1 Cattedra di Oncologia, Universita di Padova, via Gattamelata 64, 35128 Padova, Italy
2 Istituto di lstologia ed Embriologia Generale, Universita di Torino, Corso M. D'Azeglio, 10126 Torino, Italy
3 Henry KaplanAward for the best poster Virological Session

A.Introduction

Abelson murine leukemia virus (A-MuLV) is a replication-defective retrovirus capable of rapidly inducing leukemia in mice as well as of transforming in vitro mouse bone marrow cells and fIbroblasts [1, 2, 14]; the A-MuL V was derived by passage in vivo of the replication-competent Moloney leukemia virus (M-MuLV) and its genome codes for a single polypeptide which is a hybrid molecule containing a portion of the parental M-MuLV genome (gag gene) and a portion of the cellular gene termed abl [ 18], This protein, which varies in size from 160 to 90 kilodaltons, depending on the specific A-MuL V strain, is associated mainly with the detergent-insoluble cell fraction and possesses a tyrosine kinase activity [ 4, 15, 19], In this regard, the A-MuLV protein resembles the pp60src encoded by the Rous sarcoma virus (RSV) [10] and the protein kinases encoded by other retroviruses such as the feline sarcoma virus (FeSV) [3, 17] and the Fujinami sarcoma virus (FuSV) [9], The fact that the RSV pp60,\'rc is linked with specialized cellular areas such as cell-cell junctions and adhesion plaques has suggested that the alteration of these structures is involved in the origin of the transformed phenotype [12, 13] (Marchisio et al., Exp. Cell Res., in press). However, it has also been demonstrated that, in the same cell type, different cellular substrates are phosphorylated at tyrosine residues by diverse protein kinases (Di Renzo et al., submitted). Accordingly, we found it of interest to study the cellular substrates of the A-MuL V protein kinase in cells having different cytoskeletal architecture and adhesion properties such as fibroblasts and lymphocytes.

B.Materials and Methods

I. Cell Lines

TA-3 and TA-4, non-B lymphoma cell lines were established from two independent thymic lymphomas induced by inoculating intrathymically (i.t.) newborn BALB/c mice with the complex A-MuLV(MMuLV). MZ-5, a pre-B lymphoma cell line, was established from a splenic lymphoma induced by inoculating the complex A-MuLV(M-MuLV) subcutaneously (s.c.) in newborn BALB/c micc. ABC-I, a A-MuLV-transformed line of pre-B cells, was kindly provided by Dr. Natalie Teich []6]. ANN-] is an A-MuLV-transformed line of fibroblasts [ 14]. As control, a T cell lymphoma line (TB-5) induced by M-MuLV in BALB/c mice was also included in this study. Lymphoma cell lines were cultured in complete medium consisting or Dulbecco-MEM (Gibco Europe, Glasgow, Scotland) supplemented with L-glutamine, HEPES, 2-mercaptoethanol, antibiotics, and 10% heat-inactivated fetal calfserum (FCS Gibco), ANN-l fibroblasts were cultured in Dulbecco-MEM plus 10% FCS.

Fig. 1. Immunoprecipitation by anti-phosphotyrosine (anti-P- Tyr) antibodies of 32p-Iabeled detergent-insoluble proteins from A-MuLVtransformed lymphocytes and fibroblasts. Lane a molecular weight markers; lanes b and f pre-E lymphoma cells MZ-5 and AEC-I ; lanes c and e non-E lymphoma cells TA-4 and TA-3; lane d TB-5, M-MuLV-transformed T -cells; lane g ANN-I fibroblasts

II. Specific Antisera

Antibodies against phosphotyrosine residues (anti-P- Tyr antibodies) were produced as previously described [6], AntiM-MuLV serum (Lot No, 71S1161) was obtained from the Office of Program Resources and Logistics, NCI, Bethesda, Maryland,

