acid was used as negative control. Cells were irradiated with UV light at 200 mJ per cm 2 .…”

Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains

“…It is presently unknown if a similar process prevails in mammals, but recent studies hint at possible mechanisms: The cytosolic, catalytic, effector domain of yeast IRE1 can be directly activated by flavonols, which engage a hydrophobic pocket at the dimer interface stabilizing the active form of yeast IRE1 (16). It is unclear, however, if other hydrophobic ligands, such as lipids, can regulate IRE1's effector domain by a similar mechanism; and the kinase domain of mammalian PKR binds palmitate directly (17); however, it is unknown if the highly related kinase effector domain of PERK is also regulated by lipids.…”

Binding site multiplicity with fatty acid ligands: Implications for the regulation of PKR kinase autophosphorylation with palmitate

“…The experiments were performed as described previously . Briefly, WT and mutant plasmids were transformed into E. coli BL21(DE3) pLysS (Novagen) and the proteins were expressed in the cells with addition of IPTG (isopropyl β‐ d −1‐thiogalactopyranoside).…”

“…In previous work from our group, we found evidence that palmitate binds to the kinase domain of double‐stranded (ds) RNA‐dependent protein kinase (PKR), subsequently inhibiting the autophosphorylation of PKR at the two residues T446 and T451 . PKR is an interferon‐induced serine/threonine kinase that plays a variety of central roles in cellular functions such as cell growth, differentiation, signal transduction, tumorigenesis, and apoptosis .…”

“…In a first computational study, we complemented the experimental data with in silico docking results. Because of the nature of standard docking methods, we focused on finding specific binding sites in the vicinity of hypothesized regions where palmitate may interact and identified a possible site near the ATP‐binding site . We consequently hypothesized that palmitate may modulate PKR activity simply through competition with ATP binding.…”