Moloney murine sarcoma virus encoded p37mos expressed in yeast has protein kinase activity

VIROLOGY

152.502506

(1986)

Moloney

Murine

Sarcoma

in Yeast BALRAJ

SINGH,*

CURT

Virus Encoded

Has Protein

WITTENBERG,~

STEVEN

Kinase

~37~“’

Expressed

Activity

I. REED,?

AND RALPH

*Department

B. ARLINGHAIJS*~’

of Molecular Biology, Research Institute cjf‘Scripps Clinic, Lo Jolln, Cul~fhwiz tBiochemistry and Molecular Biology Sectiw, IIepartment oj’Biolo&crl Sciences, University

of California,

Barbara,

Santa

Chl~&vin

9W,o37,

SS106

We describe the expression of the Moloney murine sarcoma virus enrl-nlos protein (p37”O”) in yeast under the control of the yeast GAL1 promoter. Consistent with our previous results concerning the p37”“” protein kinase made in virus infected mouse cells, ~37”“’ produced in yeast possesses autophosphorylation activity in an immune complex kinase assay using anti-mos (37-35) serum. Cc’19% Academic Press. lnc

The transforming protein encoded by the viral mos (v-mos) gene of Moloney murine sarcoma virus (MO-MuSV)-124 is synthesized as an enwnos fusion polypeptide with an apparent molecular weight of 37,000 (p37”““) (1). Based on nucleic acid sequence data, the env-mos polypeptide shares homology with the catalytic subunit of cyclic AMP-dependent protein kinase (2) as well as with the src family of protein kinases [reviewed in Ref. (@I. Previously, using immune complex assays, we have shown that a serine protein kinase activity is associated with ~37”“” (4, 5). To provide additional evidence that this protein kinase activity is intrinsic and not caused by specific association between a mammalian cellular protein kinase and the env-mos protein, we have cloned the v-mos gene of MO-MuSV into a Saccharomyces cerevisiae expression vector. The DNA segment containing the v-mos gene excised from the plasmid pKlOl(mos) (6) was inserted into the expression site of the plasmid, YEpG2 (C. Wittenberg and S. I. Reed, unpublished). The structure of the resultant plasmid, YEpG2mo.s is shown in Fig. 1. The expression of the v-m,os gene in this plasmid is expected to be under the control of the S. cerevisiae GAL1 promoter ‘To whom dressed.

requests

for

reprints

0042-6822/86 $3.00 Copyright All rights

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

should

be ad-

and to utilize the env gene initiator codon to produce ~37”““. Therefore, the primary structure of the v-mos protein produced in yeast is expected to be identical to that produced in Mo-MuSV-infected animal cells. Since the plasmid contains a poorly complementing LEU. gene (12) which has been demonstrated to result in plasmid copy numbers of 200-600 copies per cell (13, 14) when selection for leucine prototrophy is maintained, a high gene dosage of v-mos is assured and a high level of expression anticipated. In addition, since the yeast GAL4 gene product is necessary for the derepression of the genes required for utilization of galactose (IS), YEpGBmos was cotransformed into a recipient yeast strain along with plasmid pSJ4 (11) which contains the S. cerevisiae GAL4 gene. The presence of pSJ4 along with YEpGBmos enhances the expression of the v-mos gene product greater than fivefold over the strain carrying YEpGBmos alone (B. Singh, unpublished results). The cotransformant strain was used for the experiments discussed below. The v-mm proteins expressed in yeast directed by the YEpGBmos plasmid were analyzed by immunoblotting with either of two anti-peptide v-mos antibodies (Fig. 2). Authentic ~37”“” produced in viral infected NIH 3T3 cells is shown in Fig. 2, lanes ‘7 and 15. Sera prereacted with excess v-mos 502

