May 8, 1987 - Parsons. Department of Microbiology, University of Virginia School of ... Parsons, unpublished data) activate tyrosine protein kinase activity.
The EMBO Journal vol.6 no.8 pp.2359-2364, 1987
Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus
Albert B.Reynolds, Jordi Vila', Timothy J.Lansing, William M.Potts, Michael J.Weber and J.Thomas Parsons Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA 'Present address: Department de Microbiologia, Facultat de Medicine, Hospital Clinic i Provincial, Villarroel, 132 Barcelona, Spain Communicated by R.R.Friis
The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp65CSrc. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c- C terminus by inducing premature termination of the c-src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that Cterminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity. Key words: cellular src/transformation/tyrosine kinase/src regulation/site-directed mutagenesis Introduction Expression of the src gene of Rous sarcoma virus (RSV) leads to cellular transformation (Hanafusa, 1977), increases in tyrosinespecific phosphorylation of cellular proteins (Erikson and Erikson, 1980; Radke et al., 1980; Cooper and Hunter, 1981; Cooper et al., 1983a), and the synthesis of a tyrosine protein kinase, pp6Ovsrc (Brugge and Erikson, 1977; Collett and Erikson, 1978; Levinson et al., 1978; Hunter and Sefton, 1980). In contrast, over-expression of the cellular homologue of the RSV src gene product, pp6Ocsrc, does not induce morphological transformation (Iba et al., 1984; Parker et al., 1984; Shalloway et al., 1984), at levels of expression equal to or greater than that required to effect transformation by the v-src product. Cells containing elevated levels of pp6Ocsrc do not exhibit increased tyrosine phosphorylation of cellular proteins, suggesting that the tyrosine protein kinase activity of the c-src protein is restricted in vivo (Iba et al., 1985; Coussens et al., 1985). Comparison of the amino acid sequences of the v-src and c-src proteins reveals some seven (Schmidt Ruppin strain of RSV) or 17 (Prague strain of RSV) amino acid changes throughout the protein. In addition the IRL Press Limited, Oxford, England
carboxy-terminal 19 residues of the c-src protein have been replaced with 12 unrelated residues in v-src (Takeya and Hanafusa, 1983). Substitution of either amino-terminal or carboxy-terminal sequences of pp60c-src with the cognate sequences from v-src activates the tyrosine protein kinase activity of the hybrid protein and leads to cellular transformation (Iba et al., 1985; Kato et al., 1986). In addition, point mutations within either the kinase domain (Levy et al., 1986) or an amino-terminal domain (Kato et al., 1986; W.M.Potts, A.B.Reynolds, T.J.Lansing and J.T. Parsons, unpublished data) activate tyrosine protein kinase activity and promote cellular transformation. Therefore structural alterations in pp6Ocsrc appear to contribute directly to changes in oncogenic potential as well as to the activation of tyrosine protein kinase activity. Tyrosine phosphorylation appears to play an important role in the regulation of pp60`src kinase activity (Courtneidge, 1985; Cooper et al., 1986). The major site of tyrosine phosphorylation in RSV-encoded pp60-src is Tyr 416 (Smart et al., 1981; Patschinsky et al., 1982). In contrast, the major site of in vivo phosphorylation of pp6OW-src is a carboxy-terminal tyrosine residue, Tyr 527 (Cooper et al., 1986; Laudano and Buchanan, 1986). Evidence for the negative regulation of pp6Ocsrc kinase activity was presented by Courtneidge (1985) who showed that pp6Oc-src kinase activity was inhibited by tyrosine phosphorylation. Cooper and King (1986) used in vitro phosphatase treatment of pp6Ocsrc to show that dephosphorylation of Tyr 527 activated tyrosine protein kinase activity. The analysis of pp6ocsrc in polyomatransformed cells (Cartwright et al., 1986) further demonstrated that the activated form of pp6Jcsrc found associated with polyoma middle T antigen contained no detectable phosphorylation of Tyr 527 whereas free pp60`,src not associated with middle T antigen, was phosphorylated on Tyr 527. These experiments indicate that the activity of pp6Ocsrc may be regulated by the phosphorylation state of Tyr 527. To test the role of tyrosine phosphorylation in the regulation of protein kinase activity, we have used site-directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60)'src. We show that the single alteration of Tyr 527 to Phe or Ser activates in vivo tyrosine protein kinase activity of pp6Ocsrc and induces cellular transformation of cultured chicken embryo cells. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c-src do not induce transformation or increase in vivo tyrosine protein kinase activity. Analysis of amber mutations which alter the structure of the pp6(`csrc C terminus by inducing premature termination of the c-src protein reveals that truncation at either residue 518 or 523 results in morphological transformation and increases in in vivo tyrosine phosphorylation. In contrast, removal of the last four residues of c-src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c-src protein. We conclude from these studies that C-terminal alterations which either remove or replace Tyr 527 serve to activate the c-src protein resulting in morphological transformation and increased tyrosine protein kinase activity. 2359
A.B.Reynolds et al.
