University of New York at Stony Brook, Stony Brook,. New York ..... Kelly, 1'. M., and Schlesinger, M. J. (1978) Cell 15, 1277-1286. 12. Kawai, S. (1980) J. Virol.
Communication
THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 257, No. 15, Issue of August 10,pp. 8604-8607,1982 Printed in U.S.A.
A Major Cytoplasmic Glucoseregulated ProteinIs Associated with theRous Sarcoma Virus pp60"'"Protein*
revealed that thevirus-encoded transforming protein, pp60"", is complexed with two cellular proteins (6, 7). One of these proteins (pp90) has recently been shown to be an abundant cytoplasmic protein of M , = 90,000. The bulk of this protein is not complexed with pp60"" but, rather, sediments as a monomer in glycerol gradients (7). The functional significance (Received for publication, March 8, 1982) of the association between pp60"" and pp90is not clear. However, there is evidence suggesting that the thirdmember Karl W. Lankss, Efthimios J. Kasambalidess, of this complex (Mr= 50,000) is phosphorylated by pp60"" at Michael ChinkersE, and Joan S. Bruggeg tyrosine. Interestingly, the proportion of pp60"" in the comFrom the +Department of Pathology, State University of plex is increased in lysates from cells transformed by viruses New York-Downstate Medical Center, Brooklyn, New containing a temperature-sensitive defect in the src gene (7). York 11203 and the Q Department of Microbiology, State University of New York atStony Brook, Stony Brook, In addition, it has been found that the M , = 90,000 protein New York 11794 associated with pp60"" is identical with a polypeptide which is preferentially synthesized in cells cultured at elevated temPrevious studies have established that glucose dep- peratures (8). Because the functions of the proteins whose rivation of murine cells suppresses the synthesis of an synthesis is induced are unknown, the physiological signifiM, = 85,000 polypeptide. A protein of approximately cance of this "heat shock" effect is obscure. However, the fact the same molecular weight has been found to be asso- that analogous responses have been observed in organisms as ciated with the transforming protein of Rous sarcoma different as Drosophila (9), Tetrahymena (lo), and chick virus, pp60"".The present study compares the M, = fibroblasts (11)suggests participation in a significant meta85,000 glucose-regulatedprotein with the pp60"'"-asso- bolic process. ciated protein. By the criteria of mobility in sodium Knowing that both glucose deprivation andheat shock dodecylsulfate-polyacrylamide gels andone-dimenaffected synthesis of major cytoplasmic proteins in the same sional partial proteolytic peptide mapping, the two promolecular weight range, it seemed reasonable to use murine teins appear to be identical. It has previouslybeen shown that the pp60e"-associated proteinis also iden- cells to compare the affected proteins with regard to apparent tical with one of several proteins whose synthesis is molecular weight in SDS-polyacrylamide gels and peptide induced after growth ofcells at elevated temperatures maps. This report describes the results of this studyin which or in the presence of arsenite and canavanine. Consid- we show bythe criteria of apparent molecular weight in SDSering that it is involved in a number of complex re- polyacrylamide gels and one-dimensional peptide mapping sponse patterns, the name syndmmin is proposed for that the M, = 85,000 glucose-regulated protein is identical this protein. These findings open the possibility of a with the pp608"-associated protein that is induced by heat fundamental interrelationship among the heat shock shock, i.e. pp90.
effect, regulation of protein synthesis by glucose deprivation, and oncogenic transformation by Rous sarcoma virus.
