The most abundant viral transcript in human papillomavirus (HPV) 11 - infected xenograft tissue has been shown to encode the E1/\E4 protein. The function of E1/\E4 protein has not been determined. Several potential phosphorylation sequence motifs were identified in the HPV 11 El/\E4 protein, including potential sites of phosphorylation by mitogen-activated protein kinase (MAPK), cAMP-dependent protein kinase (PKA), casein kinase II, and protein kinase C. To test phosphorylation of the HPV 11 E1/\E4 protein, a soluble maltose binding protein (MBP) fusion was produced in Escherichia coli. Only MAPK and PKA phosphorylated the E1/\E4 protein. Phosphoamino acid analysis showed that one or more threonine residues were phosphorylated by MAPK, and both serine and threonine residues were phosphorylated by PKA. MBP- E1/\E4 mutant proteins were designed to delineate the E1/\E4 phosphoacceptor residues. MAPK was shown to phosphorylate E1/\E4 on threonine 53 within a MAPK consensus phorphorylation sequence motif. PKA was shown to phosphorylate E1/\E4 at two residues: threonine 36 within a consensus motif and serine 44 within a variant of the PKA consensus phosphorylation sequence motif. HPV 11- infected human genital tissue grown as a xenograft in an athymic mouse was labeled with [32P]orthophosphate. Phosphoamino acid analysis of E1/\E4 protein immunoprecipitated from 32P-labeled tissue revealed that both serine and threonine residues were phosphorylated. Analysis by liquid chromatography-mass spectrophotometry was consistent with phosphorylation of residues within the PKA and MAPK phosphorylation sequence motifs. Expression of E1/\E4 protein containing phosphorylation substitution mutations showed that the PKA mutant did not differ from wild-type E1/\E4 protein in intracellular distribution. In contrast, the MAPK mutant did not localize exclusively to the cytoplasm nor did it colocalize with wild-type E1/\E4 protein. We conclude that HPV 11 E1/\E4 protein is phosphorylated in vitro and in vivo. Our data are consistent with phosphorylation of HPV 11 E1/\E4 protein by MAPK and PKA in infected tissue. (C) 2000 Academic Press.
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