Detection of phosphorylated forms of Moloney murine leukemia virus major capsid protein p30 by immunoprecipitation and two-dimensional gel electrophoresis.
We detected phosphorylation of the major Moloney murine leukemia virus (M-MuLV) capsid polypeptide, p30, by using 32Pi-labeled virions. This was observed both on two-dimensional polyacrylamide gels directly or on one-dimensional gels of viral lysates that had been immunoprecipitated with monospecific goat anti-p30 serum. The phosphorylation event had been difficult to detect because pp12 the major virion phosphoprotein incorporates almost all of the 32P label added to infected cells (Y. Yoshinaka and R. B. Luftig, Virology 116:181-195, 1982). When immunoprecipitates from M-MuLV lysates labeled with 32Pi were compared with those labeled with [35S]methionine, it was calculated that the degree of phosphorylation at the p30 domain of Pr65gag was only 0.22 to 0.54% relative to phosphorylation at the p12 domain. Two-dimensional gel electrophoresis of the 32P-labeled p30 immunoprecipitates showed that there were three phosphorylated p30 forms with isoelectric points (pIs) of 5.7, 5.8, and 6.0. These forms were generally more acidic than the [35S]methionine-labeled p30 forms, which had pIs of 6.0, 6.1, 6.3 (the major constituent with greater than 80% of the label), and 6.6. The predominant phosphoamino acid of the major phosphorylated p30 form (pI 5.8) was phosphoserine. Further, tryptic peptide analysis of this p30 form showed that only one peptide was predominantly phosphorylated. Based on a comparison of specific labeling of p30 tryptic peptides with [14C]serine, [35S]methionine, and 32Pi, we tentatively assigned the phosphorylation site to a 2.4-kilodalton NH2-terminal peptide containing triple tandem serines spanning the region from amino acids 4 to 24.
Ikuta
K
K
Department of Microbiology, Immunology and Parasitology, Louisiana State University Medical Center, New Orleans 70112-1393.
Luftig
R B
RB
D002213:Capsid / Q000276:immunology / Q000378:metabolism*; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000378:metabolism*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D010768:Phosphoserine / Q000378:metabolism
D010750:Phosphoproteins; D010768:Phosphoserine