MATERIALS ANDMETHODS
Cell and Virus Stocks"L929 cells were obtained from Microbiological Associates (Bethesda, MD). The wild type SR (subgroup D) strain of RSV and its temperature-sensitive mutant LA90 were from Dr. P. Vogt (University of Southern California). Murine BALB/c/ Work from several laboratories has established that cul- 3T3 cells were a gift from Dr. A. Levine (State University ofNew tured cells regulate the rate of synthesis of at least four York, Stony Brook) andwere transformed by the SRD-and SR-LA90 polypeptides depending on the availability of glucose in their strains of RSV using the polyethylene glycol fusion method of Kawai (12).Although the cells were not subcloned, they appearedto be 100% culture medium (1-4).The rate of synthesis of one of these transformed. L929 cells were maintained in Dulbecco's modified Eapolypeptides, an M , = 85,000 species, is inhibited when the gle's medium containing 10% newborn calf serum; the transformed extracellular glucose concentration is reduced to very low 3T3 cells were maintained in the same medium containing 5% newlevels and is correspondingly increased when cells are re-fed born calf serum. The L1210V murine lymphoma cells were obtained with glucose. In murine L-cells, these alterations in rate of from Dr. J. Broome (State University of New York-Downstate) and polypeptide synthesis result in significant changes in the abun- were passed by intraperitoneal inoculation of BDFI mice. Protein Purification-The M , = 85,000 glucose-regulated protein dance of the M , = 85,000 polypeptide relative to total cell was purified as previously described (5) except that 6 M guanidine protein. Purification and partial characterization of this pro- hydrochloride was substituted for 8 M urea in the final Sephadex Gtein showed it to be a major cytoplasmic constituent that 200 step (13). This modification greatly diminished aggregation, thereby increasing the yield of the homogeneous protein from 20 to appears tosediment as a monomer in sucrose gradients (5). Completely unrelated studies directed toward understand- 60% of that present in the initial cell lysate. The free cytoplasmic ing the mechanism of oncogenic transformation by RSV' have form of the murine pp6F-associated protein was partially purified from RSV-3T3 cells by DE52-cellulose (Whatman) chromatography * This workwas supported by grants GM26167,CA27951, and essentially as described for the M , = 85,000 protein. ["2P]orthophosCA28146 from the National Institutes of Health. The costs of publi- phate-labeled pp6F-associated protein was prepared by immunoprecation of this article were defrayed in part by the payment of page cipitation from SR-LA90-3T3 cell lysate using TBR serum (described charges. This article must therefore be hereby marked "aduertise- below). SR-LA90-3T3 cells were used in this analysis because they ment" in accordance with 18 U.S.C. Section 1734 solely to indicate contain high levels of the pp50. p p 6 F . pp90 complex. Antisera and Immunoprec~pitation-The rabbit serum (TBR)was this fact. ' The abbreviations used are: RSV, Roussarcoma virus; SDS, obtainedfromanimals bearing RSV-induced tumors as described previously (13). For preparationof cell extracts, cultureswere labeled sodium dodecyl sulfate.
8604
Glucose-regulated Protein Associated with
RSV pp6@" Protein
8605
for 4 h with ["'Plorthophosphate (500 pCi/ml; ICN) in phosphate-free Dulbecco's modified Eagle's medium, washed, lysed in 1% Triton X100, 1% deoxycholate, 0.1% SDS, 0.15 M NaCI, 0.1 M Tris-HCI, pH 7.2 (13), containing 1% Trasylol (FBA Pharmaceuticals, NY), and clarified a t 30.000 X g. The supernatant was immunoprecipitated using protein A-containing Staphylococcus aureus (Cowan strainI) in 20fold excess over the antiserum (13, 14). Peptide Mapping-""P-labeled protein bands were obtained from unfixed gels following autoradiography (7). and unlabeled bandswere cut from briefly stained gels as previously described (15). One-dimensional peptide mapping by limited proteolysis was performedin 12.5% SDS-polyacrylamide gels as described by Cleveland et al. (16) using S. aureus V8 protease (Miles Laboratories Inc.). Unlabeled peptide bands were detected using the silver stain of Oakley et al. (17) including the initialprefixing step. After staining, gels were immersed in Kodak Rapid Fixer and lightly wiped to remove surface precipitates. SDS-Polyacrylamide Gel Electrophoresis-5-15% SDS-polyacrylamide gradient gels were prepared as previously described (15) and used for the one-dimensional separations unless otherwise indicated.
glucose-regulated protein from L1210V lymphoma cells eluted from DEAE-Sephadex (A-50) a t 0.17-0.18 M NaCl (5), while DE52 chromatography of the soluble proteins from a total lysate of RSV-3T3 cells (Fig. 1, lune 3) yielded a peak of pp90 eluting a t approximately 0.2 M NaCl (Fig. 1, lune 4). Not only is this elution position similar to that obtainedduring purification of the M , = 85,000 glucose-regulated protein, but the degree of purity is also similar to that obtained a t a comparable stagein the purification scheme. Despite their somewhat different reported molecular weights, the two proteins have identical mobilities in SDS-polyacrylamide gels (Fig. 1, lunes 2 and 4 ) . Fig. 1shows that these bands have mobilities corresponding to that of the L929 M, = 85,000 polypeptide whose synthesis is suppressed by glucose deprivation (lane 7). Coomassie bluestained (lane 1) and [3sS]methionine-labeled (lune 6) patterns of total L929 cell proteins are shown for comparison. Also shown in this figure is an immunoprecipitate of pp60"" with RESULTS its associated pp90 and pp50 proteins (lune 5).The mobility Protein Purification and Molecular Weight in One-dimen- of the pp90 is identicalwith that of the proteinpartially sionul SDS-Polyacrylamide Gels-Attention was focused on purified from SR-RSV-3T3 cells (lune 4 ) . Thus, within the the possible relationship between the glucose-regulated pro- limits of resolution of the one-dimensional SDS-polyacryltein of apparent M , = 85,000and the pp6jO"""associated protein amide gel technique, it appears that theM , = 85,000 glucose(pp90). Besides the similarity in their reported apparent mo- regulated protein and the free form of the pp60""-associated lecular weights, both the M, = 85,000 protein and the free protein are indistinguishable. Peptide Mapping-In order to compare further the strucform of pp90 were shown to be abundant cellular proteins that sedimented as monomers in sucrose or glycerol gradients (5, tural relatedness of these two proteins, we felt that the abun7,8). Although the procedures used in their purification were " not identical, the proteins behaved similarly when subjected to ion exchange chromatography. That is, the M, = 85,000
ma?
