Proc. NatL Acad. Sci. USA Vol. 79, pp. 282-286, January 1982 Biochemistry Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line (phosphotyrosine/pp6O c) JOHN E. CASNELLIE*, MARIETTA L. HARRISONtt, LINDA J. PIKE*, KARL ERIK HELLSTROMtt, AND EDWIN G. KREBS* *Howard Hughes Medical Institute Laboratories, Department of Pharmacology, University of Washington, Seattle, Washington 98195; tDivision ofTumor Immunology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104; and MDepartment of Pathology, University of Washington, Seattle, Washington 98195 Contributed by Edwin G. Krebs, September 14, 1981 ABSTRACT The particulate fraction from a lymphoma cell In this paper, the synthesis ofpeptides related to the tyrosine line, LSTRA, was found to contain an apparent high level of ty- phosphorylation site in pp6Osrc and their phosphorylation by a rosine protein kinase activity. When this fraction was incubated detergent extract ofa particulate fraction from a lymphoma cell with [Y-32PIATP in thepresence of10mM MnCl2, hydrolyzed, and line are reported. assayed, 70-80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins MATERIALS AND METHODS showed that a large portion ofthe 32p was in a single protein with a molecular weight ofapproximately 58,000. The phosphorylated Materials. All serum and cell culture supplements were pur- residue in this protein was identified as phosphotyrosine. Deter- chased from GIBCO except sodium pyruvate (Microbiological gent extracts of the particulate fraction from LSTRA cells con- Associates, Bethesda, MD) and potassium penicillin G (Eli tained both the Mr 58,000 protein and the enzyme responsible for Lilly). Phosphothreonine, phosphoserine, Triton X-100, and its phosphorylation. These extracts were found to catalyze the Hepes were purchased from Sigma. Phosphotyrosine was syn- phosphorylation of the tyrosine residue in the synthetic peptide, thesized according to Rothberg et aL (12). The t-butyloxycar- Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to bonyl derivatives ofamino acids were obtained from Peninsula the sequence around the tyrosine that is phosphorylated in pp60SC; Laboratories (San Carlos, CA)orVegaBiochemicals (Tucson, AZ). the Km for the peptide in this reaction was 5 mM. High-perfor- Cell Culture. Rous sarcoma virus-transformed mouse fibro- mance liquid chromatography was used to assay for this phos- blasts were kindly provided by L. Rohrschneider (Fred Hutch- phorylation. A second peptide was synthesized that contained two inson Cancer Research Center, Seattle, WA). They were main- additional arginine residues whose presence permitted the phos- tained in minimal essential medium supplemented with 10% phorylation ofthe peptide to be measured by a simple assay using heat-inactivated fetal calf serum, potassium penicillin G (100 phosphocellulose paper. The Km for this peptide was 3-4 mM, in- units/ml), and streptomycin (100 mg/ml). The Moloney lym- dicating that the presence of the additional arginine residues did phoma cell line LSTRA was originally induced in BALB/c mice not alter the apparent affinity of the kinase for the peptide. by the Moloney murine leukemia virus (13) and maintained in culture as described (14). Cells were washed once with phos- Recently, a class of protein kinases has been identified that phate-buffered saline (pH 7.2) before homogenization. phosphorylates tyrosine residues in proteins (1-4). This type of Preparation of Cell Fractions and Extracts. Approximately reaction appears to be relatively uncommon because in com- 4 X 107 cells were suspended in 4 ml ofcold 5 mM Hepes, pH parison with the amounts of phosphoserine and phosphothre- 7.4/1 mM MgCl2/5 mM 2-mercaptoethanol. After a 10-min onine, the amount of phosphotyrosine recovered from most incubation on ice, the cells were broken open with 20 strokes cells is quite low (1, 5). Protein kinases that phosphorylate ty- ofthe A pestle in a Wheaton Dounce homogenizer. Sucrose was rosine residues were initially found associated with transform- then added to a final concentration of0.25 M. The broken cell ing proteins ofRNA tumor viruses. Further work demonstrated suspensions were centrifuged for 30 sec at 1300 X g at 2°C in that the protein kinase associated with the epidermal growth order to remove nuclei. The supernatants were removed and factor receptor phosphorylated tyrosine residues (6). centrifuged at 300,000 x g for 45 min at 2°C. The pellets were When the amino acid sequence around the site of phos- resuspended in 0.3 ml of25 mM Hepes, pH 7.4/5 mM 2-mer- phorylation of the tyrosine residue in the Rous sarcoma virus captoethanol. To prepare detergent extracts of this fraction, became known it was of Triton.X-100 was added to a final concentration of 1% and the transforming protein, pp603'C, (7-9), material was centrifuged at 4°C for 