We have analysed the protein kinase ac- tivities in two murine ... several virally transformed cells (Burr et al., 1980; Boss et al.,. 1981; Gacon et al., 1982).
The EMBO Journal Vol.1 No. 12
pp. 1579-1582, 1982
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High level of tyrosine protein kinase in a murine lymphoma cell line induced by Moloney leukemia virus
G. Gacon*, S. Gisselbrecht1, J.P. Piau, J.P. Boissel, J. Tolle2, and S. Fischer3 Institut de Pathologie Moleculaire, INSERM U.15, 24 rue du Faubourg StJacques, 'INSERM U. 152, 27 rue du Faubourg St-Jacques, 75014 Paris, France, 2Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115, USA, and 3INSERM U.49, Hopital de Pontchaillou, Rue Henri-le-Guilloux, 35011 Rennes, France Communicated by B. Friis Received on 18 October 1982
Several sarcoma-inducing viruses encode protein kinases that phosphorylate tyrosine residues. Such enzymatic activities can be detected within the detergent-insoluble matrix of transformed fibroblasts. We have analysed the protein kinase activities in two murine lymphoma cell lines (MBL2 and LSTRA) induced by Moloney murine leukemia virus (MoMuLV). After incubation of the detergent-insoluble matrix of these cells with ['y-32P]ATP, several alkali-resistant phosphoproteins, including a very heavily labelled 55 000 mol. wt. protein (p55), have been detected in LSTRA, reflecting the activity of a protein kinase specific to this ceil line. This protein kinase activity shares some of the distinctive properties of the protein kinases of transfonning viruses, i.e., specificity for tyrosine residues, association with membranous and/or cytoskeletal structures, and inhibition by a synthetic peptide derived from the phosphorylation site of pp6(Src. In view of the absence of a transfonning gene in MoMuLV, it is likely that the high level of protein kinase detected in the LSTRA cell line arises from the expression of a cellular gene. Key words: tyrosine phosphorylation/cytoskeleton/lymphoma Introduction Protein kinases that possess the unique capacity to phosphorylate tyrosine residues (Hunter and Sefton, 1980; Collett et al., 1980) were initially identified as the products of the transformming genes of several RNA tumor viruses (Collett and Erikson, 1978; Witte et al., 1980). Subsequently, low levels of such enzymes were detected in non-transformed cells (Oppermann et al., 1979). The cellular genes encoding these protein kinases are considered as potentially oncogenic; however, so far, the involvement of cellular tyrosine protein kinases in transformation has not been firmly established. The active forms of these enzymes are mainly localized in the plasma membrane and/or associated with the cytoskeletal structure (Hynes, 1980; Burr et al., 1980). Moreover, the largest part of these protein kinases and some of their substrates can be recovered in the detergent-insoluble fraction of several virally transformed cells (Burr et al., 1980; Boss et al., 1981; Gacon et al., 1982). We have now detected in the detergent-insoluble material (DIM) from one murine lymphoma-induced by Moloney murine leukemia virus (MoMuLV) several phosphoproteins containing phosphotyrosine, *To whom reprint requests should be sent. © IRL Press Limited, Oxford, England. 0261-4189/82/0112-1579$2.00/0.
which reflect the high level of tyrosine kinase in this cell line. A similar kinase activity has recently been solubilized by detergent treatment from a particulate fraction obtained by hypotonic extraction of LSTRA cells (Casnellie et al., 1982). In view of the absence of transforming gene in the Mo-MuLV genome, the cellular origin of this enzyme is discussed. Results When the patterns of in vitro phosphorylation (the labelling of cellular extracts and of intact cells are referred to as in vitro and in vivo phosphorylation, respectively) of the DIM from normal mouse lymphocytes, MBL2 and LSTRA lymphoma cells are compared (Figure IA), a heavily labelled phosphoprotein mol. wt. _55 000 is visible in the LSTRA cells (Figure IA, lane b), which cannot be detected in control or in MBL2 cells. This phosphoprotein (p55) is sometimes seen as a doublet between mol. wts. of 55 000 and 60 000 similar to the phosphorylated protein (p58) reported by Casnellie et al. (1982). When the gel is treated with strong alkali, the labelling of p55 remains unchanged and several less intensely phosphorylated bands are detected in LSTRA DIM. Conversely, in both control and MBL2 cells, the phosphorylation pattern is almost completely abolished by alkali treatment (Figure iB). The alkali stability of these phosphoproteins suggests that
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Fig. 1. Phosphorylation patterns of the DIM from murine lymphoid cells. Panel A: untreated gel; panel B: alkali-treated gel. a: control lymphocytes; b: LSTRA lymphoma cells; c: MBL2 lymphoma cells.