SHORT

H

X

E

B

COMMUNICATIONS

503

1 and 9), no specific proteins were detected. When S. cerevisiae cells containing YEpG2mos and pSJ4 were grown in a medium containing 2% glucose, again no mos protein was detected (lanes 3 and 11). Upon shifting the cells to a medium containing 2% galactose, a protein comigrating with authentic ~37”“” and reactive with both Anti-mm (37-55~)

FIG. 1. Structure of the S cerevisine v-mos expression plasmid, YEpG2mos. To construct the YEpGSmos plasmid, the v-mos gene of MO-MuSV-124 was fused at the BuI site at position -8 upstream from its initiation codon (7) to a synthetic BumHI site 56 bp downstream from the transcription initiation site and 4 bp upstream from the yeast GAL1 translation initiation codon (x) via a BarnHI-XbuI linker derived from the M13mp18 polylinker (9). v-mos sequences were derived from pKlOl(mos) (6) as a l.l-kb &IEIindIII fragment and cloned into &I-HindIII-digested M13mp18 (9). The 1.75-kb BarnHI-BgZII fragment of this recombinant clone was then excised and ligated into the BamHI expression site of vector YEpG2. The plasmid vector, YEpG2, is a recombinant plasmid constructed by inserting the 1.4-kb PstIHtrrnHI fragment of pBM150 (8) carrying the yeast (;.4Ll-10 promoter region into the BarnHI-SjohI site of the high copy number yeast plasmid pMA3 (21) via a PsfI-&‘$/I linker derived from the poly linker of M13mp18. The S. cer~isiae GALIpromoter region has been previously described (X). A recombinant plasmid having the proper orientation of the v-mos gene relative to the GAL1 promoter is depicted above (YEpG2mo.s). YEpG2 or YEpG2mo.s was then used to transform (10) S. cerevtiioe strain X100, a MATa, lm2, urtti haploid (obtained from A. J. Carpousis) which had been previously transformed to uracil prototrophy with the high copy number GAL4 plasmid, pSJ4 (11). Ltu’ IJra+ transformants were isolated and used for subsequent experiments. S ceret%iau DNA sequences, open bar; v-mos protein coding sequences, solid bar; v--mos 3’ noncoding sequences, crosshatched bar; M13mp18 and pBR322 DNA sequences, solid line. B, I_irrmHI site; Bg, Bg1II site; E, EcoRI site; H, Hind111 site; I’, I’sfI site; S, S&I site; X, .%a1 site. Restriction sites within the v-mos sequences are not shown. The arrow indicates the direction of transcription.

peptide did not detect the protein (lanes 8 and 16). When S. cerevisiae cells carrying YEpG2 (lacking the v-mos gene) and pSJ4 were grown in the presence of either glucose (not shown) or galactose (Fig. 2, lanes

12

34

5

Antims 1363-3741

678

9 10 11 12 13 14 15 16 -2oo-

-97.4-68-

-43-

-25.1ABABABAB

- 4c A B A B A B A B

FIG. 2. Detection of recombinant p37’““” produced in yeast. A yeast strain containing pSJ4 and YEpGZmos was inoculated into minimal medium lacking leucine and uracil but containing either 2% glucose or 2% galactose. At the mid-log phase of growth, 50 ml of cells were harvested, washed with ice cold water. resuspended in 0.4 ml sample buffer (0.125 MTris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% 2-mercaptoethanol, and 10% glycerol), vortexed with glass beads (450-500 pm), boiled for 5 min, and the cell lysate cleared by centrifugation in a microfuge. After electrophoresis on an 8% SDS-polyacrylamide gel, the proteins were electroblotted onto nitrocellulose filters as described (16). The filters were incubated wit.h the appropriate antibody, reacted with ‘%protein A, washed, and exposed to film for autoradiography (16). Lanes 1-8 were exposed for 48 hr and lanes 9-16 were exposed for 30 hr. In lanes 1-8 are shown lysates reacted with anti-mos (37-55) serum (16); lanes 9-16 with anti-vtos (363-374) serum which is equivalent to anti-C3 serum (I). Lanes 1, 2, I), and 10: lysates from yeast cells containing YEpG2 grown on galactose; lanes 3, 4, 11, and 12: lysates from yeast cells containing YEpG2mos grown on glucose; lanes 5, 6, I:], and 14: lysates from yeast cells containing YEpG2n~o.s grown on galactose; lanes 7,8, X5, and 16: lysates from NIH 3T3 cells infected with Mo-MuSV for 2 days. The filters were reacted with anti-peptide serum (the A lanes) or anti-peptide serum prereacted with excess amounts of the corresponding PWOS synthetic peptide (the B lanes).

