Modulation of type M2 pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein.
Journal: 1999/March - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 9990017
Abstract:
We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic flux in tumor cells. The glycolytic intermediate fructose 1, 6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.
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Proc Natl Acad Sci U S A 96(4): 1291-1296

Modulation of type M<sub>2</sub> pyruvate kinase activity by the human papillomavirus type 16 E7 oncoprotein

Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Abt. F0301, INF 242, D-69120 Heidelberg, Germany; ScheBo Tech GmbH, Bahnhofstrasse 6, D-35435 Wettenberg, Germany; Institut für Biochemie and Endokrinologie, Fachbereich Veterinärmedizin, Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Germany; and International Center for Genetic Engineering and Biotechnology, Padriciano 99, Trieste, Italy
To whom reprint requests should be sent at present address: Institut f. Biomedizinische Alternsforschung der Oesterreichischen Akademie der Wissenschaften, Rennweg 10, A-6020 Innsbruck, Austria. e-mail: ta.ca.waeo@rreuD-nesnaJ.P.
Communicated by George Klein, Karolinska Institutet, Stockholm, Sweden
Communicated by George Klein, Karolinska Institutet, Stockholm, Sweden
Received 1998 Aug 20; Accepted 1998 Dec 7.

Abstract

We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic flux in tumor cells. The glycolytic intermediate fructose 1,6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.

Keywords: glycolysis, cell transformation, cancer, virus
Abstract

Unicellular organisms have a variety of sensing mechanisms to adapt the cell proliferation rate to variations in their environmental nutrient supply. Several gene products, like the ras or cdc kinase proteins, which are involved in nutrient sensing in yeast (1, 2), are conserved during the evolution of multicellular organisms, and in mammals, these gene products often are altered in tumors. Despite our knowledge about the protein machinery regulating cell proliferation increasing tremendously over the recent years, we are still at the beginning to understand how nutrients contribute to proliferation control in multicellular organisms. There is, however, quite good evidence that phosphometabolites derived from both glycolysis (for recent review, see ref. 3) and the pentose phosphate pathway (ref. 4 and references therein) provide some of the signals linking metabolic conditions to cell proliferation. The glycolytic phosphometabolites, which are necessary for the biosynthesis of nucleic acids, phospholipids, and complex carbohydrates, are up-regulated in the G1 phase of the cell cycle (for recent review, see ref. 3), and constant high levels of phosphometabolites have been detected by P NMR spectroscopy in rapidly proliferating tumor cells (5).

The key enzymes regulating the glycolytic phosphometabolite pools and glycolytic flux rate are hexokinase, 6-phosphofructo 1-kinase, and especially pyruvate kinase (PK) (6, 7). The activity of PK, the key enzyme controlling the exit of the glycolytic pathway, determines the relative amount of glucose that is channeled into synthetic processes or used for glycolytic energy production (6, 8). Proliferating mammalian cells express the M2 type isoenzyme of PK (M2-PK) (9), and expression of M2-PK is cell cycle regulated in proliferating rat thymocytes (10). M2-PK occurs in an active tetrameric and a less active dimeric form (8, 11, 12), and the switch between both forms, which is controlled by the glycolytic phosphometabolite fructose 1,6-bisphosphate (FBP) (8), regulates the glycolytic flux in tumor cells.

In humans, cancer of the cervix is linked to infection by human papillomaviruses (HPV) of the high-risk group, whereas viruses of the low-risk group are not associated with cancers in vivo and fail to transform human cells in vitro (13). The E7 oncogene of HPV-16, a high-risk HPV type, cooperates with the HPV-16 E6 gene to immortalize human keratinocytes (14) and with an activated ras gene to transform rodent fibroblasts (15). The transforming activity of E7 is sensitive to mutations in both the N-terminal (15) and C-terminal (16) domains. Although the E7 N terminus mediates binding to proteins of the retinoblastoma gene family and thereby contributes to deregulation of the cell cycle (reviewed in ref. 17), the function of the C-terminal domain remains to be disclosed.

Control and E7-expressing NIH3T3 subclones were used for gel filtration analysis of M2-PK as indicated; the results are given as the relative amounts of the tetrameric and dimeric forms of M2-PK (equation M3± SEM, n = 4). FBP levels were determined in perchloric acid extracts. equation M4± SEM, n = 5. n.s., not significant.

The concentration of various metabolites is given for mock-treated and dexamethasone-treated 14/2 cells. In each case, the significance of dependence on E7 expression was calculated as described (8, 12). n = 5. The term (pyruvate⋅ATP):(PEP⋅ADP) was calculated from the concentrations of the individual metabolites.

The consumption and production of various key nutrients and metabolites is given for mock-treated and dexamethasone-treated 14/2 cells. In each case, the significance of dependence on E7 expression is indicated by the P value. equation M8± SEM, n = 5. n.s., not significant.

Acknowledgments

We thank R. Brent for the WI-38 cDNA library, R. Tindle and J. Braspenning for antibodies to HPV-16 E7, and M. Tommasino for critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (S.M.: Ma 1760/1–2; P.J-D.: Ja 427/5–2), the European Union (Biomed 2), and the Land Hessen (S.M.: Hessisches Ministerium für Wissenschaft und Kunst, HSP III).

Acknowledgments

ABBREVIATIONS

PKpyruvate kinase
M2-PKtype M2 PK
HPVhuman papillomavirus
FBPfructose 1,6-bisphosphate
GSTglutathione S-transferase
PEPphosphoenolpyruvate
GAPDHglyceraldehyde 3-phosphate dehydrogenase
ABBREVIATIONS

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