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they contain phosphotyrosine, since the phosphoester bond of phosphotyrosine is more resistant to alkali hydrolysis than that of other phosphoamino acids (Cooper and Hunter, 1981; Cheng and Chen, 1981). This has been confirmed by direct phosphoamino acid analysis; as illustrated in Figure 2, the major alkali-resistant phosphoprotein detected in LSTRA cells (i.e., p55) yields only phosphotyrosine. Therefore, we conclude that LSTRA cells contain an abnormally high level of tyrosine protein kinase activity. As judged from the intensity of the labelling of DIM preparations, this activity is 5- to 10-fold higher in LSTRA cells than in Rous sarcoma virus (RSV) transformed cells (data not shown). Synthetic peptides derived from the phosphorylation site of pp60src (the transforming protein of RSV), behave like competitive inhibitors of several virally encoded protein kinases (Wong and Goldberg, 1981). Therefore, in an attempt to characterize further the protein kinase detected in LSTRA cells, we examined the effects of a similar peptide (for sequence data see Materials and methods) on the phosphorylation pattern of the DIM from LSTRA cells. When the reaction is carried out in the presence of this peptide, the labelling of the alkali-resistant bands including p55 is completely abolished (Figure 3). Conversely, the main alkali labile phosphorylations are not affected. The experiments on in vitro phosphorylation reported above indicate that the DIM of LSTRA cells contains a high level of tyrosine-specific protein kinase. In an attempt to
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demonstrate such an enzymatic activity within the intact cell, cells were labelled in culture by incubation with 32Pi. The DIM was then prepared and analysed for the presence of alkali-resistant phosphoproteins. The phosphorylation pattern obtained by this procedure is much more complex than that obtained after in vitro labelling (Figure 4). However, in agreement with the data on in vitro phosphorylation, the main difference observed between the MBL2 and LSTRA cells is the presence in the latter of an extra, alkali-resistant phosphoprotein of mol. wt. 55 000, corresponding to the major band observed in vitro. Discussion We have found in the detergent-insoluble matrixes isolated from LSTRA cells, a tyrosine protein kinase, absent from the control cells. This enzymatic activity was largely inhibited by a src-derived peptide. It shares some of the distinctive properties of the protein kinases encoded by RNA tumor viruses. Firstly, as judged from the phosphoamino acid analysis of p55, it is capable of phosphorylating tyrosine residues. Secondly, this enzymatic activity is recovered in the DIM of the cells. The DIM contains cytoskeletal components, membrane integral proteins, and nuclei (Lenk and Penman, 1979; Cervera et al., 1981). Upon further fractionation of the DIM, the LSTRA protein kinase was found bound to membrane and cytoskeletal components and not the nucleus (data not shown). Since the protein kinases of several RNA tumor viruses are concentrated in this material (Burr et al., 1980; Boss et al., 1981; Gacon et al., 1982) it is plausible that, like
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4Fig. 2. Phosphoamino acid analysis of p55 from LSTRA. After alkali treatment of the gel, gel slices containing phosphorylated p55 were submitted to acid hydrolysis (HCI 6 N 100°C, 2 h), the resulting amino acid mixture was resolved in two dimensions on a cellulose thin layer plate by electrophoresis at pH 3.5 for I h at I kV in acetic acid/pyridine/H20 (50:5:945) (vertical direction); followed by chromatography in isobutyric acid/0.5 M NH40H (5:3) (horizontal direction).
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a b
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Fig. 3. Inhibition of the protein kinase activity of LSTRA by src-derived synthetic peptide. The DIM from LSTRA cells was incubated with [y-32P]ATP in standard conditions (ane a) and in the presence of 5 mM peptide (lane b). The resultant phosphoproteins were analysed by SDSpolyacrylamide gel electrophoresis Panel A: untreated gel; panel B: alkalitreated gel.
Tyrosine protein kinase in a murine lymphoma
viral protein kinases, the protein kinase detected in LSTRA cells is associated with plasma membrane and/or cytoskeletal structures. The biological significance of this fact is not clearly understood. However, in RSV-transformed cells, the association of the protein kinase (pp60src) with membranous structures plays a critical role in the tumorigenic capacity of these cells (Krueger et at., 1982); this suggests that important substrates of the kinases of transforming viruses are associated with the plasma membrane. LSTRA lymphoma cells provide a new model for searching for such substrates among the various alkali-resistant phosphoproteins detected in the DIM. Thirdly, the activity of LSTRA protein kinase is clearly inhibited by the src-derived peptide, suggesting some degree of similarity between this enzyme and the known protein kinases of transforming viruses. Moreover, the LSTRA protein kinase phosphorylates such synthetic peptides (Casnellie et al., 1982). On the basis of these similarities with the protein kinases specified by oncogenic viruses it seems plausible that the LSTRA protein kinase may be involved in the transformation process. In view of the mol. wts. of the major phosphoproteins detected in LSTRA (55 000 -60 000), we have tested the possibility that the LSTRA protein kinase is analogous to the pp6osrc. Using various anti-src antisera, we have not, however, been able to immunoprecipitate or to inhibit the protein kinase activity (data not shown). The genome of Mo-MuLV does not contain any oncogene and therefore does not express any transforming protein. The absence of kinase activity in the Mo-MuLV-induced MBL2 lymphoma is consistent with this. Thus, the high level of tyrosine protein kinase in the LSTRA cell line must arise from the unusual expression of a cellular gene. But how does the infection with Mo-MuLV lead to the increased expression of
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Fig. 4. Detection of alkali-resistant phosphoproteins after in vivo labelling of the DIM from murine lymphoma cells. After incubation of the cells with 32Pi for 3 h, the DIM was prepared and analysed as described. Lane A: LSTRA, lane B: MBL2.
