Blood platelets express high levels of the pp 60 Ocsrcspeciflc tyrosine kinase activity

We have examined human and rabbit blood platelets for expression of pp6oc-,rc, the normal cellular homolog of the transforming protein of Rous sarcoma virus. pp6Oc8rc kinase activity was determined by an immunecomplex kinase assay that uses enolase as the substrate, and pp6srC protein levels were determined by an immunoblot assay. Lysates from platelets expressed high levels of pp60c-rc-specific kinase activity and pp6O4' protein compared to the levels found in other tissues. pp6Ocsrc was also found to be one of the major proteins phosphorylated in vitro in membranes isolated from platelets. Multiple protein species other than pp6Oc " were also phosphorylated on tyrosine in the membrane phosphorylation reactions, and phosphotyrosine represented -80% of the total phosphoamino acid residues phosphorylated in the membranes. These results indicate that tyrosine kinases represent the major protein phosphorylating enzymes detected in isolated platelet membranes. Although the association of tyrosine kinase activity with many viral oncogene products and cellular growth hormone receptors has suggested a role for these enzymes in the regulation of cell proliferation, these results indicate that the expression of high levels of tyrosine kinase activity is not exclusively associated with proliferating cells. The c-src gene, which is homologous to the transforming gene, v-src, of Rous sarcoma virus (RSV), is highly conserved across widely divergent vertebrate species (1, 2), and closely related genes have been identified in several invertebrate species (3-5). An essential role for the c-src gene product is suggested by this conservation; however, the function of this protein in normal cells is not known. Both the v-src and c-src genes code for phosphoproteins of Mr 60,000 that possess tyrosine-specific kinase activity (for review see ref. 6). The c-src protein is present in very low levels in cultured fibroblasts and phosphorylation of tyrosine residues represents a rare event in these cells, accounting for only 0.03% of the total phosphate incorporated into cellular proteins (7). Transformation by most retroviruses encoding tyrosine kinase transforming proteins causes a 7to 10-fold increase in the levels of phosphotyrosine in cellular phosphoproteins (7). This observation suggests that tyrosine phosphorylation of cellular proteins is involved in transformation, however, the specific substrates that mediate transformation have not been identified, and thus the role of tyrosine kinase activity in transformation is not known (for review see ref. 8). The role of the c-src tyrosine kinase activity in normal cells has been equally elusive, and no candidate substrates of the c-src kinase activity have been reported. Several investigators have reported the detection of high levels of tyrosine kinase activity in particulate fractions from normal hematopoietic cells (9-13). However, the kinases responsible for this activity have not been identified. We report here that pp60csrc levels are very high in blood platelets and peripheral blood lymphocytes (PBL). Since c-src protein has been found to associate with the plasma membrane (14), we have examined the phosphorylation of proteins in isolated membranes from platelets to determine whether high levels of kinase activity in total cell lysates correlated with elevated levels of tyrosine phosphorylation in membranes. Tyrosine-specific phosphorylation of proteins was found to represent the major kinase activity present in platelet membranes and pp60csrc was one of the most prominent tyrosine phosphorylated species. These results suggest that pp6o0-src might serve a specific function in the membranes of platelets and that expression oftyrosine kinase activity is not limited to proliferating cells. MATERIALS AND METHODS Tissues and Cells. Blood was taken from human volunteers using 10 mM EDTA as an anticoagulant. After centrifugation of the blood at 1500 x g for 8 min, the buffy coat was removed, layered onto Ficoll/Paque (Pharmacia) gradients and centrifuged as described (15). The lymphocyte layer was removed and washed with balanced salt solution [150 mM NaCl, 10 mM EDTA, 10 mM Tris HCl (pH 7.5)] to remove contaminating