Potential Role of Phosphorylation as a Regulator of Aspartyl-(asparaginyl)-β-hydroxylase: Relevance to Infiltrative Spread of Human Hepatocellular Carcinoma

Abundant expression of aspartyl-(asparaginyl)-β-hydroxylase (AAH) correlates with infiltrative growth of hepatocellular carcinoma (HCC). Herein, we examine the role of phosphorylation in relation to AAH's protein expression, hydroxylase activity, promotion of cell motility, and activation of Notch signaling in human Huh7 hepatoma cells. Predicted glycogen synthase kinase-3β (GSK-3β), protein kinase A (PKA), protein kinase C (PKC), and casein kinase 2 (CK2) phosphorylation sites encoded by human AAH cDNA were ablated by S/T→A site-directed mutagenesis using N-Myc-tagged constructs in which gene expression was controlled by a cytomegalovirus promoter. Functional consequences were assessed in transiently transfected Huh7 cells. Cells transfected with wildtype AAH had significantly increased AAH expression, catalytic activity, HES-1 expression, and directional motility relative to controls. Single phosphorylation site mutations in the C-terminus largely abrogated these effects and further inhibited catalytic activity relative to that in cells transfected with empty vector, whereas the effects of single point mutations within the N-terminus were more varied. In contrast, AAH cDNAs carrying multiple phosphorylation site mutations exhibited wildtype levels of AAH catalytic activity suggesting that the effects of AAH phosphorylation are complex and non-uniform. AAH expression and function can be modulated by direct phosphorylation of the protein. These findings suggest additional strategies for inhibiting infiltrative growth of HCC.