504

SHORT

COMMUNICATIONS

anti-mos (37-55) and anti-mos (363-374) sera (lanes 5 and 13) was easily detectable on immunoblots. The reactivities of both these anti-peptide antisera toward the protein were abolished when the antibodies were prereacted with an excess of corresponding v-mos peptides (Fig. 2, lanes 6 and 14). Next, we characterized the biochemical activity of p37”“” produced in yeast using an immune complex protein kinase assay (4). When extracts from galactose-grown S. cerevisiae cells containing YEpG2whos were immunoprecipitated with anti-mos (3’7-55) serum and incubated with Mnzf and y-32P-labeled ATP, a polypeptide the size of p37”“” and its highly phosphorylated derivative, termed p43”““, were observed as illustrated in two different experiments (Fig. 3, lanes 1 and 7). In the second experiment (Fig. 3B), MO-MuSV infected rat myoblast cells, when assayed similarly, also yielded ~37”“” and ~43”“” (lane 11). However, no phosphorylated bands were detectable with this assay when anti-mos (37-55) antibodies were prereacted with excess v-mos peptide (Fig. 3, lanes 2,8, and 12). Neither yeast cells carrying YEpGBmos but grown on glucose (Fig. 3, lane 3), nor cells carrying YEpG2 and grown on galactose (Fig. 3, lane 5) showed any v-mos protein kinase activity when tested. We found previously that anti-mos (363374) serum immunoprecipitated p37”“” but inhibited the ~37~“” associated protein kinase activity (5). Here we show that this serum recognizes ~37”“” synthesized in yeast (Fig. 2, lane 13). However, anti-mos (363-374) serum used in the immune complex protein kinase assay instead of antimos (3’7-55) did not result in autophophorylation of ~37~“” (Fig. 3, lane 9). Finally, to identify which amino acids were phosphorylated during the in vitro reaction of enwmos proteins expressed in yeast, ~37”“” and ~43”“” were hydrolyzed and the phosphoamino acids separated by thin-layer chromatography (Fig. 4, lane 1). The results of these analyses yielded phosphoserine (80-90%)and phosphothreonine (lo-20%) for the species synthesized in yeast. Under these conditions, MO-MuSV encoded ~37”“” and ~43”“” derived from a kinase assay of immune complexes ob-