Results
A
Isolation of mutations which alter the carboxy terminus of
p6pc-src
Hind
H -termln O~~~~~O
C
511
c- s rc 518 mutatbo
t Kn I
m
519
*
527
*
*
FEYLQAFLEDYFTSTEPQYQPGENL
523
1530
511
point 519 mwutotkMs 527
----------
527 v-src
-
.F_________-----.-F------
_
_
_
_
__
--------
S-
-
-
-K----Q-LPACVLE VAE
B Mlu I
Hindm M
C
Kpn I LTR
env
PRLCV gag
pBR322
M13 mpl8
~~~Hind N
Kpn I I ~~~~~~~Miu
Fig. 1. (A) Schematic diagram of mutagenesis strategy. A HindIII-KpnI fragment containing the c-src gene was cloned into M13mpl8 as described in Materials and methods. The C-terminal 25 amino acids of pp6C-src are shown to denote the region of mutagenesis. Dashes indicate positions of sequence identity while asterisks indicate the position of mutations. Synthetic oligonucleotides containing specific codon alterations were used to construct the mutations denoted above. For the mutations 518Am, 523Am and 530Am, the codons GAC, ACA and GGA were replaced by the amber codon TAG. For the mutations 51IS, 519F, 527S and 527F, the tyrosine codon TAC was substituted with TCC (serine) or TTC (Phe). The carboxy terminus of pp60v-src from the Prague A strain of RSV is shown at the bottom for comparison. (B) DNA fragments containing carboxy-terminal mutations (C*) were subcloned into pRLCv to reconstitute the desired c-src mutant (c-src*) in a non-permuted retroviral vector.
2360
For mutagenesis studies, the v-src gene of molecularly cloned Prague A (Pr A) RSV DNA (Wilkerson et al., 1985) was replaced with the intronless derivative of the cellular src gene, p5H (Levy et al., 1986). A vector containing the c-src gene was further modified as described in Materials and methods to facilitate shuttling of the c-src gene into the single-stranded phage, M13 (Figure lA). The modified construct, termed pRLc (containing the c-src gene) or pRLV (containing the v-src gene) contains both copies of the direct-repeat sequences which flank the src gene of RSV (Schwartz et al., 1983). A third vector, pRLcV, in which the 3' half of the c-src gene (an MluI-KpnI fragment) was replaced with the cognate fragment from v-src, was used to reclone fragments containing single point mutations within the 3' portion of c-src (Figure iB). Restriction site differences between c-src and v-src sequences provided a direct test to ensure that only c-src mutants were recovered in the recloning process. Mutations within the 3' portion of the c-src gene were generated using oligonucleotide-directed mutagenesis (Figure lA). The following mutations were isolated and identified as described in Materials and methods: pmS1lS, a Tyr to Ser change at residue 511; pm5I9F, a Tyr to Phe change at residue 519; pm527F and pm572S, a Tyr to Phe or Tyr to Ser change at residue 527 respectively; pm5I8Am, a termination codon inserted at residue 518; pm523Am, a termination codon inserted at residue 523; and pm53OAm, a termination codon inserted at residue 530
(Figure 1A). Biological activity of C-terminal variants Plasmid derivatives of pRLc containing the individual mutations as well as pRLV and pRLc, were used to transfect chicken embryo (CE) cells as described in Materials and methods. Foci of transformed cells appeared 6-8 days after transfection in cultures transfected with mutagenized plasmid DNA from pmiS8Am, pm523Am, pm527F, pm527S and pRLV (Figure 2, panels A, B, F, G and J). Continued propagation of the cells led to full transformation of the cultures by 10 days. Transformed CE cells, seeded in soft agar, formed macroscopic colonies readily visible within 14 days (data not shown). No morphological transformation was observed in cultures of CE cells transfected with the mutants pm53OAm, pmSl lS, pm5l9F or with pRLc (Figure 2, panels C, D, E and H), although by day 10 the cultures were resistant to superinfection with Pr A RSV, indicating substantial virus replication. Repeated passage of pRLc-infected cells or cells infected with the non-transforming variants of pRLc yielded small foci of transformed cells 18-21 days after transfection. These foci did not expand within the culture and likely result from the generation of spontaneous transforming variants, as described by Iba et al. (1984). Synthesis and in vivo tyrosine protein kinase activity of variant