m I
b m m 2
3
4
5
6
7
1 2 3 4 5 6 FIG.2. Autoradiograms of peptide maps produced by lim-
FIG. 1. SDS-polyacrylamide gradient gels of murine cell proteins. Coomassie blue-stained gels of lune 2 , 120 pg of total L929 cell
ited proteolysis and electrophoresis in SDS-polyacrylamide gels of pp60""-associated and pp90 proteins. SR-LA90-3T3 cells
lysate; lune 2,5pg of purified M,= 85,000 glucose-regulated protein; lune 3,120pg of total RSV-3T3cell lysate; and lune 4,5 pg of partially purified pp90. Autoradiograms of [%]methionine-labeled proteins from immunoprecipitate using TBR serum (lune 5);total lysates of L929 cells grown in glucose-supplemented (4 g/liter) and glucose-free medium (lunes 6 and 7,respectively).
werelabeledwith["'P]orthophosphate and either immunoprecipitated with TBR serum or fractionated by DE52 chromatography as described under "Materials and Methods." Lunes 1-3, pp90 co-precipitated with pp60'"; lunes 4-6, pp90 fractionated by DE52 chromatography. Lunes 1 and 6,0.5 ng of S.uureus V8 protease; lunes 2 and 5.5 ng of protease; lanes 3 and 4,50 ngof protease.
Glucose-regulated Protein Associated with
8606
0.5 0.2
1.25
0.2 1.25 0.5
RSV pp60”” Protein
0.2
0.5
1.25 FIG. 3. Peptidemapping by limited proteolysis and electrophoresis in SDS-polyacrylamide slab gels. Lanes 1-3, 7.5 p g of purified M , = 85,000 glucose-regulated protein; lanes 4-6, partially purified free cytoplasmic form of pp60”‘-associated protein: lanes 7-9, M , = 85,000 band from total L929 cell lysate. Lanes 1, 4 and 7, 0.2 pg of S. aurczs V8 protease; lanes 2.5, and 8.0.5 p g of protease; lanes 3 , 6, and 9. 1.25 pg of protease. Peptide bands were visualized by silver stainingas described under “Materials and Methods.”
I
2
3
4
5
6
7
8
9
dant cytoplasmic form of pp90 could be used since previous weight may be reported for the same protein in different work (7) had shown it to be identical with the pp60““’-associ- experimentalsystemshas also obscuredthemoregeneral ated protein. This was confirmed when analysis of the phos- question of the relationship between the glucose-regulated phopeptide maps of free pp90 and pp6WrC-associatedproteins and heat shock proteins. Although we have systematically examined only the protein whose synthesis is suppressed by yielded identical fragment patterns (Fig. 2). The [%]methionine-labeled fragment patterns were also identical (data not glucose deprivation, the appearance of the one-dimensional shown). gel patterns and preliminary identification of two-dimensional On this basis, we compared thepurified M , = 85,000 glucose- gel spots strongly suggests that, under certainconditions, the regulated protein with the corresponding band in total L929 M, = 95,000 and 82,000 proteins whose synthesis is induced lysates and in the partially purified preparation of free pp90. by glucose deprivation may also be induced by heat shock. Fig. 3 showsthatpeptidemaps of thesebandscut from In order to avoid focusing attention on specific molecular preparative gels of all three preparations are very similar at weights or ononly one of the many response patterns in which eachproteaseconcentration. The intensities of the lower it is involved, we propose the name syndromin for the M , = molecular weight fragments also increase to a similar extent 85,000 protein characterizedin this study. The namederived is in each case with increasing enzymeconcentration, indicating from the roots syn(mv) + drom (6pjpr) having the meanings similarsusceptibility totheprotease.Thesedata provide of “together” and “path”, respectively, and is intended to additional support for the previous conclusion (5) that the convey the sense of many different stimuli converging in a protein purified from L1210V lymphoma cells is, indeed, the response that affects the same polypeptide. It is also approsame entity as that