It is well recognized that there are proteins that bind phospholipids at the membrane surface, with varying specificity, to play essential roles in cellular signaling and function. Several lipid binding domains on kinases were recently discovered, suggesting that direct binding of lipid molecules on kinases could be important in controlling kinase activity. Unsaturated fatty acids and fatty acid-CoA can bind to nuclear receptors to initiate signaling. However, palmitate, a saturated fatty acid, is best known for their ability to bind proteins that facilitate their transport into cells for oxidation. Recently we found that palmitate mediates signaling by regulating the enzymatic activity of diverse kinases, thus suggesting that palmitate binds not only proteins involved in transport but perhaps also those involve in signaling. To investigate how palmitate regulates the kinase activity at the biophysical and molecular levels, two kinases, PKR (double-stranded RNA-activated protein kinase) and an ER-localized transmembrane protein IRE1 (Inositol-reqiring enzyme 1), were selected as model kinases. Our group previously suggested that palmitate is involved in PKR-mediated apoptosis by reducing the autophosphorylation of PKR at the Thr451 residue. In contrast, palmitate increased both the kinase and RNase activity of IRE1, thereby inducing the downstream UPR (unfolded protein response) signaling. Using a fluorescence polarization-based palmitate binding assay, we showed that palmitate can directly bind to the kinase domain of both PKR and IRE1 to regulate their enzymatic activity. To further confirm that palmitate can indeed bind kinase proteins, we performed computational docking experiments, which suggested that palmitate may bind near the ATP-binding site on PKR and IRE1. Mutation binding studies confirmed the computational results, suggesting that several amino acid residues around the ATP-binding site are important for stabilizing the palmitate-kinase interaction. We further confirmed that palmitate interacts with other kinases, likely, due to the conserved ATP-binding site on the kinases. A better understanding of how palmitate differentially regulates the enzymatic activity of PKR and IRE1 would provide a more comprehensive view of the diverse roles palmitate plays as well as insight into designing more effective drugs to inhibit/activate kinase activities.
This preliminary program is subject to change.
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Molecular Mechanism by Which Palmitate Regulates Kinase Activity
It is well recognized that there are proteins that bind phospholipids at the membrane surface, with varying specificity, to play essential roles in cellular signaling and function. Several lipid binding domains on kinases were recently discovered, suggesting that direct binding of lipid molecules on kinases could be important in controlling kinase activity. Unsaturated fatty acids and fatty acid-CoA can bind to nuclear receptors to initiate signaling. However, palmitate, a saturated fatty acid, is best known for their ability to bind proteins that facilitate their transport into cells for oxidation. Recently we found that palmitate mediates signaling by regulating the enzymatic activity of diverse kinases, thus suggesting that palmitate binds not only proteins involved in transport but perhaps also those involve in signaling. To investigate how palmitate regulates the kinase activity at the biophysical and molecular levels, two kinases, PKR (double-stranded RNA-activated protein kinase) and an ER-localized transmembrane protein IRE1 (Inositol-reqiring enzyme 1), were selected as model kinases. Our group previously suggested that palmitate is involved in PKR-mediated apoptosis by reducing the autophosphorylation of PKR at the Thr451 residue. In contrast, palmitate increased both the kinase and RNase activity of IRE1, thereby inducing the downstream UPR (unfolded protein response) signaling. Using a fluorescence polarization-based palmitate binding assay, we showed that palmitate can directly bind to the kinase domain of both PKR and IRE1 to regulate their enzymatic activity. To further confirm that palmitate can indeed bind kinase proteins, we performed computational docking experiments, which suggested that palmitate may bind near the ATP-binding site on PKR and IRE1. Mutation binding studies confirmed the computational results, suggesting that several amino acid residues around the ATP-binding site are important for stabilizing the palmitate-kinase interaction. We further confirmed that palmitate interacts with other kinases, likely, due to the conserved ATP-binding site on the kinases. A better understanding of how palmitate differentially regulates the enzymatic activity of PKR and IRE1 would provide a more comprehensive view of the diverse roles palmitate plays as well as insight into designing more effective drugs to inhibit/activate kinase activities.