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, M. E., Andresson, T., McLean, P., Smith, K., Bubb, V. J., and Schlegel, R. (1991) Nature 352, 347-349; Conrad, M., Bubb, V. J., and Schlegel, R. (1993) J. Virol. 67, 6170-6178; Goldstein, D. J., Toyama, R., Schlegel, R., and Dhar, R. (1992) Virology 190, 889-893). We generated mutations within 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.
Highlights
From the Departments of tFathology and §Obstetrics and Gynecology, Georgetown University Medical School, Washington, D
The vacuolar proton ATPase is a large enzyme complex that is present in several intracellular membrane compartments and drives the unidirectional flux of protons from the cytoplasmic to lumenal sides of these organelles [5,6,7]
Activation and altered processing of epidermal growth factor receptors [21], as well as activation of PDGF1 receptors [22, 23], is a characteristic of cell transformation induced by the bovine papillomavirus type-l E5 oncoprotein
Summary
From the Departments of tFathology and §Obstetrics and Gynecology, Georgetown University Medical School, Washington, D. Mutants in TM4 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. E5 has recently been shown to exist in an in vivo complex with the PDGF receptor [22, 26, 27], the epidermal growth factor receptor [26], and the 16K subunit of vacuolar H+ -ATPase [1, 2, 22, 28] It appears that E5 may actively recruit 16K into a ternary complex with PDGF receptor, suggesting a role for 16K in signal transduction. Our studies indicate that the glutamic acid residue of 16K participates in the binding of E5, that its mutagenic alteration converts the 16K protein into a transforming protein, and that wild-type 16K can suppress the oncogenic activity of E5
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