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Phosphorylation of HIV-1 gag proteins by protein kinase C
B Burnette1, G Yu, R L Felsted
1Laboratory of Biological Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
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.
Insights
Protein kinase C (PKC) phosphorylates the HIV-1 p17gag matrix protein. This phosphorylation, occurring at Ser111, is crucial for gag protein function and can be modulated by PKC activity.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- The 17-kDa N-terminal matrix protein (p17gag) is a component of the HIV-1 Pr55gag precursor polyprotein.
- Protein kinase C (PKC) is a family of enzymes involved in various cellular signaling pathways.
Purpose of the Study:
- To investigate whether the HIV-1 p17gag protein is a substrate for PKC.
- To identify the role and mechanism of PKC-mediated phosphorylation of HIV-1 gag proteins.
Main Methods:
- Infection of COS-7 cells with a recombinant vaccinia virus expressing HIV-1 gag-pol.
- Treatment with PKC inhibitor H-7 and phorbol ester PMA.
- Phosphorylation assays using bacterially expressed p17gag and purified PKC.
- V8 protease digestion and phosphoamino acid analysis.
Main Results:
- Basal gag protein phosphorylation was significantly inhibited by H-7 and stimulated by PMA.
- p17gag and Pr55gag showed dramatic phosphorylation in MCF-7 cell clones with high PKC activity.
- Bacterially expressed p17gag was efficiently phosphorylated by purified PKC in a Ca2+- and phosphatidylserine-dependent manner.
- Identical phosphopeptide maps and phosphoamino acid analysis (phosphoserine) indicated conserved PKC phosphorylation sites.
Conclusions:
- HIV-1 p17gag is a substrate for PKC, with phosphorylation occurring at a conserved Ser111 site.
- PKC-mediated phosphorylation plays a significant role in the function of HIV-1 gag proteins.