platelets. Platelets were retrieved from these washes and also from the blood plasma (after the removal of the buffy coat) by centrifugation at 2000 x g for 10 min. Erythrocytes were retrieved after the first centrifugation. The purity of different cell fractions was checked by direct microscopic examination. Rabbits were exsanguinated by cardiac puncture, and muscle, brain, spleen, thymus, lymph node, and bone marrow were excised. Lymphocytes, erythrocytes, and platelets were separated from the peripheral blood as described above. SRD 3T3 cells were derived from RSV (subgroup D) transformation of mouse BALB 3T3 cells. pMc-srcB cells were derived from NIH 3T3 cells by Johnson et al. (16) by transformation with a plasmid carrying the chicken' c-src gene. These cells express 20to 40-fold higher levels of the c-src protein than NIH 3T3 cells. Immunoprecipitations and in Vitro Kinase Reactions. pp6c-src was immunoprecipitated from lysates with 0.0i ,41 of monoclonal antibody (mAb) 327 (obtained from ascites fluid) by using formalin-fixed Staphylococcus aureus bacteria as described (17). In vitro kinase reactions were performed after immunoprecipitation of the pp60c-rc by the addition of 2 ,ug of acid-treated enolase (18), 5 ,Ci of [y-32P]ATP (1 Ci = 37 GBq), 1 ,uM ATP, 10mM Tris HCl (pH 7.2), and 5 mM MgCl2 for 15 min at room temperature. The reactions were stopped with the addition of NaDodSO4 sample buffer and then subjected to electrophoresis on 7.5% NaDodSO4/polyacrylamide gels as described by Laemmli (19). The dried gels were Abbreviations: RSV, Rous sarcoma virus; PBL, peripheral blood lymphocyte(s); mAb, monoclonal antibody. 852 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 83 (1986) 853 then exposed to XAR-5 film (Kodak) and enhanced with Cronex Lightning Plus intensifying screens. Immunoblot Assay. Immunoprecipitated pp60csrc was subjected to electrophoresis as above and the 60-kDa region of the gel was electroblotted onto nitrocellulose filter paper (20). The filter was then incubated with mAb 327 followed with '25I-labeled antimouse immunoglobulins. All antibody reactions were performed in 50 mM Tris HCl (pH 7.5), 150 mM NaCl, and 0.5% Tween 20. pp60v-src expressed in Escherichia coli (21) was used as a marker. Membrane Isolation and in Vitro Phosphorylation. Membrane preparation from human platelets and erythrocytes was carried out as described by Thom et al. (22), including 10 AM sodium orthovanadate (Na3VO4) in all buffers and employing an additional step of Dounce homogenization of the membrane pellet before layering onto the sucrose cushion. The use of nylon gauze was omitted. Each membrane protein preparation was incubated 15 min at room temperature with 50 ,Ci [y-32P]ATP, 5 ,M ATP, 5 mM MgCl2, 10 mM Hepes (pH 7.4), and 1o (vol/vol) glycerol. Reactions were stopped with NaDodSO4 sample buffer. Alkali treatment of gels was carried out as described by Cooper and Hunter (23). Phosphoamino Acid Analysis. Phosphoproteins were eluted from the gels by digestion with 50 ,g of trypsin in 1 ml of0.05 M ammonium carbonate, pH 8.5. After lyophilization, samples were hydrolyzed with 6 M HCl for 1.75 hr as described (24). Phosphoamino acid analysis was performed on Whatman Grade No. 3MM Chrom paper for 1.75 hr at 4000 V at pH 3.5. After autoradiography, the spots were excised and solubilized, and the amount of radioactivity in each spot was determined. In Vivo Labeling of Platelets. Approximately 109 platelets were isolated from freshly drawn human blood as described earlier and washed with platelet labeling medium (150 mM NaCl/15 mM Tris HCl, pH 7.4/5.5 mM glucose) (25). Cells were labeled with [32P]orthophosphate (ICN, 1 mCi/ml) in vivo in platelet labeling medium for 1 hr at 37°C. Cells were then pelleted, washed twice with phosphate-buffered saline [150 mM NaCl, 2.5 mM KCl, 1.5 mM KH2PO4, 10 mM Na2HPO4 (pH 7.4)], and cell lysates were prepared for pp60csrC immunoprecipitation as described above. Partial Proteolytic Peptide Analysis. In vitro and in vivo phosphorylated proteins were localized by autoradiography, excised from the gel, and subjected to electrophoresis on 12.5% NaDodSO4/polyacrylamide gels in the presence of 150 ng of V8 protease (Miles) following the procedure developed by Cleveland et al. (26).