The E1^E4 protein of human papillomavirus type 16 (HPV16) causes cytokeratin reorganization in the middle and upper epithelial layers and is thought to contribute to multiple facets of the virus life cycle. Although little is known as to how HPV16 E1^E4 (16E1^E4) functions are controlled following the first expression of this protein, the finding that low-risk E1^E4 proteins can be phosphorylated in vivo suggests an important role for kinases. Here, we show that 16E1^E4 is phosphorylated by cyclin-dependent kinase 1 (CDK1) and CDK2, extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and PKC α, with CDK1/2 serine 32 and ERK threonine 57 phosphorylations representing the two primary events seen in cells in cycle. Interestingly, T57 phosphorylation was found to trigger a structural change in the 16E1^E4 protein that compacts the central fold region, leading to an increase in 16E1^E4 stability and overall abundance in the cell. When compared to wild-type 16E1^E4, a T57D phosphomimic was found to have greatly enhanced keratin-binding ability and an ability to modulate the binding of the unphosphorylated form, with keratin binding protecting the T57-phosphorylated form of 16E1^E4 from proteasomal degradation. In HPV16 genome-containing organotypic rafts, the T57-phosphorylated form was specifically detected in the intermediate cell layers, where productive infection occurs, suggesting that T57 phosphorylation may have a functional role at this stage of the viral life cycle. Interestingly, coexpression with 16E5 and ERK activation enhanced T57 phosphorylation, suggesting that E1^E4 and E5 may work together in vivo. Our data suggest a model in which the expression of 16E5 from the major E1^E4-E5 mRNA promotes T57 phosphorylation of E1^E4 and keratin binding, with dephosphorylation occurring following the switch to late poly(A) usage. Other forms of E1^E4, with alternative functional roles, may then increase in prevalence in the upper layers of the epithelium
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