PROTEIN-KINASE C-MEDIATED PHOSPHORYLATION OF HIV-I NEF IN HUMAN CELL-LINES

Stable human cell lines expressing the human immunodeficiency virus ty pe I (HIV-I) Nef protein from inducible promoters were used to analyze the phosphorylation status of Nef in vivo. Nef phosphorylation in bot h HeLa and Jurkat cells was stimulated by phorbol ester treatment, Pho sphoamino acid analysis revealed a predominance of phosphoserine with a small proportion of phosphothreonine. Treatment of cells with select ive protein kinase inhibitors revealed that Nef phosphorylation was ma rkedly reduced by bisindolylmaleimide, an inhibitor of protein kinase C, but was unaffected by inhibitors of mitogen-activated protein kinas e 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 i mpaired, suggesting that membrane targeting of Nef was required for ph osphorylation, This was expected given that activated protein kinase C translocates from the cytosol to the plasma membrane. However, analys is of the subcellular localization of phosphorylated wild-type Nef rev ealed that both the cytosolic and membrane-associated pools of Nef wer e phosphorylated to an equivalent extent, Thus the significance of myr istoylation for Nef function may be in influencing protein conformatio n, although these data could be explained by a transient and dynamic i nteraction between myristoylated Nef and the plasma membrane.