Endoplasmic reticulum (ER) is a central organelle for cellular homeostasis through its myriad of functions including protein and lipid biosynthesis, protein folding and secretion and calcium homeostasis. When protein folding or secretion is disrupted, ER elicits a unique signaling response initiated at its membranes called the unfolded protein response (UPR). UPR attempts to restore cellular homeostasis and survival via reducing unfolded protein levels, however, if this cannot be achieved or the stress is prolonged the UPR could lead to apoptosis. Three specific ER membrane proteins, inositol-requiring enzyme-1 (IRE1), Protein Kinase R-resemble like ER kinase (PERK) and activating transcription factor 6 (ATF6), act as ER stress sensors and initiate distinct but interlaced signaling pathways to restore ER homeostasis. Recently, studies demonstrated that over nutrition, especially high amount of saturated fatty acids or cholesterol in the circulation, leads to the induction of ER stress in metabolic tissues, resulting in the activation of UPR signaling pathways. Furthermore, ER stress was shown to play a causal role in the pathogenesis of metabolic diseases such as obesity, insulin resistance, type 2 diabetes and atherosclerosis. Interferon inducible double strand RNA activated protein kinase R (PKR) is also known to be activated during ER stress. Recent studies showed it can be activated by lipids during ER stress in cells and in metabolic tissues of obese mice. Genetic ablation or inhbition of PKR enhances systemic glucose homeostasis and insulin sensitivity in obesity in rodent models. However, it is not known how PKR becomes activated by overnutrition or by ER stress. In fact, many of the specific cellular components and molecular mechanisms in lipid induced cellular stress or death, namely lipotoxicity, is not completely understood. PKR is one of the serine/threonine kinase that is known to be activated during lipid induced ER stress, but only a few specific downstream substrates are known and these fall short of explaining PKR’s role in lipotoxicity in chronic metabolic disease pathogenesis. PKR also plays a crucial role in activation of inflammasomes through interacting with the inflammasome components. There is a gap in our knowledge regarding PKR’s specific molecular actions in nutrient-induced inflammation and metabolism in chronic metabolic diseases. In this thesis study, my major goal was to develop specific tools to modulate PKR’s activity and search for its specific substrates in lipotoxicity and its role in mediating lipid-induced ER stress response. For this purpose, I developed a novel chemical-genetic approach to specifically modify PKR’s kinase activity during ER stress. In this approach, the bulky sidechain of a gatekeeper amino acid (such as methionine) in the ATP binding cavity of PKR has been altered to a smaller side-chain amino acid (such as glycine) in order to slightly enlarge the cavity to accommodate bulky ATP analogs (activating or inhibiting). This mutant of the PKR has been named the analog sensitive kinase allele (ASKA) of PKR and was shown to utilize normal ATP as well as the bulky ATP analogs in kinase reactions. Furthermore, I demonstrated the specific inhibition of PKR kinase with the inhibitory, bulky ATP analogs such as4-Amino-1-tert-butyl-3-(1’-naphthyl)pyrazolo[3,4-d]pyrimidine (NAPP1) or 4-Amino- 1-tert-butyl-3-(1’-naphthylmethyl)pyrazolo[3,4-d]pyrimidine (1-NMPP1). In order to move one step closer to identification of potential PKR substrates, I also optimized kinase reactions for immunoprecipitated PKR ASKA mutant and visualized several potential downstream substrates in my initial experiments Finally, I studied a unique relationship between two ER stress related kinases IRE1 and PKR in lipid induced ER stress conditions. I observed specific inhibition of IRE1’s endoribonuclease activity with an inhibitor, but not its kinase activity, completely blocks PKR activation by lipids. These findings strongly support hat IRE1’s RNAse activity is necessary for PKR kinase activation by lipids. This function of IRE1 RNAse domain is novel and unsuspected. The future goals of this research should be directed to discovering the RNA mediators of IRE1-PKR coupling and understanding their role in mediating the inflammatory and metabolic pathologies associated with chronic metabolic diseases. In conclusion, in my thesis study, I developed novel chemical-genetic approach to specifically modify PKR kinase activity that could be useful in discovering novel PKR substrates. Based on the preliminary findings in this thesis, PKR appears to have many unidentified substrates regulated during lipid induced ER stress. Furthermore, using the chemical-genetic PKR as a tool as well as several other approaches I demonstrated the existence of a unique, functional relationship between IRE1 and PKR in lipotoxicity. In addition, the results in my thesis shows that IRE1’s endoribonuclease activity is required and sufficient for PKR kinase activation by lipids. These findings and tools developed during my studies can be further utilized for analyzing the specific role of PKR in lipotoxicity, which is important for the health consequences of metabolic diseases.