Regulation of Aspartyl-(Asparaginyl)-β-Hydroxylase Protein Expression and Function by Phosphorylation in Hepatocellular Carcinoma Cells.

Abstract

Background: Asparaginyl-β-hydroxylase (AAH) promotes cell adhesion, migration, and invasion via Notch activation. AAH's expression is up-regulated by insulin/IGF signaling through PI3K-Akt, but its protein is independently regulated by GSK-3β. The multiple predicted GSK-3β phosphorylation sites suggest post-translational mechanisms may regulate AAH protein expression.

Methods: Human Huh7 hepatoma cells were transfected with recombinant plasmids that expressed full-length N-terminal Myc-tagged (N-Myc-AAH) or C-terminal HA-tagged (C-HA-AAH) cDNA. Effects of IGF-1 on AAH protein were examined using cellular ELISAs, immunofluorescence, and Western blotting. Effects of kinase inhibitors relevant to AAH's predicted phosphorylation sites were studied.

Results: IGF-1 stimulation increased AAH protein expression and shifted AAH's localization from the perinuclear zone to the cell periphery, including podocytes. Subsequently, Notch-1 intracellular domain was translocated to the nucleus, which is critical for Notch- modulated gene expression. Besides GSK-3β, inhibition of PKC, PKA, and CK2, which could potentially phosphorylate AAH, increased IGF-1 stimulated AAH protein. Finally, insulin and LiCl independently and additively increased long-term AAH protein expression.

Conclusion: Insulin/IGF-1 stimulation of AAH and Notch are enhanced by inhibiting kinases that could phosphorylate AAH protein. Targeted manipulation of AAH's phosphorylation state may have therapeutic value for reducing AAH-Notch activation and attendant infiltrative growth of hepatocellular carcinomas.