p6pcysrc
To quantitate the level of c-src expression in transfected cultures, cells were labeled 10 days post-transfection with [35S]methionine and extracts immunoprecipitated with rabbit anti-bp6O(rc, a polyclonal antiserum that recognizes both viral and cellular forms of the src protein (Parsons et al., 1984). Resolution of the labeled src proteins by polyacrylamide gel electrophoresis indicated that the overall level of c-src expression was very similar in the cell populations transfected with pRLV, pRLc and the individual Cterminal variants (Figure 3). Lanes 1-3 of Figure 3 illustrate
Activation of c-src transforming activity
4~~~~~~~~~~~4
~~~~~*0
Ai'e
.4>w _t.; sos
,--
dop,~~~~~~~~
~~~Jd~~~~~~~d~~~~~-4
4f
Xw
-
4.40~~~~4 Fig. 2. Morphology of transfected CE cells. CE cells were transfected with DNA and subcultured as described in Materials and methods. Cultures were photographed 12 days post-transfection. Panel A, pm5l8Am; panel B, pm523Am; panel C, pm53OAm; panel D, pm5l IS; panel E, pm5l9F; panel F, pm527F; panel G, pm527S; panel H, pRLC; panel I, CE cells; panel J, pRLV.
the resolution of the shortened form of pp60csrc, consistent with the premature termination of the src protein encoded by pm518Am, pm523Am and pm53OAm. Comparable levels of src protein were also observed when immune complexes were subjected to immunoblotting with the monoclonal antibody 327 (data not shown), indicating that both the rate of synthesis and the stability of src proteins in the transfected cells were similar. Since previous experiments have ascribed a regulatory function to the phosphorylation of Tyr 527 (Courtneidge, 1985; Cooper et al., 1986), we assessed the extent of in vivo tyrosine kinase activity in cells transfected with pRLc, pRLv and C-terminal variants using two-dimensional gel electrophoretic separation of 32P-labeled cellular proteins. Figure 4 shows the pattern of 32plabeled proteins following treatment of the gels with alkali to selectively enhance for phosphotyrosine-containing proteins. In each panel the arrows designate the position of two major in vivo targets for pp6(Yrc, enolase [two forms designated 1 and 2 (Cooper et al., 1983b)] and p36 [designated 3 (Radke et al., 1980; Erikson and Erikson, 1980)]. A comparison of the individual gel patterns reveals an increase in the phosphorylation of p36 and enolase, a pattern consistent with increased levels of tyrosine phosphorylation in cells infected with pRLv (panel C) or the transforming C-terminal variants pm5l8Am (panel D), pm523Am (panel E) or pm527F (panel I). Uninfected cells (panel A), cells infected with pRLc (panel B) or the non-transforming variants pm53OAm (panel F), pmSl IS (panel G) or pmS19F (panel H) showed only low levels of phosphorylation of p36 or the tyrosine phosphorylated form of enolase (spot 2). Inspection of the gel patterns shown in Figure 4 also reveals a new alkali-resistant spot (designated 4) which appears only in extracts prepared from cells infected with pRLc or the non-transforming variants of c-src containing an intact C-terminal sequence (panels B, G and H). The origin of this phosphoprotein is unclear at this time but could represent a protein whose phosphorylation is mediated either directly or indirectly by the over-expression of functional pc-src pp6Ocred
The increased in vivo tyrosine protein kinase activity indicated
ai
F
-
-
!-
as os
dm >. ---W
*-
1
2
3
1
5
-
-g
6 7
8
9 10 11
Fig. 3. Immunoprecipitation of pp6O-src in infected CE cells. Cells were metabolically labeled for 6 h with [35S]methionine. Extracts were immunoprecipitated with rabbit anti-bp6Osrc serum and analysed by SDS-polyacrylamide gel electrophoresis. Lane 1, pm5l8Am; lane 2, pm523Am; lane 3, pm53OAm; lane 4, pm5llS; lane 5, pm5l9F; lane 6, pm527F; lane 7, pm527S; lane 8, pRLC; lane 9, uninfected CE cells; lane 10, pRLV; lane 11, pRLv immunoprecipitated with control normal rabbit serum. ([), Denotes the position of src proteins.
