Nck1-Dependent Modulation of PERK Activation and Insulin Levels in Pancreatic β Cells

Abstract

PKR-like endoplasmic reticulum (ER) kinase (PERK) is an ER transmembrane serine/threonine protein kinase that mediates the unfolded protein response (UPR) activated under ER stress conditions and pancreatic β cell homeostasis. Inactivating mutations in the PERK gene result in Wolcott-Rallison syndrome, an early infancy form of insulin-dependent diabetes in humans. Our goal is to elucidate the role of the Src-homology domain-containing adaptor protein, Nck1, in PERK regulation and β cell function. We demonstrate that Nck-/- mouse embryonic fibroblasts display higher levels of PERK activation and signalling in normal and ER stress conditions compared to Nck+/+ cells. Using recombinant proteins, we show that Nck1, through its SH2 domain, binds PERK and identifies phosphorylated PERK Tyr561 (pY561) as the Nck1 binding site. We demonstrate PERK phosphorylation on Y561 in pervanadate (PV)-treated MIN6 cells and observe that PERK phosphorylation on Y561 delays PERK activation and signalling in response to ER stress. In agreement, we found that the PERK (Y561F) mutant is catalytically more active than the wild-type PERK in vitro and in cultured cells. Interestingly, PV-treated MIN6 cells expressing shNck1 undergo faster pY561 dephosphorylation upon ER stress compared to control cells. Finally, we report that shNck1 MIN6 cells contain more insulin than control cells. Similarly, isolated pancreatic islets from Nck1-/- mice display increased insulin content compared to Nck1+/+ islets. Altogether, these data provide strong evidence that Nck1 limits PERK activation by promoting PERK phosphorylation on Y561 and this impacts pancreatic β cell insulin biogenesis by limiting physiological PERK activity. Supported by CDA and CIHR