The role of Protein Kinase R in lipotoxicity
Author(s)
Advisor
Erbay, EbruDate
2013Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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Abstract
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.