title: Protein kinase C-mediated phosphorylation of HIV-I nef in human cell lines.
abstract: Stable human cell lines expressing the human immunodeficiency virus type I (HIV-I) Nef protein from inducible promoters were used to analyze the phosphorylation status of Nef in vivo. Nef phosphorylation in both HeLa and Jurkat cells was stimulated by phorbol ester treatment. Phosphoamino acid analysis revealed a predominance of phosphoserine with a small proportion of phosphothreonine. Treatment of cells with selective protein kinase inhibitors revealed that Nef phosphorylation was markedly reduced by bisindolylmaleimide, an inhibitor of protein kinase C, but was unaffected by inhibitors of mitogen-activated protein kinase kinase or cAMP-dependent kinase. These data implicate protein kinase C in Nef phosphorylation in vivo, and thus confirm and extend earlier in vitro data. Phosphorylation of a nonmyristoylated Nef mutant was impaired, suggesting that membrane targeting of Nef was required for phosphorylation. This was expected given that activated protein kinase C translocates from the cytosol to the plasma membrane. However, analysis of the subcellular localization of phosphorylated wild-type Nef revealed that both the cytosolic and membrane-associated pools of Nef were phosphorylated to an equivalent extent. Thus the significance of myristoylation for Nef function may be in influencing protein conformation, although these data could be explained by a transient and dynamic interaction between myristoylated Nef and the plasma membrane.
authors.affiliation: MRC Retrovirus Research Laboratory, Department of Veterinary Pathology, University of Glasgow, Glasgow G61 1QH, Scotland, United Kingdom.
authors.forename: K
authors.initials: K
authors.lastname: Coates
authors.forename: S J
authors.initials: SJ
authors.lastname: Cooke
authors.forename: D A
authors.initials: DA
authors.lastname: Mann
authors.forename: M P
authors.initials: MP
authors.lastname: Harris
mesh_terms: D003600:Cytosol / Q000378:metabolism; D015693:Gene Products, nef / Q000378:metabolism*; D015497:HIV-1; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D019169:Jurkat Cells; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus
chemical_list: D015693:Gene Products, nef; D009227:Myristic Acids; D010703:Phorbol Esters; D054311:nef Gene Products, Human Immunodeficiency Virus; D019814:Myristic Acid; D012694:Serine; D011493:Protein Kinase C