Alternate AChE-R variants facilitate cellular metabolic activity and resistance to genotoxic stress through enolase and RACK1 interactions.
Tumorogenic transformation is a multifaceted cellular process involving combinatorial protein-protein interactions that modulate different cellular functions. Here, we report apparent involvement in two independent tumorogenic processes by distinct partner protein interactions of the stress-induced acetylcholinesterase AChE-R and N-AChE-R variants. Human testicular tumors showed elevated levels of N-terminally extended N-AChE-R compared with healthy tissue, indicating alternate promoter usage in the transformed cells. Two-hybrid screens demonstrate that the C-terminus common to both N-AChE-R and AChE-R interacts either with the glycolytic enzyme enolase or with the scaffold protein RACK1. In vitro, the AChE-R C-terminal peptide ARP elevated enolase's activity by 12%, suggesting physiological relevance for this interaction. Correspondingly, CHO cells expressing either human AChE-R or N-AChE-R but not AChE-S showed a 25% increase in cellular ATP levels, indicating metabolic significance for this upregulation of enolase activity. ATP levels could be reduced by AChE-targeted siRNA in CHO cells expressing AChE-R but not AChE-S, attributing this elevation to the AChE-R C-terminus. Additionally, transfected CHO cells expressing AChE-R but not N-AChE-R showed resistance to up to 60 microM of the common chemotherapeutic agent, cis-platinum, indicating AChE-R involvement in another molecular pathway. cis-Platinum elevates the expression of the apoptosis-regulator p53-like protein, p73, which is inactivated by interaction with the scaffold protein RACK1. In co-transfected cells, AChE-R competed with endogenous RACK1 for p73 interaction. Moreover, AChE-R-transfected CHO cells presented higher levels than control cells of the pro-apoptotic TAp73 as well as the anti-apoptotic dominant negative DeltaNp73 protein, leading to an overall decrease in the proportion of pro-apoptotic p73. Together, these findings are compatible with the hypothesis that in cancer cells, both AChE-R and N-AChE-R elevate cellular ATP levels and that AChE-R modifies p73 gene expression by facilitating two independent cellular pathways, thus conferring both a selective metabolic advantage and a genotoxic resistance.
Mor
Inbal
I
The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Bruck
Tal
T
Greenberg
David
D
Berson
Amit
A
Schreiber
Leticia
L
Grisaru
Dan
D
Soreq
Hermona
H
D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D009153:Mutagens / Q000633:toxicity*; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D018384:Oxidative Stress; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013737:Testis / Q000201:enzymology / Q000378:metabolism; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D009153:Mutagens; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins; C000606369:delta Np73 protein, human; D000110:Acetylcholinesterase; D019204:GTP-Binding Proteins; D010751:Phosphopyruvate Hydratase