Function and Regulation of PKD2 and PKD2L1

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited genetic disorder, inducing cysts in kidney, liver and pancreas. It is caused by pathogenic mutations in the PKD1 or PKD2 gene encoding PKD1 or PKD2 respectively. Cellular abnormalities in ADPKD include cell over-proliferation and apoptosis. PKD2 is a Ca2+-permeable non-selective cation channel and functions as an anti-apoptotic and anti-cell proliferation protein. Endoplasmic reticulum (ER) stress induces the phosphorylation of eukaryotic initiation factor 2α (eIF2α) by pancreatic ER-resident eIF2α kinase (PERK), which inhibits global protein synthesis and up-regulates the translation of selected target proteins. In Chapter 2, using cultured mammalian cell lines, we found that PKD2 represses cell proliferation through promoting the eIF2α phosphorylation by PERK. PKD2 knock-down repressed ER stress-induced eIF2α phosphorylation, indicating that PKD2 facilitates the eIF2α phosphorylation by PERK. We also found that PKD2 interacts with PERK and eIF2α. Together, we demonstrate that PKD2 down-regulates cell proliferation by promoting PERK-mediated eIF2α phosphorylation, presumably through a physical interaction. In Chapter 3, using cultured mammalian cell lines, we showed that PKD2 protein expression is up-regulated by cellular stresses which all increase P-eIF2α without increasing PKD2 mRNA. Increasing P-eIF2α by salubrinal or by over-expression of eIF2α up-regulates the PKD2 level. Over-expression of Gadd34 or eIF2α knock-down suppressed ER stress-induced PKD2 up-regulation. We also found that ER stress increases binding of PKD2 mRNA molecules with ribosomes, and the regulation of PKD2 by P-eIF2α is mediated by the upstream open reading frame (uORF) in the PKD2 5’-untranslated region. Therefore, this study demonstrates that P-eIF2α translationally up-regulates PKD2 protein expression through the uORF of PKD2. PKD2L1 is a Ca2+-modulated non-selective cation channel. In Chapter 4, we found, using Xenopus oocyte, that the receptor for activated C kinase 1 (RACK1) binds with the PKD2L1 N-terminus and inhibits its channel activity. This was supported by our finding that over-expressed N-terminal fragment Met1–Pro45 acts as a blocking peptide and abolishes the inhibitory effect of RACK1. Therefore, we demonstrate that RACK1 binds with Met1–Pro45 of PKD2L1 to inhibit its channel activity. Therefore, our studies constitute valuable contributions to understanding the function and regulation of PKD2 and PKD2L1.