Aspartyl (Asparaginyl) β-Hydroxylase (AAH) is a Type 2 transmembrane protein that has a predicted molecular mass of ~86 kD . AAH is a member of the α-ketoglutarate-dependent dioxygenase family of molecules [2, 3], and catalyzes the hydroxylation of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins [4, 5]. The consensus sequence for AAH hydroxylation is present in Notch, Jagged, and extracellular matrix molecules such as laminin and tenascin, which have demonstrated roles in cell motility or adhesion [4, 5]. The proposed AAH hydroxylation reaction uses molecular oxygen to form succinate, carbon dioxide, and 3-hydroxyaspartic acid . The catalytic domain resides within the carboxyl terminus and corresponding ~52 kD cleavage product of AAH .
The ~200 kB AAH gene encodes 3 proteins, AAH, Humbug, and Junctin [5, 8, 9], which are generated by alternative splicing and exon sharing . There are two AAH mRNA transcripts that encode identical proteins, which differ only in length of the 3'-untranslated region [1, 5]. Humbug is derived from the first 13 exons of the AAH gene, and lacks the C-terminal region that is responsible for catalytic activity in AAH [4, 5, 9, 10]. Junctin is the smallest of the 4 transcripts, and contains Exons 1A, 2, 3, 4A, and 5A of the AAH gene . Therefore, all 3 AAH-related 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.
AAH is abundantly expressed in a broad range of malignant neoplasms and transformed cells lines, including those of hepatic, biliary, breast, intestinal, pulmonary, pancreatic, and neural origin, whereas most normal mature tissues have relatively low levels of AAH [1, 11–14]. However, placenta is a notable exception in that motile and invasive trophoblasts express high levels of AAH [1, 15]. Initial studies established a convincing role for AAH in malignancy by demonstrating transformation of NIH3T3 cells that were stably transfected with the human AAH cDNA, and partial reversal of the transformed phenotype in cells that were transfected with a dominant negative AAH mutant that lacked catalytic activity . In situ studies demonstrated that the highest levels of AAH immunoreactivity were localized at the infiltrating margins of malignant neoplasms, rather than in their centers [1, 13, 14]. The peripheral distribution of prominent AAH immunoreactivity was not correlated with zonal differences in cell viability or proliferation , and correspondingly, proliferation states that were un-related to transformation, such as hepatocyte or bile duct regeneration, and pre-malignant conditions such as primary sclerosing cholangitis, were found to have low (normal) levels of AAH . Therefore, enhanced AAH expression is not correlated with cell proliferation per se. Instead, the findings of increased AAH immunoreactivity along the infiltrating margins of tumors and in metastatic foci [1, 13, 14], together with the high levels of AAH in trophoblastic cells, which are normally motile and invasive, led us to hypothesize that AAH has a functional role in cell motility [14, 16].
Humbug is also abundantly expressed in malignant neoplasms of diverse histogeneses, including carcinomas of hepatic, biliary, colonic, and pulmonary origin, as well as various transformed cell lines [4, 5, 9, 17]. Humbug can bind calcium, and over-expression of Humbug results in increased intracellular levels of calcium due to its release from intracellular stores [9, 10]. Thus far, Junctin expression has been characterized in skeletal and cardiac muscle [5, 9], but not in malignant neoplastic cells. Like Humbug, Junctin has a role in regulating calcium release from the sarcoplasmic reticulum [4, 5, 9, 18, 19]. In addition, Junctin can physically associate with the ryanodine receptor complex, and may have an important role in stabilizing the complex [4, 5, 9, 18, 19]. Compared with AAH, less is known about the possible function and expression of Humbug and Junctin in relation to malignancy, tumor progression, and motility.
In previous studies, a role for AAH in relation to motility was demonstrated in part by the significantly reduced levels of both AAH and directional motility observed in cells that were transfected with antisense oligodeoxynucleotides that targeted the 5'end of AAH mRNA [14, 16]. However, the molecular characterization of Humbug, its structural relationship to AAH, high-level expression in malignant neoplasms, and the realization that the antisense oligodeoxynucleotides used in those experiments would have also inhibited Humbug, prompted us to further examine the expression and regulation of AAH, Humbug, and Junctin, and determine if Humbug has a role in cell motility. The strategy for examining the regulation and function of AAH and related genes evolved from a series of independent experiments demonstrating that: 1) IGF-1 promotes migration of immature neuroblastic and neuroblastoma cells [20–22]; 2) IGF-I can stimulate AAH expression [17, 23]; and 3) cyclin dependent kinase-5 (Cdk-5) is an important regulator of neuronal migration in the developing central nervous system (CNS) [24–28]. The present work characterizes IGF-I regulation and downstream signaling pathways through Erk MAPK, PI3 Kinase-Akt, and Cdk-5 that modulate AAH, Humbug, and Junctin expression and directional motility in SH-Sy5y human neuroblastoma cells.