by the two-dimensional gel analysis was confirmed by measurement of total phosphotyrosine levels in infected cells. Phosphoamino acid analysis, following labeling of the cultures for 16 h with 32pi, revealed a 4- to 6-fold increase in the amount of pTyr in proteins from cells infected with pRLV, pm527F, pmS18Am and pm523Am (data not shown). Cells infected with either pRLc or the non-transforming variants pmS l S, pmS l9F orpm530Am showed little if any increase in total phosphotyrosine levels compared with CE cells (data not shown). This analysis further confirms the relationship between mutations which activate the oncogenic potential of src and increased tyrosine phosphorylation in vivo. 2361
A.B.Reynolds et al.
,.
I
A A
I
Ow
40
...
I_
$p 4.
_0 16
A
B
*a .
,4
1.
O. .
C
G v 4k,
,W _
_w
O
[.
H
.@
e
Fig. 4. Two-dimensional gel analysis of alkali-stable phosphoproteins in infected CE cells. Twelve days post-transfection, cells were labeled with 32p for 12 h and processed for two-dimensional gel analysis as described in Materials and methods. Proteins were separated using ampholytes in the pH 3.5-10 range (first dimension) and 10% polyacrylamide (second dimension). The gels were incubated in alkali to reduce the signal from phosphoserine and RNA. Panels A-C represent complete gels. Brackets in panel A designate the portion of the gels shown in panels D-I. Panel A, uninfected CE cells; panel B, pRLC; panel C, pRLV; panel D, pm5l8Am; panel E, pm523Am; panel F, pm53OAm; panel G, pmSl IS; panel H, pm 519F; panel I, pm527F. Arrows correspond to: 1, enolase phosphorylated at serine; 2, enolase phosphorylated at serine and tyrosine; 3, p36; and 4, a novel phosphoprotein (see text). The identity of enolase and p36 was based on relative mol. wt and pl. The phosphoprotein pattern for pm527S was identical to that for pm527F.
Discussion The results presented above show that substitution of Tyr 527 with either Phe or Ser activates the transforming potential of the c-src gene product, pp6oc-src. Cells infected with these variants are morphologically transformed and form colonies in soft agar at efficiencies similar to that of cells infected with RSV. Other mutations which result in structural alterations within the carboxy terminus of pp6i{`src do not activate transformation, i.e. replace2362
ment of Tyr 519 with Phe and Tyr 511 with Ser. In addition we find that the premature truncation of the src gene product
resulting from the replacement of the codons for amino acids 518 and 523 with amber codons results in the activation of the oncogenic phenotype and increases in in vivo phosphorylation of known src targets. These results are consistent with the proposed role of Tyr 527 in the regulation of pp6csrc activity (Cooper et al., 1986) and suggest that the inability to phosphor-
Activation of c-src
ylate Tyr 527 can contribute directly to the oncogenic activation of pp6O(src. Our results also indicate that other tyrosine residues present in the C terminus do not contribute substantially to the regulation of pp6O("src activity. Structural modifications involving the truncation or substitution of amino acid sequences containing Tyr 527 are a hallmark of a number of retroviral tyrosine kinases (Takeya and Hanafusa, 1983; Semba et al., 1985; Nishizawa et al., 1986; Sukegawa et al., 1987). Recombination of viral and cellular sequences leading to the alteration of C-terminal sequences is consistent with the model that loss of the regulatory phosphorylation site, Tyr 527, contributes to the oncogenic activation of the tyrosine kinase family of proteins. The observation that premature termination of the src protein at amino acid residue 530 does not activate the oncogenic activity of pp60src suggests that the carboxy terminus of c-src encoded by pm53OAm still retains a sequence sufficient to mediate the regulatory phosphorylation of Tyr 527. Interestingly, this variant does