PKN binds and phosphorylates human papillomavirus E6 oncoprotein.
The high risk human papillomaviruses (HPVs) are associated with carcinomas of cervix and other genital tumors. Previous studies have identified two viral oncoproteins E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high risk HPV E6 protein to immortalize human mammary epithelial cells has provided a single gene model to study the mechanisms of E6-induced oncogenic transformation. In recent years, it has become clear that in addition to E6-induced degradation of p53 tumor suppressor protein, other targets of E6 are required for mammary epithelial cells immortalization. Using the yeast two-hybrid system, we have identified a novel interaction of HPV16 E6 with protein kinase PKN, a fatty acid- and Rho small G protein-activated serine/threonine kinase with a catalytic domain highly homologous to protein kinase C. We demonstrate direct binding of high risk HPV E6 proteins to PKN in wheat-germ lysate in vitro and in 293T cells in vivo. Importantly, E6 proteins of high risk HPVs but not low risk HPVs were able to bind PKN. Furthermore, all the immortalization-competent and many immortalization-non-competent E6 mutants bind PKN. These data suggest that binding to PKN may be required but not sufficient for immortalizing normal mammary epithelial cells. Finally, we show that PKN phosphorylates E6, demonstrating for the first time that HPV E6 is a phosphoprotein. Our finding suggests a novel link between HPV E6 mediated oncogenesis and regulation of a well known phosphorylation cascade.
Gao
Q
Q
Department of Radiation Oncology, New England Medical Center, Boston, Massachusetts 02111, USA.
Kumar
A
A
Srinivasan
S
S
Singh
L
L
Mukai
H
H
Ono
Y
Y
Wazer
D E
DE
Band
V
V
D019943:Amino Acid Substitution; D001940:Breast / Q000166:cytology; D002460:Cell Line; D002461:Cell Line, Transformed; D002474:Cell-Free System; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D015723:Gene Library; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae; D014162:Transfection
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D012097:Repressor Proteins; C061570:protein kinase N; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C