III. Immunoprecipitation Assay

Detergent-insoluble fractions were labeled according to Burr [5] by incubating detergent-insoluble proteins with 32p-Iabeled y-ATP (specific activity 5000 mCilmM, Amersham) in conditions allowing phosphorylation catalyzed by the kinase coded by A-MuLV [4,5], After phosphorylation, the proteins were immunoprecipitated either by anti-P- Tyr antibodies or by anti-M-MuLV serum, as previously described [6], After elution from protein A-Sepharose with Laemmli buffer [II], proteins were separated by SDS-PAGE, Dried gels were exposed to Kodak X-Omat film and processed for auforadiography.

C.Results and Discussion

The natural targets for in vivo transformation by A-MuLV are pre-B lymphocytes [I, 2]; however, the A-MuLV, if inoculated i.t., is also able to induce thymic lymphomas [7], Thus, in addition to ABC-l cells we also studied some lines derived from A-MuLV induced thymic lymphomas; these cells do not express either T cell or B cell markers, even after stimulation with Con-A or LPS, respectively, and accordingly were putatively defined as non-B cells, In order to investigate whether a specific substrate for A-MuLV kinase could be detected in different A- MuLV transformed cell types, the detergent-insoluble cellular fraction was studied with the aid of monospecitic antibodies directed against the phosphorylated form of protein tyrosine residues (anti-P- Tyr antibodies), As shown in Fig, 1, two main proteins of 70 and 120 kilodaltons were precipitated from ANN-1 fibroblasts (Fig, 1, lane g),

Fig.2. Immunoprecipitation of phosphotyrosine proteins by anti M-MuLV serum and anti P-lyr antibodies in TA-3 lymphoma cells and ANN-I fibroblasts. Lane c molecular weight markers; lanes a and b detergcnt-insoluble traction from TA-3 lymphoma cells immunoprecipitatcd by anti P- Tyr antibodies and anti M-MuLV serum, respectively; lanes d and e detergent-insoluble traction from ANN-I fibroblasts immunoprecipitated by anti-P- Tyr antibodies and anti-M-MuL V serum, respectively

whereas three different tyrosine phosphoryla ted proteins of 150, 100, and 65 kilodaltons were immunoprecipitated from pre-B and non-E lymphoma cells (Fig. I, lanes b, c, e,f). No phosphorylated proteins were detected in nontransformed fibroblasts and normal thymus cells (data not shown in Fig. I). On the contrary, in the TB-5 M-MuLV lymphoma cells, two proteins of 55 and 30 kilodaltons and an additional one of 100 kilodaltons, comigrating with the corresponding protein of A- MuLV lymphoma cells, were detected (Fig. I, lane d). Moreover, in a preliminary experiment, using the anti-M-MuLV serum, which is able to recognize the gag-coded portion of the kinase protein, we observed only one immunoprecipitate band of 120 kilodaltons in ANN-I fibroblasts (Fig. 2, lane e) and of 150 kilodaltons in lymphoma cells (Fig. 2, lane b). The anti-M-MuLV serum was not able to precipitate either the 70 kilodaltons phosphoprotein in fibroblasts (Fig. 2, Jane d) or the 100 and 65 kilodaltons phosphoproteins in lymphoma cells (Fig. 2. lane b), suggesting that these latter are probably cellular substrates and not degradation products of the A-MuLV tyrosine itself. These data seem to indicate that A-MuL V protein kinase, under identical experimental conditions, is able to phosphorylate at tyrosine residues different substrates in cells possessing diverse cYtoskeletal architecture. It is known that A-MuLV-coded protein kinase, like other transforming protein kinases, does not discriminate for substrate phosphorylation [8, 18]; consequently, the different pattern of phosphorylation induced by A-MuL V protein kinase in fibroblasts and lymphocytes is probably imputable to a different association of the tyrosine kinase with the cytoskeletal macromolecules. Acknowledgments This work was supported by the Consiglio Nazionale delle Ricerche, Progetto Finalizzato Oncologia, and Assoziazione Italiana per la Ricerca suI Cancro. D.S. is a recipient of a Fondazione Assicurazioni Generali training grant.

