Phosphorylation of the human papillomavirus type 1 E4 proteins in vivo and in vitro.
Following incubation of HPV 1-induced warts in the presence of [32P] phosphate several of the E4-encoded proteins were found to be radiolabeled. Two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 17K E4 polypeptides had incorporated [32P]phosphate whereas those of 16K were unlabeled. Purified E4 gene products were separated by ion exchange chromatography into a large number of different species, which were of similar size but of different charge due to varying extents of phosphorylated peptides have been isolated and identified. Phosphoserine and phosphothreonine were identified in all 16/17K E4 fractions but not phosphotyrosine. Both HPV 1 E4 16K and 17K fractions were phosphorylated in vitro by cAMP-dependent protein kinase but not by myosin light chain kinase or by phosphorylase kinase. Incubation with cAMP PK gave incorporation of approx. 0.5 mole phosphate/mol of protein indicating that the cAMP-dependent protein kinase site(s) was partially phosphorylated in vivo. This view was supported by the fact that species which were more heavily phosphorylated in vivo incorporated less phosphate after cAMP-dependent protein kinase phosphorylation. HPV 1 E4 was also phosphorylated at serine and threonine residues by a crude cytoplasmic extract prepared from cultured human keratinocytes and cultured human retinoblasts. These results are discussed in the light of the known effects of phosphorylation on the interactions of other keratinocyte-specific proteins.
Grand
R J
RJ
Department of Cancer Studies, University of Birmingham, Medical School, United Kingdom.
Doorbar
J
J
Smith
K J
KJ
Coneron
I
I
Gallimore
P H
PH
D002474:Cell-Free System; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007633:Keratins / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D027383:Papillomaviridae / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010750:Phosphoproteins / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*; D014860:Warts / Q000382:microbiology*
D010446:Peptide Fragments; D010750:Phosphoproteins; D014764:Viral Proteins; D007633:Keratins; D011494:Protein Kinases; D010764:Phosphorylase Kinase; D009219:Myosin-Light-Chain Kinase