Per-Arnt-Sim kinase regulates pancreatic duodenal homeobox-1 protein stability via phosphorylation of glycogen synthase kinase 3β in pancreatic β-cells

Background: The enzyme PASK regulates the expression of PDX-1 and insulin in pancreatic β-cells via unknown mechanisms. Results: PASK enhances PDX-1 protein stability via phosphorylation of GSK3β on Ser⁹. Conclusion: PASK regulates insulin gene expression at least in part through inactivation of GSK3β and stabilization of PDX-1 protein. Significance: We identified GSK3β as a novel target of PASK in the regulation of pancreatic β-cell function. In pancreaticβ-cells, glucose induces the binding of the transcription factor pancreatic duodenal homeobox-1 (PDX-1) to the insulin gene promoter to activate insulin gene transcription. At low glucose levels, glycogen synthase kinase 3β (GSK3β) is known to phosphorylate PDX-1 on C-terminal serine residues, which triggers PDX-1 proteasomal degradation. We previously showed that the serine/threonine Per-Arnt-Sim domain-containing kinase (PASK) regulates insulin gene transcription via PDX-1. However, the mechanisms underlying this regulation are unknown. In this study, we aimed to identify the role of PASK in the regulation of PDX-1 phosphorylation, protein expression, and stability in insulin-secreting cells and isolated rodent islets of Langerhans. We observed that glucose induces a decrease in overall PDX-1 serine phosphorylation and that overexpression of WT PASK mimics this effect. In vitro, PASK directly phosphorylates GSK3β on its inactivating phosphorylation site Ser⁹. Overexpression of a kinase-dead (KD), dominant negative version of PASK blocks glucose-induced Ser⁹ phosphorylation of GSK3β. Accordingly, GSK3βSer⁹ phosphorylation is reduced in islets from pask-null mice. Overexpression of WT PASK or KDGSK3β protects PDX-1 from degradation and results in increased PDX-1 protein abundance. Conversely, overexpression of KD PASK blocks glucose-induction of PDX-1 protein. We conclude that PASK phosphorylates and inactivates GSK3β, thereby preventing PDX-1 serine phosphorylation and alleviating GSK3β-mediated PDX-1 protein degradation in pancreatic β-cells.