Phosphorylation Modulates Aspartyl-(Asparaginyl)-β Hydroxylase Protein Expression, Catalytic Activity and Migration in Human Immature Neuronal Cerebellar Cells

Abundant aspartyl-asparaginyl-β-hydroxylase (ASPH) expression supports robust neuronal migration during development, and reduced ASPH expression and function, as occur in fetal alcohol spectrum disorder, impair cerebellar neuron migration. ASPH mediates its effects on cell migration via hydroxylation-dependent activation of Notch signaling networks. Insulin and Insulin-like growth factor (IGF-1) stimulate ASPH mRNA transcription and enhance ASPH protein expression by inhibiting Glycogen Synthase Kinase-3β (GSK-3β). This study examines the role of direct GSK-3β phosphorylation as a modulator of ASPH protein expression and function in human cerebellar-derived PNET2 cells.

Inhibition of ASPH phosphorylation enhances ASPH protein expression, but attendant alterations in intra-cellular trafficking may govern the functional consequences in relation to neuronal migration, adhesion and Notch activated signaling.

Predicted phosphorylation sites encoded by human ASPH were ablated by S/T→A site-directed mutagenesis of an N-Myc-tagged wildtype (WT) cDNA regulated by a CMV promoter. Phenotypic and functional features were assessed in transiently transfected PNET2 cells.

Cells transfected with WT ASPH had increased ASPH protein expression, directional motility, Notch-1 and Jagged-1 expression, and catalytic activity relative to control. Although most single- and multi-point ASPH mutants also had increased ASPH protein expression, their effects on Notch and Jagged expression, directional motility and adhesion, and catalytic activity varied such that only a few of the cDNA constructs conferred functional advantages over WT. Immunofluorescence studies showed that ASPH phosphorylation site deletions can alter the subcellular distribution of ASPH and therefore its potential interactions with Notch/Jagged at the cell surface. Conclusions: Inhibition of ASPH phosphorylation enhances ASPH protein expression, but attendant alterations in intra-cellular trafficking may govern the functional consequences in relation to neuronal migration, adhesion and Notch activated signaling.