References

1. Abelson HT, Rabstein LS ( 1970) Lymphosarcoma. virus-induced thymic-independent disease in mice. Cancer Res 30.2213-2222
2. Baltimore D, Rosemberg N, Wittc ON ( 1979) transformation of immaturc lymphoid cells by Abelson murine leukcmia virus.lmmunol Rev 48. 1-22
3. Barbacid M, Beemon K, Devarc SG ( 1980) Origin and functional properties of the major genc product of the Snyder- Theilen strain of feline sarcoma virus. Proc Natl Acad Sci USA77:5158-5162
4. Boss MA, Dreyfuss G, Baltimore D (1981) Localization of the Abelson murine leukemia virus protein in a detergent-insoluble subcellular matrix. architecture of the protein. J ViroI40:472-481
5. Burr JG, Dreyfuss G, Penman S, Buchanan JM (1981) Association of the src gene product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts. Proc Natl Acad Sci USA 77:3484-3488
6. Comoglio PM, DiRenzo MF, Tarone G, Giancotti FG, Naldini L, Marchisio PC (1984) Detection of phosphotyrosine-containing proteins in the detergent-insoluble fraction of RSV-transformed fibroblasts by azobenzene phosphonate antibodies. EMBO J 3:483-489
7. Cook WD (1982) Rapid thymomas induced by Abelson murine leukemia virus. Proc Natl Acad Sci USA79.2917-2921
8. Erikson RL, Collett MS, Erikson E, Purchio AF ( 1979) Evidence that the avian sarcoma virus transforming gene product is acyclic AMP-independent protein kinase. Proc Natl Acad Sci USA 76: 6260-6264
9. Feldman RA, Wang E, Hanafusa H (1983) Cytoplasmic localization of the transforming protein of Fujinami sarcoma virus: saltsensitive association with subcellular components. J Virol 45: 782- 791
10. Hunter T, Sefton BM (1980) Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci USA77.1311-1315
11. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680-685
12. Rohrschneider LR (1979) lmmunofluorescence on avian sarcoma virus transformed cells. localization of the src gene product. Cell 16.11-14 13. Rohrschneider LR ( 1980) Adhesion plaq ues of Rous sarcoma virus-transformed cells contain the src gene product. Proc Natl Acad Sci USA77:3514-3518
14. Scher CD, Siegler R (1975) Direct transformation of 3T3 cells by Abelson murine leukaemia virus. Nature 253.729- 731
15. Sefton BM, Hunter T, Raschke WC (1981) Evidence that the Abelson virus protein functions in vivo as a protein kinase that phosphorylates tyrosine Proc Natl Acad Sci USA78.1552-1556
16. Teich N, Boss M, Dexter TM (1979) Infection of mouse bone marrow cells with Abelson murine leukemia virus and establishment of producer cells. In: Neth R, Gallo RC, Hofscheider PM, Mannweiler K (eds) Modern trends in human leukemia III. Springer, Heidelberg, pp 487-490
17. Van de Ven WJM, Reynolds FH, Stephenson JR ( 1980) The nonstructural components of polyproteins encoded by replication-defective mammalian transforming retroviruses are phosphorylated and have associated protein kinase activity. Virology 101.185-197
18. Wang JYJ, Queen C, Baltimore D (1982) Expression of an Abelson murine leukemia virus-encoded protein in Escherichia coli causes extensive phosphorylation of tyrosine residues. J Bioi Chem 257: 13181-13 184
19. Witte ON, Rosemberg N, Paskind M, Shields A, Baltimore D ( 1978) Identification of an Abelson murine leukemia virus-encoded protein present in transformed fibroblasts and lymphoid cells. Proc Natl Acad Sci USA 75.2488-2492