not alter the highly conserved structural motif . . . ProGln-Tyr-Gln-Pro . . . a sequence present at the carboxy terminus of several cellular tyrosine protein kinases (Takeya and Hanafusa, 1983; Parker et al., 1985; Semba et al., 1985; Kawakomi et al., 1986; Nishizawa et al., 1986; Sukegawa et al., 1987). Two-dimensional gel analysis as well as measurement of total phosphotyrosine content demonstrates that C-terminal variants that induce morphological alteration of CE cells in culture also exhibit increased levels of tyrosine phosphorylation in vivo. The pattern of alkali-resistant phosphoproteins observed upon analysis of cells infected with transforming variants is remarkably similar to that of cells infected with viral src. At this level of discrimination pp6(Yrc encoded by the C-terminal transforming variants appears to phosphorylate the same constellation of proteins as pp61v-src. These data are again consistent with the model that increases in tyrosine protein kinase activity occur as a result of loss of pp6Osrc regulation and are sufficient to initiate the events leading to morphological transformation. However, a second possibility is that C-terminal changes also alter the target specileading to the phosphorylation of a new celluficity of lar substrate(s). The identification of an alkali-resistant phosphoprotein (spot 4, Figure 4) in cells infected with pRLc or the non-transforming variants, pmiS I S, pm5 19F, but not in cells infected with pRLv, or the C-terminal transforming variants, indicates that other proteins may be modulated by tyrosine phosphorylation, perhaps by the action of pp6o-src. The role of pp6c`src in the phosphorylation of spot 4 is not clear. However, the fact that soot 4 is not phosphorylated (Figure 4, panel F) in cells infected by the pmS3OAm, a non-transforming variant lacking the C-terminal four residues of c-src, points to a possible interaction between this protein and pp6o-src. In summary, we have presented data showing that mutations which result in structural alterations within the C-terminal region of pp6c-src, including the alteration of a single Tyr residue, Tyr 527, activate the transforming potential of the c-src gene product and lead to increases in in vivo tyrosine protein kinase activity. These results are consistent with a model that the phosphorylation of the C-terminal Tyr 527 contributes to the regulation of the in vivo protein kinase activity of pp6Oc-src. After the submission of this manuscript, three groups (Cartwright et al., 1987; Kmiecik and Shalloway, 1987; PiwnicaWorms et al., 1987) showed that the expression of the avian c-src gene, containing the site-specific alteration of Tyr 527 to Phe, in rodent cells induced cellular transformation. These results are consistent with the results presented above using the natural host cell, avian chicken embryo cells.
pp6c-src,
transforming activity
Materials and methods Cells, viruses and plasmids Cultures of primary chicken embryo cells (CE cells) were prepared from gsnegative embryos (Spafas) and maintained in culture as previously described (Bryant and Parsons, 1984). RSV DNA employed in the mutagenesis experiments was derived from a non-permuted molecular clone of Pr A RSV inserted into a pBR322 plasmid vector (pJD100; Wilkerson et al., 1985). The modification and subsequent mutagenesis of this plasmid vector are described below. For transfection experiments, DNA (1-2 Ag) was applied to cells using standard CaPO4 transfection techniques and cell morphology was routinely monitored over a period of 4-21 days. Replication of transformation-defective virus was assessed by resistance to super-infection with Pr A RSV as described previously (Bryant and Parsons, 1982). Vector construction and mutagenesis To facilitate the cloning of the c-src gene into the single-stranded coliphage vector M13mp18, a plasmid vector containing the intronless c-src gene, pJD100 