Phosphorylation of the human papillomavirus type 16 E1^E4 protein
Human papillomaviruses (HPVs) are linked to over 99 % of cervical cancers and HPV16 is found in approximately half of all cases. The 16E1AE4 protein is abundantly expressed in productive infections but is downregulated or lost in cancers. The protein colocalises with and induces the collapse of cytokeratin networks, and in doing so, may facilitate virus release from cells. 16E1AE4 also causes cell cycle arrest by binding to and affecting the function of CDKl/cyclin B, which may enhance the virus' ability to use cellular DNA replication machinery to amplify its viral genome. 16E1AE4 has also been shown to colocalise with CDK2/cyclin A. Phosphorylation of 1E1AE4 and 11E1AE4 has been shown in vivo. In this study, phosphorylation of 16E1AE4 was investigated. When 16E1AE4 is expressed in cell culture, a slower-migrating, phosphorylated form is seen after SDS-PAGE. 16E1AE4 is phosphorylated by CDK1, CDK2, p42MAPK, PKA and PKCct in vitro. p42MAPK in vitro phosphorylation results in a slower-migrating form of 16E1AE4 similar to that seen in cell culture. Mass spectrometry and mutagenesis was used to map some in vitro phosphorylation sites. Mutagenesis, isoelectric focusing and the use of kinase inhibitors, suggest that when 16E1AE4 is expressed in cell culture, serine 32 is phosphorylated by CDK1/2 and threonine 57 by p42/p44MAPK. Threonine 57 phosphorylation causes the observed change in migration with SDS-PAGE. A bacterially-expressed 16E1AE4 with threonine 57 mutated to aspartic acid to mimic phosphorylation, was shown to bind keratin more strongly than the unphosphorylated, wild-type 16E1AE4. Phosphorylation may therefore have a role in regulating 16E1AE4 activity in vivo. Coexpression of the HPV16 E5 protein was found to enhance threonine 57 phosphorylation by activating p42/44MAPK, suggesting another function for E5. By studying the regulation and functions of HPV proteins, the virus life cycle will be better understood, expanding the opportunity to develop new diagnostic and therapeutic tools.