Transforming gene product of Rous sarcoma virus phosphorylates tyrosine

Amino Acid Substrate Specificity of Viral pp60^src.

The protein kinase activity associated with pp60^src phosphorylates the heavy chain of immunoglobulin when immunoprecipitates containing pp60^src are incubated with Mg²⁺ and ATP (2, 4, 6, 7). The linkage of the phosphate incorporated into the heavy chain was completely stable to treatment with 1 M HCl for 2 hr at 55°C. This ruled out the possibility that the phosphate was attached to the protein via histidine or as an acyl phosphate (24). The phosphate linkage was much more stable to alkali (60% resistance to 1 M KOH for 2 hr at 55°C) than expected for either phosphoserine or phosphothreonine (24). This suggested that the phosphate acceptor in the heavy chain was not threonine as reported (6) but rather some other amino acid. Hydrolysis of the isolated heavy chain with 6 M HCl for 2 hr at 110°C and two-dimensional separation of the products revealed that about 25% of the radioactivity was released as Tyr(P) which, in turn, comprised >95% of the radioactivity in phosphoamino acids (Fig. 1). Identification of the phosphorylated amino acid as Tyr(P) was corroborated by the comigration of the phosphate label with authentic Tyr(P) during electrophoresis at pH 3.5 (see Fig. 2) and also on chromatography in saturated ammonium sulfate/1 M sodium acetate/isopropanol, 40:9:1 (23) (data not shown). It seems unlikely that the occurrence of Tyr(P) in the heavy chain is an artifact of acid hydrolysis because Tyr(P) was released from the phosphorylated heavy chain by exhaustive digestion with Pronase (data not shown).

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Fig. 1.

Identification of phosphoamino acids in phosphorylated immunoglobulin heavy chain. Immunoglobulin heavy chain phosphorylated in an immunoprecipitate conzaining Schmidt–Ruppin RSV-D pp60^src was isolated and subjected to partial acid hydrolysis as described in Materials and Methods. A sample (2500 cpm) of the products was subjected to electrophoresis toward the anode at pH 1.9 and chromatography in isobutyric acid/ammonia. The origin is indicated with an arrow. Here and in subsequent figures, the positions of the stained marker phosphoamino acids are indicated by broken lines. The orthophosphate was run off the plate during electrophoresis. The minor spots are probably incompletely hydrolyzed phosphopeptides. Autoradiography was performed with a fluorescent screen for 12 hr. Thr(P), phosphothreonine; Ser(P), phosphoserine.

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Fig. 2.

Identification of amino acids phosphorylated by viral pp60^src and endogenous pp60^sarc. Immunoglobulin heavy chains phosphorylated by pp60^src from SR-RSV-D infected cells, pp60^src of SR-RSV-D synthesized in vitro (7), and pp60^sarc from chicken and human cells were isolated and subjected to partial acid hydrolysis. The hydrolysates were analyzed on a single plate by electrophoresis toward the anode at pH 3.5. The origin is indicated by arrows. Lanes, the exposure times (with a fluorescent screen), and the protein that phosphorylated the heavy chain were: A, pp60^src from infected cells, 25,000 cpm applied, 1 hr; B, pp60^src synthesized in vitro, 1400 cpm; 2 days; C, human pp60^sarc, 800 cpm, 10 hr; D, chicken pp60^sarc, 4000 cpm, 10 hr.

Strong support for the idea that the protein kinase activity associated with pp60^src isolated from transformed cells is due to pp60^src itself and not to a contaminating protein kinase is the observation that pp60^src isolated from a reticulocyte lysate after synthesis by in vitro translation has protein kinase activity (7, 25). We therefore examined which amino acid became phosphorylated when the protein kinase activity associated with pp60^src produced by in vitro translation of RSV virion RNA was assayed in the immunoprecipitate. Tyr(P) was again the principal phosphorylated amino acid (Fig. 2).

Amino Acid Substrate Specificity of Endogenous pp60^sarc.

Tryptic peptide analysis has shown that although the pp60^sarc and pp60^src polypeptides are closely related they are not identical (21). We examined whether the protein kinase activity associated with chicken and human pp60^sarcs shared with that associated with viral pp60^src the ability to phosphorylate tyrosine when assayed in the immunoprecipitate. In both cases the heavy chain of the phosphorylated immunoglobulin was found to contain predominantly Tyr(P) (Fig.2).

Phosphoamino Acids in pp60^src.

pp60^src contains two sites of phosphorylation: one in the NH₂-terminal half of the molecule (which has been shown clearly to be a phosphoserine), and one in the COOH-terminal half (26). The phosphoamino acid in the COOH-terminal half was identified as phosphothreonine on the basis of its electrophoretic mobility at pH 1.9 (26). Stimulated by indications that pp60^src could undergo autophosphorylation and knowing that Tyr(P) and phosphothreonine are difficult to resolve by electrophoresis at pH 1.9, we decided to reexamine the question of what phosphorylated amino acids were contained in pp60^src.

In addition to pp60^src, two other polypeptides, of 80,000 and 50,000 daltons, are found in immunoprecipitates made from ³²P-labeled transformed chicken cells with antitumor antiserum (Fig. 3) (3). Neither of these is related to pp60^src in amino acid sequence (ref. 3; see below). Both appear to be cellular proteins that coprecipitate with pp60^src because they are in physical association with some fraction of the pp60^src molecules in a transformed chicken cell (our unpublished observations; J. Brugge, personal communication). We analyzed the phosphotryptic peptides and the phosphorylated amino acids present in all three phosphoproteins.