c-src, was modified by insertion of a HindHI linker at an XwoI site, 238 bp 5' to the start of the src coding sequence (Schwartz et al., 1983). In addition, using oligonucleotide-directed mutagenesis, a KpnI site was introduced 14 nucleotides 3' of the src termination codon. The modified vector, designated pRLC (containing the intronless c-src gene) or pRLV (containing the v-src gene), permitted the shuttling of a 1.75-kb HindIm-KpnI fragment into the HindIII and KpnI sites of M13mp18. The complete sequence for both the v-src and c-src HindIll-AKpnI fragments was determined by Sanger dideoxy sequencing (Sanger et al., 1977). Mutagenesis experiments were carried out using oligonucleotides (21 mers) containing the desired codon changes (Figure 1). Purified oligonucleotides were annealed with recombinant M13 phage DNA and double primer extension reactions were carried out as described by Zoller and Smith (1984). Mutant plaques were identified by plaque hybridization with 32P-end-labeled mutagenic oligonucleotides. Mutant phage were plaque purified, the mutations confirmed by DNA sequencing and recloned into the avian retroviral vector, pRL (Figure 1). Inmuunoprecipitation and polyacrylamide gel analysis of labeled cell proteins Metabolic labeling of cultures with [35S]methionine was carried out as described previously (Parsons et al., 1984). For immunoprecipitation of src proteins, extracts were prepared using RIPA' buffer (Cooper and King, 1986) and labeled cell extracts were incubated with rabbit antiserum to bacterial-p60", anti-bp6O'" (Gilmer and Erikson, 1983). Immune complexes were collected on formalin-fixed Staphylococcus aureus (Pansorbin, Calbiochem), washed, dissolved in sample buffer and the labeled proteins were resolved by polyacrylamide gel electrophoresis. Extracts containing equal amounts of protein were immunoprecipitated to allow relative quantitation of src proteins. Two-dimensional analysis of 32P-labeled cellular proteins Two-dimensional gel analysis of labeled cellular proteins was carried out essentially as described by Garrels (1979). Cultures were incubated with 32p; for 12-16 h, the labeled cells lysed and incubated with staphococcal nuclease, RNase and DNase as described (Garrels, 1979). The labeled cell extracts were adjusted to 9.5 M urea, 4% Nonidet P-40, 0.3% SDS, 0.1 M dithiothreitol and 2% ampholytes (pH 3.5-10). Isoelectric focusing was carried out for 14 h at 500 V followed by 1 h at 1000 V. The second-dimension gel contained 10% acrylamide, 0.26% bis acrylamide and 0.1 % SDS. For detection of alkali-resistant phosphoprotein, the dried gels were incubated in 1 M KOH at 550C for 2 h, neutralized and dried (Cooper and Hunter, 1981). Phosphoamino acid analysis Analysis of total cell phosphotyrosine was carried out by two-dimensional thin layer electrophoresis as described previously (Hunter and Sefton, 1980).
Acknowledgements We wish to thank Betty Creasy for excellent technical support, Drs M.Payne and S.Parsons for many helpful discussions and critical reading of the manuscript. We are indebted to Dr S.J.Parsons for providing rabbit antiserum to pp6Osrc, Dr J.Brugge for providing the monoclonal antibody 327 and to Dr H.Hanafusa for providing the c-src clone, p5H. J.T.P. was a recipient of a Faculty Research Award from the American Cancer Society. A.B.R. and W.M.P. were supported by fellowships from the American Cancer Society and National Cancer Institute, respectively. J.V. was supported by a fellowship from CIRiT, Barcelona, Spain. This investigation was supported by Public Health Service grants CA29243 and CA40042 awarded by the National Cancer Institute and grant NP462 from the American Cancer Society.
References Brugge,J.S. and Erikson,R.L. (1977) Nature, 269, 346-348. Bryant,D. and Parsons,J.T. (1982) J. Virol., 44, 683-691. Bryant,D. and Parsons,J.T. (1984) Mol. Cell. Biol., 4, 862-866.
2363
A.B.Reynolds et al.