A 1

2

3

6 4

5

6

7

8

9

IO

II

12

A

B

-zoo--97.4-

-66-

c

=“j;-

P -

-25.7ABABAB

A

II

A

9

FIN;. 3. Protein kinase activity associated with ~37”“” produced in yeast. Yeast cells containing pSJ4 and YEpG2 or YEpG2mo.s were grown as in Fig. 2. At mid-log phase, the cells were harvested and washed with water. The pellet was then resuspended in lysis huffer (20 mM sodium phosphate, pH 7.2, 1% NP-40, 150 mMNaC1, 1 mMEDTA and 100 kIU/ml aprotinin). The cells were broken by vortexing with glass beads, and the cell extract was clarified by centrifuging at 60,000 g for 20 min. The cell extracts were preabsorbed with normal rabbit IgG complexed with Pansorbin (10% w/v suspension of Formalin fixed Stn~~h&coec-~s uure~s cells; Calbiochem, La Jolla, Calif.) and immunoprecipitated with the appropriate anti-rncjs serum as described (4). Immune complex protein kinase assays were done as described for ~37”“” in the presence of 1 mM sodium pyrophosphate and 2 mM quercetin (A), and reaction products were separated on an 8% SDS-polyacrylamide gel. The film (X-RP, Kodak) was exposed for 12 hr. Lanes 1, 2, and 7-10: lysates from yeast cells with YEpGZmos grown on galactose; lanes 3 and 4: lysates from yeast cells with YEpG2v~js grown on glucose; lanes 5 and 6: lysates from yeast cells with YEpG2 grown on galactose; lanes 11 and 12: lysate from LGEY rat myoblasts infected with MO-MuSV for 3 days. For immunoprecipitation, anti-rrtos (37-55) serum was used for lanes 1-8, 11, and 12; anti-mos (363-374) serum was used for lanes 9 and 10. The A lanes: anti-peptide serum alone; the B lanes: anti-peptide serum prereacted with excess peptide. Panels A and B represent two separate experiments.

tained from rat myoblast cells yielded predominantly phosphoserine also (Fig. 4, lane 2). Lane 3 shows the location of phosphotyrosine obtained from the in vitro labeled EGF receptor kinase. Thus, the physical, immunological and

SHORT 1

2

!iO5

COMMUNICATIONS

3

P-SerP-ThrP.Tyr-

Orlgin-

Frc:. 4. Phosphoamino acid analysis of p37m”“-p43m”” phosphorylated in vitro. “P-Labeled p37m”“-p43m”” bands were excised from the dried polyacrylamide gel, eluted with 0.1 MNH4HC03, pH 8.6,0.5% SDS, protein precipitated with 20% trichloroacetic acid in the presence of bovine serum albumin (10 fig/ml), and washed with cold 80% ethanol. The protein was hydrolyzed in 6 N HCl under N2 for 90 min at 110”. The hydrolysate was lgophilized and mixed with phosphoamino acid markers, The phosphoamino acids were separated by electrophoresis at pH 3.5 (acetic acid: pyridinewater, 50:5:945) on a thin-layer cellulose plate (1:). The plate was exposed to film with an enhancing sc’reen for 4X hr and then treated with ninhydrin at 100” for 5 min to localize the phosphoamino acid markers. Lane 1: p37”““-~43”“” from yeast; lane 2: I,57”““-p,43”‘“” from MO-MuSV infected LsE, rat myoblasts; lane 3: itr vitro labeled EGF receptor (18) showing phosphotgrosine as a control.

functional properties of ~37”“” from yeast are identical to that found with ~37”~” produced in MO-MuSV infected mammalian cells. The size of the yeast protein, as measured by SDS-polyacrylamide gel electrophoresis, is indistinguishable from authentic ~37”““. In addition, two anti-peptide antisera made against different peptide sequences specifically recognized this same size yeast protein. Moreover, the pattern of proteins labeled in the kinase reaction from yeast extracts duplicates that obtained with extracts from MO-MuSV infected rat myoblast cells. Finally, the pat-

terns of phosphoamino acids produced by the protein kinase activities associated with p37”‘“” prepared in yeast and animal cells are indistinguishable. We feel that these data provide strong evidence that env-mos-associated protein kinase activity is an intrinsic property of the protein. However, we cannot exclude the formal possibility that some yeast kinase coprecipitates with p37”“” and is responsible for the in vitro phosphorylation that we have observed. The data reported in this paper contradict earlier reports that p37”“” lacks intrinsic protein kinase activity. Papkoff et al. (1) were unable to demonstrate that p37”O”, produced in MO-MuSV infected NIH-3T3 cells, possessed protein kinase activity. This observation can be explained by the fact that the immune complex assay was performed with anti-mos (363-374) serum which has been shown to inhibit the protein kinase activity (5, Fig. 3). These results are in agreement with the recent findings that removal of 23 amino acids from the carboxy terminus of the v-Amos protein abolishes its biological activity as assayed by focus forming ability on NIH 3T3 cells (19). More recently, neither Seth et nl. (20) nor we were able to demonstrate protein kinase activity associated with a XcU-v-mos (HTl strain) fusion protein produced in Escherichia coli. However, the protein binds an ATP analog, p-fluorosulfonyl benzoyladenosine, and possesses ATPase activity (20). The lack of protein kinase activity may be explained by the harsh conditions required for solubilization. ACKNOWLEDGMENTS The work was supported in part by NIH Grants CA 36714 and GM 28005 to RBA and SIR, respectively. SIR was supported in part by a Faculty Research Award (FRA-248) from the American Cancer Society. The authors gratefully acknowledge Dr. James Hopper, Dr. Mark Johnston, and Dr. Alan Kingsman for making available the plasmids used in this study, and Steven Gould for his excellent technical assista.nce. REFERENCES 2. PAPKOFF, J., VERMA, I., and HIINTER, T., CM 29, 412-426 (1982).