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Fig. 3.

Analysis of immunoprecipitates from cells infected with RSV and labeled with [³⁵S]cysteine (lane A) and [³²P]orthophosphate (lane B). Infected cells were labeled for 15 hr at 41°C with [³⁵S]cysteine at 40 μCi/ml (New England Nuclear; 665 Ci/mmol) or [³²P]orthophosphate at 1 mCi/ml and immunoprecipitates were prepared by using antitumor antiserum preabsorbed with disrupted RSV virions. Electrophoretic separation was on a 15% NaDodSO₄/polyacrylamide gel. ³⁵S was detected by fluorography for 2 days; ³²P was detected by autoradiography with a fluorescent screen for 10 hr. Numbers are size in daltons. The 52,000-dalton polypeptide in lane A is related to pp60^src(3).

pp60^src contained two phosphorylated tryptic peptides (Fig. 4), designated here as α and β (9), as was first observed by Collett et al. (26). The 50,000-dalton protein contained two major and one minor phosphotryptic peptides; the 80,000-dalton protein contained an indeterminate number of phosphotryptic peptides that were poorly resolved. The phosphotryptic peptides of the three polypeptides were obviously unrelated. Hydrolysis of each protein and two-dimensional separation of the phosphorylated amino acids revealed that both pp60^src and the 50,000-dalton protein contained both Tyr(P) and phosphoserine and that the 80,000-dalton protein contained only phosphoserine (Fig. 4).

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Fig. 4.

Analysis of ³²P-labeled pp60^src and the 80,000- and 50,000-dalton proteins. ³²P-Labeled proteins were isolated from the preparation in Fig. 3 and part of each sample was subjected to tryptic digestion and analyzed. The remainder was subjected to partial acid hydrolysis and analyzed as in Fig. 1. The origins were all on the right and are indicated with arrows. All exposures were with a fluorescent screen. (A) pp60^src; tryptic digest; 6400 cpm applied; 5 hr exposure. (B) 80,000 protein; tryptic digest; 2000 cpm; 20 hr. (C) 50,000 protein; tryptic digest; 1500 cpm; 20 hr. (D) pp60^src; acid hydrolysate; 1300 cpm; 2 days. (E) 80,000 protein; acid hydrolysate; 900 cpm; 4 days. (F) 50,000 protein; acid hydrolysate; 500 cpm; 4 days.

We had previously determined (9) that pp60^src phosphopeptide α is derived from the 34,000-dalton NH₂-terminal fragment generated from pp60^src by partial proteolysis with Staphylococcus aureus V8 protease, whereas peptide β is derived from the COOH-terminal 24,000-dalton fragment (9). Partial acid hydrolysis of the purified peptides revealed that peptide α, as expected, contained phosphoserine and β contained Tyr(P). In the 50,000-dalton protein, peptide I contained Tyr(P) and peptide II contained phosphoserine (data not shown).

Tyr(P) in Normal and Transformed Cells.

Because Tyr(P) had not been detected in normal cells (28, 29) and because we now knew that at least two proteins that contained Tyr(P) were present in RSV-transformed cells, a comparison of the amounts of Tyr(P) in uninfected and RSV-transformed chicken cells seemed warranted. We isolated proteins from cells that had been labeled for 18 hr with [³²P]orthophosphate and carried out partial acid hydrolysis. RSV-transformed chicken cells contained 7- to 8-fold more Tyr(P) than did uninfected cells or cells infected with RSV carrying a deletion in the src gene (Fig. 5; Table 1).

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Fig. 5.

Analysis of phosphoamino acids of whole cells. ³²P-Labeled proteins were extracted and subjected to partial acid hydrolysis; 1.5 × 10⁶ cpm of each hydrolysate was resolved in two dimensions by electrophoresis toward the anode at pH 1.9 followed by electrophoresis toward the anode at pH 3.5. Autoradiography with a fluorescent screen was for 10 hr. The radioactive spots corresponding to the markers are indicated. The origins are designated by arrows. The material migrating toward the cathode at pH 1.9 corresponds to incompletely hydrolyzed phosphopeptides. (A) Uninfected chicken cells. (B) Chicken cells transformed with Schmidt–Ruppin RSV-A.

The hydrolysis conditions chosen here provide a compromise between the efficient release of phosphoamino acids and the competing hydrolysis of the phosphomonoester linkage. Under our conditions, approximately 50% of the ³²P was released as orthophosphate and 20–25% as phosphoamino acids. Because of this, we have expressed the radioactivity in the individual phosphoamino acids as a percentage of the sum of the radioactivity recovered as phosphoamino acids. These ratios will deviate from those in whole cells if the three phosphoamino acids are released or destroyed by acid at different rates. Although free Tyr(P) has been reported to be somewhat more acid labile than phosphoserine or phosphothreonine (27), this may not be true for Tyr(P) in a polypeptide. We have not found a major difference between the recovery of phosphoserine and Tyr(P) from pp60^src and the 50,000-dalton protein. Therefore, we think that we are not underestimating seriously the relative abundance of Tyr(P). Both the rate of release of any particular amino acid residue and the stability of the phosphomonoester bond will be affected by its polypeptide environment. However, there is no reason to assume a priori that the rate of release of Tyr(P) from proteins in transformed cells is significantly greater than the rate of release from proteins in normal cells. We believe therefore that we detect significantly more Tyr(P) in RSV-transformed cells because there is increased phosphorylation of tyrosine in these cells and not because the yield of Tyr(P) is greater from these cells.