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.

Keywords:
Aspartyl-(asparaginyl)-β-hydroxylase, Cell motility, Hepatocellular carcinoma, Notch, Phosphorylation
1.
El-Serag HB, Rudolph KL: Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2557-2576.
2.
Mittal S, El-Serag HB: Epidemiology of hepatocellular carcinoma: consider the population. J Clin Gastroenterol 2013;47(Suppl):S2-S6.
3.
Bressac B, Kew M, Wands J, Ozturk M: Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature 1991;350:429-431.
4.
Galle PR, Krammer PH: CD95-induced apoptosis in human liver disease. Semin Liver Dis 1998;18:141-151.
5.
Wright WE, Shay JW: The two-stage mechanism controlling cellular senescence and immortalization. Exp Gerontol 1992;27:383-389.
6.
Chang L, Karin M: Mammalian MAP kinase signalling cascades. Nature 2001;410:37-40.
7.
Desbois-Mouthon C, Cacheux W, Blivet-Van Eggelpoël MJ, Barbu V, Fartoux L, Poupon R, Housset C, Rosmorduc O: Impact of IGF-1R/EGFR cross-talks on hepatoma cell sensitivity to gefitinib. Int J Cancer 2006;119:2557-2566.
8.
Tovar V, Alsinet C, Villanueva A, Hoshida Y, Chiang DY, Solé M, Thung S, Moyano S, Toffanin S, Mínguez B, Cabellos L, Peix J, Schwartz M, Mazzaferro V, Bruix J, Llovet JM: IGF activation in a molecular subclass of hepatocellular carcinoma and pre-clinical efficacy of IGF-1R blockage. J Hepatol 2010;52:550-559.
9.
Lee JW, Soung YH, Kim SY, Lee HW, Park WS, Nam SW, Kim SH, Lee JY, Yoo NJ, Lee SH: PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene 2005;24:1477-1480.
10.
Hu TH, Huang CC, Lin PR, Chang HW, Ger LP, Lin YW, Changchien CS, Lee CM, Tai MH: Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer 2003;97:1929-1940.
11.
Cantarini MC, de la Monte SM, Pang M, Tong M, D'Errico A, Trevisani F, Wands JR: Aspartyl-asparagyl beta hydroxylase over-expression in human hepatoma is linked to activation of insulin-like growth factor and notch signaling mechanisms. Hepatology 2006;44:446-457.
12.
de la Monte SM, Tamaki S, Cantarini MC, Ince N, Wiedmann M, Carter JJ, Lahousse SA, Califano S, Maeda T, Ueno T, D'Errico A, Trevisani F, Wands JR: Aspartyl-(asparaginyl)-beta-hydroxylase regulates hepatocellular carcinoma invasiveness. J Hepatol 2006;44:971-983.
13.
Maeda T, Sepe P, Lahousse S, Tamaki S, Enjoji M, Wands JR, de la Monte SM: Antisense oligodeoxynucleotides directed against aspartyl (asparaginyl) beta-hydroxylase suppress migration of cholangiocarcinoma cells. J Hepatol 2003;38:615-622.
14.
Sepe PS, Lahousse SA, Gemelli B, Chang H, Maeda T, Wands JR, de la Monte SM: Role of the aspartyl-asparaginyl-beta-hydroxylase gene in neuroblastoma cell motility. Lab Invest 2002;82:881-891.
15.
Ince N, de la Monte SM, Wands JR: Overexpression of human aspartyl (asparaginyl) β-hydroxylase is associated with malignant transformation. Cancer Res 2000;60:1261-1266.
16.
Xian ZH, Zhang SH, Cong WM, Yan HX, Wang K, Wu MC: Expression of aspartyl beta-hydroxylase and its clinicopathological significance in hepatocellular carcinoma. Mod Pathol 2006;19:280-286.
17.
Wang K, Liu J, Yan ZL, Li J, Shi LH, Cong WM, Xia Y, Zou QF, Xi T, Shen F, Wang HY, Wu MC: Overexpression of aspartyl-(asparaginyl)-beta-hydroxylase in hepatocellular carcinoma is associated with worse surgical outcome. Hepatology 2010;52:164-173.
18.
Lavaissiere L, Jia S, Nishiyama M, de la Monte S, Stern AM, Wands JR, Friedman PA: Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma. J Clin Invest 1996;98:1313-1323.
19.
Jia S, VanDusen WJ, Diehl RE, Kohl NE, Dixon RA, Elliston KO, Stern AM, Friedman PA: cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase. J Biol Chem 1992;267:14322-14327.
20.
Wang QP, VanDusen WJ, Petroski CJ, Garsky VM, Stern AM, Friedman PA: Bovine liver aspartyl beta-hydroxylase. Purification and characterization. J Biol Chem 1991;266:14004-14010.
