Abstract
BackgroundAspartyl (asparaginyl)-β-hydroxylase (AAH) hydroxylates Asp and Asn residues within EGF-like domains of Notch and Jagged, which mediate cell motility and differentiation. This study examines the expression, regulation and function of AAH, and its related transcripts, Humbug and Junctin, which lack catalytic domains, using SH-Sy5y neuroblastoma cells.ResultsReal time quantitative RT-PCR demonstrated 8- or 9-fold higher levels of Humbug than AAH and Junctin, and lower levels of all 3 transcripts in normal human brains compared with neuroblastic tumor cells. AAH and Humbug expression were significantly increased in response to insulin and IGF-I stimulation, and these effects were associated with increased directional motility. However, over-expression of AAH and not Humbug significantly increased motility. Treatment with chemical inhibitors of Akt, Erk MAPK, or cyclin-dependent kinase 5 (Cdk-5) significantly reduced IGF-I stimulated AAH and Humbug expression and motility relative to vehicle-treated control cells. In addition, significantly increased AAH and Humbug expression and directional motility were observed in cells co-transfected with Cdk-5 plus its p35 or p25 regulatory partner. Further studies demonstrated that activated Cdk-5 mediated its stimulatory effects on AAH through Erk MAPK and PI3 kinase.ConclusionAAH and Humbug are over-expressed in SH-Sy5y neuroblastoma cells, and their mRNAs are regulated by insulin/IGF-1 signaling through Erk MAPK, PI3 kinase-Akt, and Cdk-5, which are known mediators of cell migration. Although AAH and Humbug share regulatory signaling pathways, AAH and not Humbug mediates directional motility in SH-Sy5y neuroblastoma cells.
Highlights
Aspartyl-β-hydroxylase (AAH) hydroxylates Asp and Asn residues within EGF-like domains of Notch and Jagged, which mediate cell motility and differentiation
Profiles of AAH, Humbug, and Junctin Expression in SHSy5y Neuroblastoma Cells Real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) studies were used to examine the expression profiles and levels of AAH, Humbug, and Junctin mRNA in SH-Sy5y cells
In PNET1 cells, Humbug and Junctin mRNA transcripts were abundant and both were more highly expressed than AAH (P < 0.001) (Figure 1)
Summary
Aspartyl (asparaginyl)-β-hydroxylase (AAH) hydroxylates Asp and Asn residues within EGF-like domains of Notch and Jagged, which mediate cell motility and differentiation. This study examines the expression, regulation and function of AAH, and its related transcripts, Humbug and Junctin, which lack catalytic domains, using SH-Sy5y neuroblastoma cells. The ~200 kB AAH gene encodes 3 proteins, AAH, Humbug, and Junctin [5,8,9], which are generated by alternative splicing and exon sharing [5]. All 3 AAHrelated proteins share common N-terminal exons that encode a trans-membrane domain in addition to a portion of the cytoplasmic domain [4,9] but differ in the length and function of the C-terminus
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