Cartwright,C.A., Kaplan,P.L., Cooper,J.A., Hunter,T. and Eckhart,W. (1986) Mol. Cell. Biol., 6, 1562-1570. Cartwright,C.A., Eckhart,W., Simon,S. and Kaplan,P.L. (1987) Cell, 49, 83-91. Collett,M.S. and Erikson,R.L. (1978) Proc. Natl. Acad. Sci. USA, 75, 2021 -2024. Cooper,J.A. and Hunter,T. (1981) Mol. Cell. Biol., 1, 165-178. Cooper,J.A. and King,C.S. (1986) Mol. Cell. Biol., 6, 4467-4477. Cooper,J.A., Nakamura,K., Hunter,T. and Weber,M.J. (1983a) J. Virol., 46, 15-28. Cooper,J.A., Reiss,N.A., Schwartz,R.J. and Hunter,T. (1983b) Nature, 302, 218-223. Cooper,J.A., Gould,K.L., Cartwright,C.A. and Hunter,T. (1986) Science, 231, 1431-1434. Courtneidge,S.A. (1985) EMBO J., 4, 1471-1477. Coussens,P.M., Cooper,J.A., Hunter,T. and Shalloway,D. (1985) Mol. Cell. Biol., 5, 2753-2763. Erikson,E. and Erikson,R. (1980) Cell, 21, 829-836. Garrels,J.I. (1979) J. Biol. Chem., 254, 7961-7977. Gilmer,T.M. and Erikson,R.L. (1983) J. Virol., 45, 462-465. Hanafusa,H. (1977) In Fraenkel-Conrat,H. and Wagner,R.R. (eds), Comprehensive Virology. Plenum, New York, pp. 401-483. Hunter,T. and Sefton,B. (1980) Proc. Natl. Acad. Sci. USA, 77, 1311-1315. Iba,H., Takeya,T., Cross,F.R., Hanafusa,T. and Hanafusa,H. (1984) Proc. Natl. Acad. Sci. USA, 81, 4424-4428. Iba,H., Cross,F.R., Garber,E.A. and Hanafusa,H. (1985) Mol. Cell. Biol., 5, 1058-1066. Kato,J.-Y., Takeya,T., Grandori,C., Iba,H., Levy,J.B. and Hanafusa,H. (1986) Mol. Cell. Biol., 6, 4155-4160. Kawakomi,T., Pennington,C. and Robbins,K. (1986) Mol. Cell. Biol., 6, 41954201. Kmiecik,T.E. and Shalloway,D. (1987) Cell, 49, 65-73. Laudano,A.P. and Buchanan,J.M. (1986) Proc. Natl. Acad. Sci. USA, 83, 892896. Levinson,A.D., Oppermann,H., Levintow,L., Varmus,H.E. and Bishop,J.M. (1978) Cell, 15, 561-572. Levy,J.B., Iba,H. and Hanafusa,H. (1986) Proc. Natl. Acad. Sci. USA, 83, 4228-4232. Nishizawa,M., Semba,K., Yoshida,M.C., Yamamoto,T., Sasaki,M. and Toyoshima,K. (1986) Mol. Cell. Biol., 6, 511-517. Parker,R.C., Varmus,H.E. and Bishop,J.M. (1984) Cell, 37, 131-139. Parker,R.C., Mardon,G., Lebo,R.V., Varmus,H.E. and Bishop,J.M. (1985) Mol. Cell. Biol., 5, 831-838. Parsons,S.J., McCarley,D.J., Ely,C., Benjamin,D.C. and Parsons,J.T. (1984) J. Virol., 51, 272-282. Patschinsky,T., Hunter,T., Esch,F.S., Cooper,J.A. and Sefton,B.M. (1982) Proc. Natl. Acad. Sci. USA, 79, 973-977. Piwnica-Worms,H., Saunders,K.B., Roberts,T.M., Smith,A.E. and Cheng,S.H. (1987) Cell, 49, 75-82. Radke,K., Gilmore,T. and Martin,G.S. (1980) Cell, 21, 821-828. Sanger,F., Nicklen,A. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Schwartz,D.E., Tizard,R. and Gilbert,W. (1983) Cell, 32, 853-869. Semba,K., Yamanashi,Y., Nishizawa,M., Sukegawa,J., Yoshida,M., Sasaki,M., Yamamoto,T. and Toyoshima,K. (1985) Science 227, 1038-1040. Shalloway,D., Coussens,P.M. and Yaciuk,P. (1984) Proc. Natl. Acad. Sci. USA, 81, 7071-7075. Smart,J.E., Oppermann,H., Czernilofsky,A.P., Purchio,A.F., Erikson,R.L. and Bishop,J.M. (1981) Proc. Natl. Acad. Sci. USA, 78, 6013-6017. Sukegawa,J., Semba,K., Yamanashi,Y., Nishizawa,M., Miyajima,N., Yamamoto,T. and Toyoshima,K. (1987) Mol. Cell. Biol., 7, 41-47. Takeya,T. and Hanafusa,H. (1983) Cell, 32, 881-890. Wilkerson,V.W., Bryant,D.L. and Parsons,J.T. (1985) J. Virol., 55, 314-321. Zoller,M.J. and Smith,M. (1984) DNA, 3, 497-488. Received on March 23, 1987; revised on May 8, 1987
2364
