In vitro phosphorylation of murine leukemia virus proteins: Specific phosphorylation of Pr65 gag, the precursor to the internal core antigen

Abstract

In vitro phosphorylation of Nonidet P-40 disrupted murine leukemia virus with [γ- 32P]ATP showed that Pr65 gag, which is present as a relatively minor component (14% of total Coomassie blue-stained virus proteins), was the major (80% labeling) phosphorylated species. A small amount of 32P label (i.e., 3–9%) was associated with proteins whose mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was the same as Pr40 gag, Pr27 gag, p12, and p10. In contrast, long-term (20 hr) in vivo phosphorylation with [ 32P]orthophosphate showed that p12 was the most highly phosphorylated viral protein species. In neither case did we find any label associated with the major virion proteins, p30, p15, or gp70. These results suggest that phosphorylation is specific for certain viral proteins and, further, that if Pr65 gag is packaged as an uncleaved polyprotein into virions it remains relatively unphosphorylated. We have also found that the kinase activity which phosphorylates Pr65 gag in vitro is tightly associated with immature core subparticles and can phosphorylate the exogenous substrate phosvitin. The same or an additional kinase activity can also be detected in the soluble fraction of detergent-disrupted virus using endogenous viral proteins or phosvitin as substrate. Based on an analysis of the tryptic peptide patterns of pPr65 gag phosphorylated in vitro and in vivo, it appears that the in vitro phosphorylation of Pr65 gag occurs at serine residues which are identical to or similar to those found for pPr65 gag in vivo, i.e., on pp12, as well as at another site. This other site is also probably on p12 since in vitro phosphorylated pPr65 gag when cleaved in vitro by the murine Pr65 gag proteolytic factor, produces as its initial product a phosphorylated vand that has the molecular weight of pPr27 gag and the antigenic determinants of p15 and p12.