5 min in a Microfuge. The interest to synthesize a peptide corresponding to this region to supernatant was collected and the pellet was discarded. determine if it would serve as a substrate. Synthetic peptides Peptide Synthesis. Solid-phase peptide synthesis was carried have been of great value in elucidating the specificity deter- out with the Beckman 990B automated instrument as described minants of substrates for the cyclic AMP-dependent protein (15). Glutamine and asparagine residues were protected with kinase (10, 11), and it was hoped that they might also be ben- the xanthyl group, and tyrosine was protected as the dichlo- eficial for similar studies involving tyrosine protein kinases. robenzyl ether. Peptides were cleaved from the resin and de- Moreover, a synthetic peptide would be more readily available protected by incubating the resin in 75% HF/25% anisole for than pp6Osrc itselfand might serve as a useful substrate for other 30 min at 0°C (16). The peptide Ile-Glu-Asp-Asn-Glu-Tyr-Thr- enzymological studies. Abbreviations: SRC-peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg- The publication costs ofthis article were defrayed in part by page charge Gln-Gly; R-R-SRC-peptide, Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala- payment. This article must therefore be hereby marked "advertise- Ala-Arg-Gly; HPLC, high-performance liquid chromatography; p58, nent" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Mr 58,000 protein. 282 Downloaded by guest on September 28, 2021 Biochemistry: Casnellie et aL Proc. Natl. Acad. Sci. USA 79 (1982) 283 Ala-Arg-Gln-Gly (SRC-peptide) was purified on DEAE-Seph- Gel Electrophoresis. Samples were analyzed on 6-12% gra- adex with a linear gradient of0. 1-1.0 M ammonium acetate (pH dient gels as described by Maizel (19). Molecular weights were 7.0). The peptide Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala- determined by using phosphorylase b (92,000), bovine serum Ala-Arg-Gly (R-R-SRC-peptide) was purified on SP-Sephadex albumin (68,000), ovalbumin (44,000), and chymotrypsinogen with a gradient of 0.1 M ammonium acetate at pH 3.2 to 0.75 (25,000) as standards. For quantitative experiments, the gel M ammonium acetate at pH 9.2. The peptides were desalted band was cut out and dissolved by incubation at 600C with 30% by chromatography in 30% acetic acid on Sephadex G-10. The H202 for 12 hr. The resultant material was then dissolved in 0.5 compositions of the peptides were confirmed by amino acid ml ofH20, and then 10 ml ofscintillation fluid was added. Pro- analysis and sequence determination. teins were extracted from dried gels for phosphoamino acid Phosphorylation Reactions. Protein phosphorylations were analysis as described (20). carried out in a volume of 50 p1 containing 20-40 ptCi of [y-32P]ATP (2000-4000Ci/mmol; 1 Ci = 3.7 X 10'0becquerels), RESULTS 10 mM MnCl2 or 10 mM MgCl2, 12.5 mM Hepes (pH 7.4), 2.5 mM 2-mercaptoethanol, and 40-80 1Lg of sample protein. Re- Tyrosine Protein Kinase Activity from LSTRA Cells. Pre- actions were incubated for 15 sec at 30TC. For phosphoamino liminary experiments with the particulate fraction from LSTRA acid analysis, the reactions were terminated by the addition of cells indicated that this fraction contained a relatively high 10 1ul of a solution of 100% trichloroacetic acid. For gel elec- amount of tyrosine protein kinase activity. The fraction was trophoresis, the reactions were stopped by the addition of 30 incubated with [y-32P]ATP in the presence of 10 mM MnCl2 j1d of 2.5% NaDodSOJ10% 2-mercaptoethanoV0.75 M su- and then hydrolyzed and analyzed for the formation of crose/0.025 M Tris, pH 8.0, containing pyronin Y at 2.5 mg/ [32P]phosphotyrosine. Under these conditions, 704-0% of the ml. The samples were then placed in a boiling water bath for radioactivity recovered in phosphoamino acids was in phospho- 5 min. tyrosine. This experiment was then repeated with particulate Peptide phosphorylation reactions were performed in 25 ,ul fractions from both the LSTRA cells and Rous sarcoma virus- containing approximately 25 jg of protein, 50 uM [y32P]ATP transformed cells. The fraction from the LSTRA cells incorpo- (2-5 Ci/mmol), 10 mM MnCl2 or MgCl2, and the indicated rated 8-10 times more radioactivity into phosphotyrosine than concentration ofpeptide. Reactions with the SRC-peptide were did the particulate fraction from the transformed cells (data not stopped by the addition of 10 g1 of100% trichloroacetic acid and shown). the protein was pelleted by centrifugation. The protein pellet The proteins in the fraction from LSTRA cells that were la- was washed with 100 ,l of 10% trichloroacetic acid and the su- beled with 32P during the phosphorylation reaction were ex- pernatants were combined. The phosphorylation of the SRC- amined by gel electrophoresis.