21.
Lee JH: Overexpression of humbug promotes malignant progression in human gastric cancer cells. Oncol Rep 2008;19:795-800.
22.
Treves S, Feriotto G, Moccagatta L, Gambari R, Zorzato F: Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane. J Biol Chem 2000;275:39555-39568.
23.
Dinchuk JE, Henderson NL, Burn TC, Huber R, Ho SP, Link J, O'Neil KT, Focht RJ, Scully MS, Hollis JM, Hollis GF, Friedman PA: Aspartyl beta-hydroxylase (Asph) and an evolutionarily conserved isoform of Asph missing the catalytic domain share exons with junctin. J Biol Chem 2000;275:39543-39554.
24.
McGinnis K, Ku GM, VanDusen WJ, Fu J, Garsky V, Stern AM, Friedman PA: Site-directed mutagenesis of residues in a conserved region of bovine aspartyl (asparaginyl) beta-hydroxylase: evidence that histidine 675 has a role in binding Fe2+. Biochemistry 1996;35:3957-3962.
25.
Gundogan F, Elwood G, Longato L, Tong M, Feijoo A, Carlson RI, Wands JR, de la Monte SM: Impaired placentation in fetal alcohol syndrome. Placenta 2008;29:148-157.
26.
Silbermann E, Moskal P, Bowling N, Tong M, de la Monte SM: Role of aspartyl-(asparaginyl)-β-hydroxylase mediated notch signaling in cerebellar development and function. Behav Brain Funct 2010;6:68.
27.
Greenwald I, Kovall R: Notch signaling: genetics and structure. WormBook 2013;17:1-28.
28.
Weng AP, Aster JC: Multiple niches for Notch in cancer: context is everything. Curr Opin Genet Dev 2004;14:48-54.
29.
Boyault S, Rickman DS, de Reyniès A, Balabaud C, Rebouissou S, Jeannot E, Hérault A, Saric J, Belghiti J, Franco D, Bioulac-Sage P, Laurent-Puig P, Zucman-Rossi J: Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets. Hepatology 2007;45:42-52.
30.
Dürr R, Caselmann WH: Carcinogenesis of primary liver malignancies. Langenbecks Arch Surg 2000;385:154-161.
31.
Lahousse SA, Carter JJ, Xu XJ, Wands JR, de la Monte SM: Differential growth factor regulation of aspartyl-(asparaginyl)-beta-hydroxylase family genes in SH-Sy5y human neuroblastoma cells. BMC Cell Biol 2006;7:41.
32.
Carter JJ, Tong M, Silbermann E, Lahousse SA, Ding FF, Longato L, Roper N, Wands JR, de la Monte SM: Ethanol impaired neuronal migration is associated with reduced aspartyl-asparaginyl-beta-hydroxylase expression. Acta Neuropathol 2008;116:303-315.
33.
de la Monte SM, Tong M, Carlson RI, Carter JJ, Longato L, Silbermann E, Wands JR: Ethanol inhibition of aspartyl-asparaginyl-beta-hydroxylase in fetal alcohol spectrum disorder: potential link to the impairments in central nervous system neuronal migration. Alcohol 2009;43:225-240.
34.
Yuan ZL, Guan YJ, Chatterjee D, Chin YE: Stat3 dimerization regulated by reversible acetylation of a single lysine residue. Science 2005;307:269-273.
35.
Lizarazo D, Zabala V, Tong M, de la Monte SM: Myriocin-ceramide inhibitor modulates insulin/IGF and ER stress pathways in experimental alcohol-related steatohepatitis. J Biochem Pharmacol Res 2013;1:148-160.
36.
Zhang JH, Qi RC, Chen T, Chung TD, Stern AM, Hollis GF, Copeland RA, Oldenburg KR: Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase. Anal Biochem 1999;271:137-142.
37.
de la Monte SM, Lahousse SA, Carter J, Wands JR: ATP luminescence-based motility-invasion assay. Biotechniques 2002;33:98-100, 102, 104 passim.
38.
Gundogan F, Elwood G, Greco D, Rubin LP, Pinar H, Carlson RI, Wands JR, de la Monte SM: Role of aspartyl-(asparaginyl) beta-hydroxylase in placental implantation: Relevance to early pregnancy loss. Hum Pathol 2007;38:50-59.
39.
Lawton M, Tong M, Gundogan F, Wands JR, de la Monte SM: Aspartyl-(asparaginyl) β-hydroxylase, hypoxia-inducible factor-α and Notch cross-talk in regulating neuronal motility. Oxid Med Cell Longev 2010;3:347-356.
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