Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes.

Abstract

Phosphorylation of the src gene product pp6Ov-src was studied in plasma menmbrane fractions prepared from Rous sarcoma virus-transformed vole cells. Upon addition of [.y-32P]ATP to isolated membrane vesicles, phosphate was incorporated into a 60,000-dalton polypeptide identified as pp60vsrc. In the presence of vanadate, pp60v-src phosphorylation was stimulated ca. 30-fold. At low concentrations of ATP (1 ,uM), this reaction occurred almost exclusively on the carboxy-terminal 26,000-dalton region of pp6v-src. However, at higher ATP concentrations (100 ,uM), additional sites of phosphorylation were evident in the amino-terminal 34,000-dalton region. Kinetic analyses, performed under conditions in which ATP hydrolysis was minimal, revealed that the phosphorylation reaction at the carboxy terminus exhibited a higher Vmax and a lower Km for ATP than those occurring at the amino terminus. In addition, the amino-terminal region of pp6Ov-sc was more rapidly dephosphorylated than the carboxy-terminal region. These results indicate that interaction of pp6Ov-src with the plasma membrane may limit the extent of amino-terminal phosphorylation by lowering the rate of the reaction and the affinity for the substrate while increasing its susceptibility to phosphoprotein phosphatases. We suggest that the use of transformed-cell membrane preparations provides a model system for studying the possible regulatory roles of phosphorylation and dephosphorylation on pp60v-src function.Cellular transformation by Rous sarcoma virus (RSV) is mediated by the expression of the viral src gene product pp60v-src (21). This 60,000-dalton polypeptide (2, 14, 31, 32) exhibits protein kinase activity specific for tyrosine residues and is itself a phosphoprotein (7,8,15,18,22,28,29). Studies of pp6Ov-src immunoprecipitated from RSV-infected cells have identified a serine residue in amino-terminal region of the molecule (Ser-17) and a tyrosine residue in the carboxy-terminal domain (Tyr-416) as the major sites of phosphorylation (6,36,39). Phosphorylation of Ser-17 is mediated by a cyclic AMP-dependent protein kinase, whereas the carboxy-terminal tyrosine phosphorylation occurs through a cyclic AMP-independent reaction (6). The phosphorylation of Tyr-416 probably represents pp6Ov-src autophophorylation, since it occurs when purified preparations of pp60vsrc are phosphorylated in vitro (20,30). Interestingly, upon addition of increasing concentrations of ATP, additional sites of tyrosine phosphorylation in the amino-terminal region of pp60v-src have been observed (1,9,20). Since these sites had not been identified in in vivo-labeled cells, their physiological significance was unclear.Biochemical and cytological investigations have established that a significant fraction of the pp6Ov-src in transformed cells is associated with the plasma membrane (11,23,25,26,33) through a domain near the amino terminus (12, 24). There is a strong correlation between membrane association of pp6Ov-src and expression of cellular transformation parameters (10,17,24). Initi...

Reconstitution of the Rous sarcoma virus transforming protein pp60v-src into phospholipid vesicles.

“…Radiolabeled fractions were adjusted to RIPA buffer (10 mM Tris, 150 mM NaCl, 1% sodium DOC, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS]) at 4°C, immunoprecipitated with anti-p60 antiserum (34), and analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) as previously described (33 (34), and the amount of radiolabel incorporated into autophosphorylated pp6Ov-src in the immune complex kinase reaction (12) Phosphorylated samples employed in domain mapping were first adjusted to 1% SDS and then to RIPA before immunoprecipitation with anti-amino-or anti-carboxy-specific pp6O-src antibodies (34). Addition of 1% SDS served to enhance recognition of pp60v-src proteolytic fragments by the domain-specific antibodies.…”

In vivo effect of sodium orthovanadate on pp60c-src kinase.

“…This hyperphosphorylated form of the molecule has increased tyrosine kinase activity both in vitro (4,9) and in vivo (4), in contrast to our findings with pp6Oc-rc. Tyrosine residues in the amino-terminal half of the molecule are apparently autophosphorylated (43), suggesting that pp60v-src can enhance its own enzymatic activity. It is not known whether tyrosine 527 of pp6Ocsrc is autophosphorylated or phosphorylated by another tyrosine kinase.…”

“…Incubation of partially purified preparations of pp6Ov-src with Mg2+ and ATP (13,40), lysis of RSVtransformed cells in the presence of Mg2+ and ATP (9), incubation of intact RSV-transformed cells with vanadate (4,9), and incubation of plasma membrane fractions from RSV-transformed cells with Mg2+, ATP, and vanadate (43) facilitate the identification of hyperphosphorylated forms of pp6&v-src (at tyrosine residues in the amino-terminal half of the molecule) that are more enzymatically active. Recently, Courtneidge (17) showed that lysis of untransformed rodent cells in the presence of vanadate facilitates the recovery of a population of pp60'csrc that is hyperphosphorylated at the major tyrosine residue in the carboxyl-terminal half of the molecule (presumably tyrosine 527 [14]).…”

Evidence that Human Cytomegalovirus Assembly Protein Shares Antigenic Sites with an Uninfected Cell Membrane Protein

“…A plasma membrane-enriched membrane fraction was obtained from uninfected HEFs (1 x 108 cells/experiment) following the procedure described by Resh & Erikson (1985). Briefly, cells were washed with STE buffer (150 mM-NaCI, 50 mi-Tris-HCl, 1 mM-EDTA), removed from the plastic surface by vigorous pipetting with cold STE buffer, collected by centrifugation and resuspended in hypotonic buffer.…”

“…Identification of the IgM-reactive antigen in uninfected cell membranes using a group of 14 MA-IgM-positive and a group of 10 MA-IgM-negative sera. A plasma membrane-enriched membrane fraction was prepared from uninfected HEFs by the procedure described by Resh & Erikson (1985). Membrane proteins were denatured in the presence of SDS and 2-mercaptoethanol, and then separated by SDS-PAGE (9~ acrylamide gel) and transferred to nitrocellulose.…”