represented by the one-dimensional SDS- priate because the protein involved is inat least threedifferent polyacrylamide gel band whose synthesis is suppressed by “syndromes,” i.e. sets of concurrent effects forming an idenglucose deprivation. Furthermore, we conclude that the M , tifiable pattern. conservation (9= 85,000 glucose-regulated protein isidentical withthe ~ ~ 6 0 “ “ - Widespread involvement and evolutionary 11) point to an important role for syndromin. Our observation associated protein. that its synthesis is affected by glucose deprivation does not ilidicaw what thisrole might be. However, further studies are DISCUSSION in progress to delineate a mechanism linking the systems in The major finding of the present study is that the murine which syndromin participates. cell polypeptide suppressedby glucose deprivation is identical REFERENCES with the major cellular protein which associates with p p 6 0 in RSV-transformed cells. This conclusion is based on mobility 1. Pouyssegur. J., Shiu. R. P. C., and I’astan, I. (1977) Cell 11.941in one-dimensional SDS-polyacrylamide gels in conjunction 94 7 2. Kasarnbalides, E.J., and Lanks, K. W. (1981) Exp. Cell Res. 132, with one-dimensional partia! proteolytic fingerprints. It has 31-39 previously been shown that the pp60-bound protein from 3. Kasambalides, E. J., and Lanks, K. W. (1979) Exp. Cell Res. 118, chicken cells is identical with one of several proteins induced 269-275 after incubation of avian cells a t elevated temperatures (8). 4. Lage-Davila, A,, Hoffman-Clerg, F., Torpier, G . , and Montagnier, We have confirmed these results using mouse L929 and RSVL. (1979) Exp. Cell Res. 120, 181-189 transformed 3T3 cells (18). The fact that one protein is in5. Lanks, K. W.. and Kasarnbalides, E. J. (1979) Biochim. Biophys. Acta 578. 1-12 volved in all three experimental systems has probably been 6. Hunter, T., and Sefton, B. M. (1980) Proc. Natl. Acad. Sci. U. S. obscured by the almost bewildering variety of cell types and A. 77, 1311-1315 molecular weights previously reported. For example, depend7. Brugge, J. S.,Erikson, E., and Erikson, R. L. (1981) Cell 25,363ing on the gel system and standards employed, the reported 37’ molecular weight of this glucose-regulated protein has ranged 8. Opperrnan. H.,Levinson. W.. and Bishop, J. M. (1981) Proc. Natl. from approximately 80,000-95,000 (1, 3, 11, 19, 20). Our apAcad. Sci. U. S. A. 78. 1067-1071 9. Ashburner. M.,and Bonner, J . J. (1979) Cell 17, 241-254 proach, in which all of the relevant responses are elicited in the same or very similar systems, hasalso been usedby others 10. Yuyama. S.. and Zirnrnerrnan, A. M.(1972) Exp. Cell Res. 71, 19.7-203 to compare the polypeptides induced by heat shock with those 1 1 . Kelly, 1’. M., and Schlesinger, M.J. (1978) Cell 15, 1277-1286 induced by canavanine and arsenite (1 1,20) and be mayuseful 12. Kawai, S . (1980) J . Virol. 34, 772-77fi in identifying other polypeptides that participate in several 13. Brugge, d. S., and Erikson, R. L. (1977) Nature (Lond.)269,346diverse physiological responses. 348 14. Kessler, S. W. (1975) J . Immunol. 115, 1617-lfi24 For example, the possibility that adifferentmolecular
Glucose-regulated Protein Associated with 15. Chin, N. W., andLanks, K. W. (1980) J. Cell B i d . 85,402-413 16. Cleveland, D. S., Fischer, S., Kirschner, M., and Laemmli, U. (1977) J . Biol. Chem. 252, 1102-1106 17. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Biochem. 105,361-363 18. Lanks, K. W., Kasambalides, E. J., Chinkers, M., and Brugge, J.
RSV pp60"'" Protein
8607
S . (1982) Fed.Proc. 41, 491 19. Hightower, L. E., and Smith, J. (1978) in Negative Strand Viruses and Host Cell (Mahy, B., ed) pp. 395-405, Academic Press, New York 20. Johnston, D., Opperman, H., Jackson, J., and Levinson, W. (1980) J. Bzol. Chem. 255, 6975-6980