506

SHORT

COMMUNICATIONS

W. C., and DAYHOFF, M. O., Proc. NatL Acad. Sci. USA 79,2836-2839 (1982). 3. HUNTER, T., and COOPER, J. A., Annu. Reu. Bioch,em. 54, 897-930 (1985). k MAXWELL, S. A., and ARLINGHAUS, R. B., Viroloyy

8 BARKER,

143,321-333 (1985). 5. MAXWELL, S. A., and ARLINGHAIIS, R. B., J. Viml. 55,8’74-876 (1985). 6. JUNGHANS, R. P., MURPHY, E. C., JR., and ARLIN(;HAUS, R. B., J. Mol. Biol. 161, 229-255 (1982). 7. VAN BEVEREN, C., VAN STRAATEN, F., GALLESHAW, J. A., and VERMA, I. M., Cell 27, 97-108 (1981). 8. JOHNSTON, M., and DAVIS, R. W., Mol. Cd. Biol. 4, 1440-1448 (1984). 9. NORRANDER, J., KEMPE, T., and MESSING, J., Gene 26,101-106 (1983). 10. ITO, H., FUKU~A, Y., MURATA, K., and KIMURA, A., J. Bacterial. 153, 163-168 (1983). 11. JOHNSTON, S. A., and HOPPER, J. E., Proc. Natl. Acad. Sci. USA 79,6971-6975 (1982). 1.Z BEGGS, J. D., Nature (London) 275,104-109 (1978). 1.3’. BAKER, S. M., OKKEMA, P. G., and JAEHNINC, J. A., Mol. Cell. Biol. 4, 2062-2071 (1984).

14. YOCUM, R. R., HANLEY, S., WEST, R., and PTASHNE, M., MoL Cell. Biol. 4, 1985-1998 (1984). 15. OSHIMA, Y., In “The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression” (J. N. Strathern, E. W. Jones, and J. R. Broach, eds.), pp. 159-180. Cold Spring Harbor Laboratory, New York, N.Y., 1982. 16. GALLICK, G. E., SPARROW, J. T., SINGH, B., MAXWELL, S. A., STANKER, L. H., and ARLINGHAIJS, R. B., J. Gen. ViroL 66,945-955 (1985). 17, HIJNTER, T., and SEFTON, B., Proc. Nat!. Acad. Sci. USA 77, 1311-1315 (1980). 18. COHEN, S., CARPENTER, G., and KING, L. J., JR., BioL Chem. 255, 4834-4842 (1980). 19. BOLD, R. J., and DONOGHUE, D. J., MoL CelL Bid 5,3131-3138 (1985). 20. SETH, A., and VAN DE WO~~E, G. F., J. Viral. 56, 144-152 (1985). 21. DOBSON, M. J., TUITE, M. F., ROBERTS, N. A., KINGSMAN, A. J., KINGSMAN, S. M., PERKINS, R. E., CONROY, S. C., DUNBAR, B., and FOTHER(;ILL, L. A., Nucleic Acids Rex 10, 2625-2637 (1982).