a cellular gene? The only murine leukemia virus known to encode a tyrosine protein kinase is the Abelson virus (AbMuLV) which arose from recombination between Mo-MuLV and a mouse cellular gene. The viral protein kinase is expressed as a GAG-fusion protein of mol. wt. 120 000-160 000, according to the virus strain (Witte et al., 1980; Rosenberg and Witte, 1980). The cellular counterpart of the Abelson transforming gene is expressed in normal tissues as a 150 000 mol. wt. protein (Witte et al., 1979). No alkali-resistant phosphoprotein with a mol. wt. in this range has been detected in LSTRA cells (see Figure 1). Moreover, the phosphorylation pattern of the DIM of cells transformed by AbMuLV is different from that of LSTRA cells (J.P. Piau et al., in preparation). To test a possible recombination between the viral genome and a cellular gene, the production of virus by LSTRA cells was examined by co-cultivation of mitomycin-treated LSTRA cells with NIH/3T3 cells. Infectious Mo-MuLV could be detected by the XC test as early as 5 days following the cocultivation. However, no foci of transformed cells could be observed during an 8 week period. Furthermore, the infected NIH/3T3 cells presented no evidence of kinase activity. These results show that LSTRA produces infectious MuLV but no transforming or kinase inducing agent. The alternative possibility is that the insertion of the virus in the cellular genome results in the activation of the gene encoding the protein kinase. Work is now in progress to elucidate the mechanism of the oncogene expression and to characterize its product.
Materials and methods Cell lines The MBL-2 and LSTRA lymphoid tumor lines were originally isolated as transplantable tumors following inoculation with Mo-MuLV of C57 BL/6 and BALB/c newborn mice, respectively (Glynn et al., 1964). They were maintained in vivo as ascitic tumors by serial transplantation at weekly or biweekly intervals in syngeneic hosts. Both cell lines were grown in culture in RPMI 16 40 medium supplemented with 5%7o foetal calf serum (FCS). In vitro radiolabelling of the DIM Ascitic cells or cells grown in culture were washed twice with phosphatebuffered saline (PBS) and twice with the DIM buffer (10 mM Pipes pH 6.8, KCI 100 mM, sucrose 300 mM phenylmethylsulphonyl fluoride (PMSF) I mM, Aprotinine 100 KIIJ/ml). The cells were then extracted for 5 min at 0°C in the DIM buffer containing l1lo NP40 (I ml of buffer for 107 cells). The insoluble fraction was washed twice by centrifugation in an Eppendorf centrifuge. The DIM pellet was resuspended and incubated in 100 pl of the extraction buffer containing 10 mM MgCI2 and 150 lii/ml of [-y-32P]ATP (Amersham 5000 Ci/mmol). The protein concentration was between 0.2 and 0.8 mg/ml. The DIM was then dissolved in SDS sample buffer. In vivo labelling of the DIM Cells were incubated in phosphate-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 507o dialysed calf serum and 32po4 at mCi/ml for 3 h at 37°C. The DIM was then prepared as described. Analysis of phosphoproteins Phosphorylated samples were resolved on gradient SDS-polyacrylamide gels (7.5-1507o). The labelled proteins were detected by autoradiography of the dried gels. For the detection of the alkali-resistant phosphoproteins, the gels were soaked in 2 N NaOH for 1 h at 550C with gentle shaking, dried and exposed for autoradiography. Alkali-resistant phosphoproteins were analysed for phosphoamino acid content by electrophoresis chromatography on cellulose thin layer plates according to Hunter and Sefton (1980). Peptide synthesis Solid phase synthesis was carried out using a Beckman synthesizer according to the method of Gutte and Merrifield (1971). The purity of the peptide was checked by h.p.l.c. followed by amino acid analysis. The peptide used in this study is analogous to the phosphorylation site of pp6Osrc (the transforming protein kinase encoded by RSV); its sequence is as follows: Ac-Leu-Ile-
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Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly-NH2. The amino terminus has a covalently bonded acetyl group; the carboxyl terminus is an amide.
Acknowledgements We thiank Dr. M.Y. Fiszman for fruitful discussions and critical reading of the manuscript. This work was supported by the Association pour le Developpement de la Recherche sur le Cancer.
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