Phosphorylation of HIV-1 gag proteins by protein kinase C.
We have demonstrated that the 17-kDa N-terminal matrix protein (p17gag) of HIV-1 Pr55gag is a substrate for protein kinase C (PKC). Phosphorylation of p17gag and Pr55gag was studied in vivo by infecting COS-7 cells with a recombinant vaccinia virus containing the HIV-1 gag-pol gene. Basal gag protein phosphorylation was inhibited up to 75% with the PKC inhibitor, H-7, and stimulated 3-4-fold with phorbol 12-myristate 13-acetate. In experiments using MCF-7 cell lines, p17gag and Pr55gag were dramatically phosphorylated only in clones with high PKC activity. Bacterially expressed and purified non-myristoylated and N-myristoylated p17gag were efficiently phosphorylated in a Ca2+ and phosphatidylserine-dependent manner by purified PKC. The N-myristoylated p17gag exhibited an apparent Km = 4 microM for PKC phosphorylation. Both in vitro and in vivo phosphorylated p17gag yielded identical V8 protease digestion phosphopeptide maps, indicating identical PKC phosphorylation sites. Phosphoamino acid analysis of the in vitro phosphorylated p17gag revealed only phosphoserine. These data are consistent with the identification of a highly conserved consensus PKC phosphorylation site motif in the HIV-1 gag protein at Ser111 and suggests that PKC phosphorylation plays an important role in gag protein function.
Burnette
B
B
Laboratory of Biological Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Yu
G
G
Felsted
R L
RL
D015683:Gene Products, gag / Q000378:metabolism*; D015488:HIV Antigens / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015488:HIV Antigens; D011498:Protein Precursors; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate