Abstract 3009: Mst1 Induces the Unfolded Protein Response Through Direct Phosphorylation of PERK
Mammalian sterile 20-like kinase1 (Mst1) is a serine/threonine kinase which plays an important role in mediating apoptosis in response to ischemia/reperfusion and during cardiac remodeling in the heart. We have shown previously that Mst1 negatively regulates compensatory cardiac hypertrophy through activation of the unfolded protein response (UPR) and that PERK, a sensor of endoplasmic reticulum (ER) stress, is required for Mst1-induced suppression of cardiac hypertrophy. It remains to be elucidated, however, how Mst1 regulates PERK in cardiac myocytes. Co-immunoprecipitation and immunocytochemical analyses demonstrated that Mst1 physically interacts with PERK at the ER in cardiac myocytes. Recombinant Mst1 phosphorylated GST-PERK-T980A, a kinase-inactive mutant, suggesting that PERK is directly phosphorylated by Mst1, not autophosphorylated. Using tandem mass spectrometry analysis, we identified two Serines (862 and 1105) and two Threonines (944, and 1109) located in the cytoplasmic region of PERK, that. are phosphorylated by Mst1. Although overexpression of wild type PERK enhanced Mst1-induced phosphorylation of eIF2α, that of PERK (S862A) attenuated eIF2α phosphorylation. Overexpression of PERK (S862A) reversed Mst1-induced inhibition of phenylephrine-induced hypertrophy in cardiac myocytes, suggesting that phosphorylation of S862 plays an important role in mediating Mst1-induced activation of PERK and the UPR, and subsequent suppression of cardiac hypertrophy. Co-immunoprecipitation assays showed that, although PERK dissociates from BiP, an ER chaperone regulating dimerization of PERK, in the presence of Mst1, PERK (S862A) failed to dissociate from BiP even in the presence of Mst1. On the other hand, a PERK mutant which is constitutively active due to an inability to associate with BiP was not inhibited by Mst1. These results suggest that Mst1 phosphorylates PERK at S862, thereby inducing dissociation of BiP, thereby causing dimerization/activation of PERK. In conclusion, Mst1 induces the UPR through direct phosphorylation of PERK, which mediates suppression of compensatory hypertrophy by Mst1.
This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).