Characterization of a Mutant Pancreatic eIF-2α Kinase, PEK, and Co-localization with Somatostatin in Islet Delta Cells

Abstract

Phosphorylation of eukaryotic translation initiation factor-2␣ (eIF-2␣) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2␣ kinases including mammalian double-stranded RNA-dependent eIF-2␣ kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2␣ kinases including phosphorylation of eIF-2␣ in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2␣ kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

Proteasome Inhibition Alters Glucose-stimulated (Pro)insulin Secretion and Turnover in Pancreatic β-Cells

“…The gene encoding CHOP/GADD153 is induced by ER stress through a signaling pathway that involves activation of PERK and phosphorylation of the translation initiation factor eIF2␣. Identified as the eIF2 kinase enriched in pancreatic cells (59), PERK is an ER transmembrane protein whose activity is repressed by GRP78/BiP. When misfolded proteins accumulate in the ER, GRP78/BiP dissociates from PERK, resulting in the activation of this kinase, which then phosphorylates eIF2␣, a master regulator of translation (60 -62).…”

Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls

“…It is intriguing that both Ire1␣ and PERK are most highly expressed in pancreatic tissue Shi et al 1999). The primary function of the pancreas is to respond to blood glucose levels in order to maintain glucose homeostasis through regulation of absorption, gluconeogenesis, and glucose utilization.…”

Chapter Twenty Identification and Characterization of Endoplasmic Reticulum Stress‐Induced Apoptosis In Vivo

“…We have raised murine anti-human IRE1α antibodies that can detect endogenous levels of IRE1α in murine liver tissue by IP-Western blot analysis Tirasophon et al, 2000). In addition, a polyclonal rabbit anti-PERK PITK-289 antibody (kindly provided by Dr. Yuguang Shi, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN) (Shi et al, 1999) or a commercial antibody (Cell Signaling) can be used to detect endogenous PERK activation though IP-Western blot assay.…”