Function of Nck adaptor proteins in the Unfolded Protein Response and glucose homeostasis in mice

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the Unfolded Protein Response (UPR), an intracellular signaling network aimed at alleviating stress through the coordinated inhibition of mRNA translation and the transcriptional induction of ER chaperone genes. A few years ago, we identified an interaction between Nck signaling adaptors and the beta-subunit of the eukaryotic initiation factor 2 (eIF2beta). We ascertained that overexpression of Nck1 antagonizes ER stress-mediated inhibition of translation by decreasing phosphorylation of eIF2beta on Ser51 by the PERK ser/thr protein kinase. In this thesis, I determined that Nck overexpression prevents the induction of ATF4 and CHOP genes associated with eIF2beta phosphorylation in ER stress conditions. Conversely, mouse embryonic fibroblasts (MEFs) genetically deficient of Nck1 and Nck2 genes (Nck1-/-Nck2-/-) show increased levels of ATF4, GADD34 and CHOP in response to ER stress, further supporting a role for Nck in regulating the translational arm of the UPR. Concurrently, I found that Nck adaptors localize at the ER and are part of an eIF2beta holophosphatase complex containing PP1c, a ser/thr phosphatase that dephosphorylates eIF2beta on Ser51. Unexpectedly, however, I observed that RNA interference (RNAi)-mediated Nck1 depletion results in decreased levels of eIF2beta phosphorylation and GADD34 induction in ER-stressed cells. Moreover, I uncovered a direct and SH2-mediated interaction between Nck and PERK. I provided strong evidence that PERK is phosphorylated on tyrosine residues in vitro and demonstrated that Tyr561 located in PERK juxtamembrane domain is virtually essential for the association with Nck adaptors. Therefore, I proposed that during the process of PERK activation, Tyr561 is phosphorylated and creates a binding site for Nck adaptors. I hypothesized that Nck is required for proper targeting and phosphorylation of eIF2beta by PERK. To demonstrate this in vivo, I took advantage that obesity in mice causes ER stress in specific tissues. My data validate my hypothesis as eIF2beta phosphorylation levels in liver and adipose tissue of obese mice were decreased by 45% in mice genetically inactivated of the nck1 gene. Characterization of glucose homeostasis in normal and obese Nck-/- and Nck2-/- knockout mice revealed marked alteration in glucose tolerance and insulin sensitivity compared to controls, thereby shedding light on a new physiological function for these signaling adaptors in controlling glycaemia in vivo.