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Updated: Apr 11, 2026

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method
Published on: October 23, 2018
Regulation of Aspartyl-(Asparaginyl)-β-Hydroxylase Protein Expression and Function by Phosphorylation in
Diana L Borgas1, Jin-Song Gao1, Ming Tong1
1The Liver Research Center, Divisions of Gastroenterology and Neuropathology, and Departments of Medicine, Pathology, Neurology, and Neurosurgery, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, RI, USA.
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.
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