Vacuolar H-ATPase Mutants Transform Cells and Define a Binding Site for the Papillomavirus E5 Oncoprotein

doi:10.1074/jbc.270.12.6830

Vacuolar H-ATPase Mutants Transform Cells and Define a Binding Site for the Papillomavirus E5 Oncoprotein (*)

From the (1)Departments of Pathology and
(2)Obstetrics and Gynecology, Georgetown University Medical School, Washington, D. C. 20007

^§ To whom correspondence should be addressed:
Dept. of Pathology, Georgetown University Medical School, 3900 Reservoir Rd., NW, Washington, D. C., 20007.

Abstract

The 16K subunit of the vacuolar H-ATPase binds specifically to the bovine (BPV) and human (HPV) papillomavirus E5 oncoproteins, and it has been suggested that this interaction may contribute to cell transformation (Goldstein, D. J., and Schlegel, R.(1990) EMBO J. 9, 137-146; Goldstein, D. J., Finbow, [Abstract] M. E., Andresson, T., McLean, P., Smith, K., Bubb, V. J., and Schlegel, R.(1991) Nature 352, 347-349; Conrad, M., Bubb, V. J., [Medline] and Schlegel, R.(1993) J. Virol. 67, 6170-6178; [Abstract] Goldstein, D. J., Toyama, R., Schlegel, R., and Dhar, R. (1992) Virology 190, 889-893). We generated mutations within [Medline] the 16K protein to define binding domains for BPV-1 E5 as well as to characterize the role of 16K in cell transformation. 16K consists predominantly of 4 transmembrane (TM) domains. We showed that mutations within the TM4 domain severely inhibited E5 binding. More specifically, conversion of glutamic acid 143 to arginine within TM4 severely reduced 16K/E5 binding, suggesting that charged interactions facilitated efficient binding. This hypothesis was confirmed by demonstrating that binding to the defective 16K arginine mutant could be restored by complementary charge mutations in E5; conversion of E5 glutamine 17 to glutamic acid or aspartic acid enhanced interactions with the 16K arginine mutant. Surprisingly, mutants in TM4 not only bound poorly to wild-type E5 but were converted into an oncoprotein and induced anchorage-independent growth of NIH 3T3 cells. These data define glutamic acid 143 in the 16K TM4 domain and glutamine 17 within E5 as important contributors to E5/16K binding and suggest a role for the 16K protein in the regulation of cell proliferation.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PDGF

platelet-derived growth factor

PCR

polymerase chain reaction

DMEM

Dulbecco's modified Eagle's medium

PBS

phosphate-buffered saline

TM

transmembrane

MOPS

4-morpholinepropanesulfonic acid.
↵²T. Andresson, and R. Schlegel, unpublished results.
- Received September 23, 1994.
- Revision received December 8, 1994.