T. Andresson et al., VACUOLAR H-ATPASE MUTANTS TRANSFORM CELLS AND DEFINE A BINDING-SITE FOR THE PAPILLOMAVIRUS E5 ONCOPROTEIN(), The Journal of biological chemistry, 270(12), 1995, pp. 6830-6837
The 16K subunit of the vacuolar H+-ATPase binds specifically to the bo
vine (BPV) and human (HPV) papillomavirus E5 oncoproteins, and it has
been suggested that this interaction may contribute to cell transforma
tion (Goldstein, D. J., and Schlegel, R. (1990) EMBO J. 9, 137-146; Go
ldstein, D. J., Finbow, M. E., Andresson, T., McLean, P., Smith, K., B
ubb, V. J., and Schlegel, R. (1991) Nature 352, 347-349; Conrad, M., B
ubb, V. J., and Schlegel, R. (1993) J. Virol. 67, 6170-6178; Goldstein
, D. J., Toyama, R., Schlegel, R., and Dhar, R. (1992) Virology 190, 8
89-893). We generated mutations within the 16K protein to define bindi
ng 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 inh
ibited E5 binding. More specifically, conversion of glutamic acid 143
to arginine within TM4 severely reduced 16K/E5 binding, suggesting tha
t charged interactions facilitated efficient binding. This hypothesis
was confirmed by demonstrating that binding to the defective 16K argin
ine mutant could be restored by complementary charge mutations in E5;
conversion of E5 glutamine 17 to glutamic acid or aspartic acid enhanc
ed 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 glutami
ne 17 within E5 as important contributors to E5/16K binding and sugges
t a role for the 16K protein in the regulation of cell proliferation.