Dec 19, 1991 - 3, 31-56. 5. Cooper, J.A. (1990) in Peptide andProtein Phosphorylation, ed. ... 352-360. 27. Takeya, T. & Hanafusa, H. (1983) Cell 32, 881-890. 28. Foster, D., Levy ... Tanaka, A., Gibbs, C. P., Stephen, A., Anderson, K., Kung,.
Proc. Natd. Acad. Sci. USA Vol. 89, pp. 2190-2194, March 1992 Biochemistry
Molecular cloning and expression of chicken C-terminal Src kinase: Lack of stable association with c-Src protein (protein tyrosine kinase/src homology 2 domain)
HISATAKA SABE*, BEATRICE KNUDSEN*, MASATO OKADAt, SHIGEYUKI NADAt, HACHIRO NAKAGAWAt, AND HIDESABURO HANAFUSA* *Laboratory of Molecular Oncology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399; and tDivision of Protein
Metabolism, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan
Contributed by Hidesaburo Hanafusa, December 19, 1991
chicken csk, to study its tissue distribution, homology to other tyrosine kinases, and its association with c-Src.t
Cloning and sequencing of chicken C-termiABSTRACT nal Src kinase (CSK), a tyrosine kinase that phosphorylates the regulatory C-terminal tyrosine residue present on cytoplasmic tyrosine kinases of the Src family, demonstrated a high degree of interspecies conservation as well as src homology 2 and 3 domains N-terminal to the kinase domain. The lack of autophosphorylation sites distinguishes CSK from other tyrosine kinases. CSK is unique and does not belong to a gene family, suggesting that it may phosphorylate other members of the Src family of tyrosine kinases in addition to c-Src. Since complex formation between c-Src and CSK seemed a likely regulatory step in the control of c-Src kinase activity, such an asoation was investigated by immunoprecipitation and Western blotting as well as intracellular localization studies. Although some portions of CSK were found in a membrane fraction, no complex formation between CSK and c-Src was observed, suggesting that the src homology 2 domain of CSK does not play a role in the direct interaction of c-Src.
MATERIALS AND METHODS Proteins. CSK protein was purified as described from rat brain (7). Chicken v-Src protein was produced in the baculovirus-infected Sf-9 insect cells and purified by anti-Src monoclonal antibody (mAb) 327 affinity chromatography as described (18). Isolation of cDNA and DNA Manipulations. Chicken csk cDNAs were isolated from a AgtlO cDNA library made from mRNAs purified from 11-day-old chicken brain (19) using the 0.6-kilobase (kb) 5' end EcoRI fragment of rat csk cDNA (17) as a probe. Filters were hybridized under stringent conditions (650C, 1 M NaCl) and washed at 500C in the presence of 0.lx standard saline citrate (SSC)/0.1% SDS. The entire protein coding region was sequenced by the dideoxynucleotide chain-termination method (20) using Sequenase version 2.0 (United States Biochemical). Southern and Northern blots were carried out according to standard protocols (21). DNA fragments were radiolabeled with [a-32P]dCTP using a random hexamer as described by the manufacturer (Pharmacia). Expression, Western Blot Analysis, and Immunoprecipitation of CSK Protein. For expression in mammalian cells, cDNAs containing the entire coding region of chicken CSK were excised from AgtlO vector by EcoRI digestion and ligated into the EcoRI site of pcDNA-I vector (Invitrogen, San Diego) in both the sense and the antisense orientations to the cytomegalovirus promoter. A 3.2-kb BamHI/Bgl II chicken c-src DNA fragment from pHB5 (22) was constructed into the BamHI site of pBabePuro vector (23) for transient expression in COS cells. COS-7 cells were transfected by the DEAE-dextran method as described (24). Seventy-two hours after transfection, cells were lysed in a buffer containing 1% Nonidet P-40, 20 mM Tris'HC1 (pH 7.4), 0.14 M NaCl, 5 mM EDTA, 1 mM Na3VO4, 10 uM Na2MoO4, and protease inhibitors (phenylmethylsulfonyl fluoride, Trasylol, and leupeptin). Western blotting analyses were performed as described (25). CSK protein was detected with polyclonal rabbit anti-rat CSK antibody produced by immunization of Escherichia coli expressed full-length rat CSK protein (17). The Src proteins were detected by mAb327 (26) or by mAb2-17 (Quality Bioteck, Camden, NJ), which recognizes amino acids 2-17 of chicken Src protein. Bound antibody was detected by 125I_ conjugated anti-mouse IgG or protein A (Amersham). Subcellular Fractionation. After washing in phosphatebuffered saline and in a hypotonic buffer (5 mM Tris HCI, pH
The control of tyrosine phosphorylation represents an important regulatory mechanism during the response of cells to external stimuli. Uncontrolled tyrosine phosphorylation can result in cellular transformation (1-5). Accordingly, the tyrosine kinase activity of the cellular Src protein (c-Src) is tightly regulated in vivo. Autophosphorylation of Tyr-416 increases and phosphorylation on Tyr-527 suppresses c-Src kinase activity (4, 6, 7). However, since v-Src is truncated at its C terminus, this negative regulatory role is absent and cells infected by Rous sarcoma virus have elevated tyrosine kinase activity (4). Furthermore, substitution of Tyr-527 by other amino acids (e.g., phenylalanine) causes elevated kinase activity and cellular transformation (8). A variety of p60CSrc mutants that transform cells also exhibit decreased phosphorylation on Tyr-527 (9-11). Other members of the src family tyrosine kinases also contain a C-terminal tyrosine residue that corresponds functionally to Tyr-527 of c-Src (5, 10, 12, 13). Initial experiments pointed to a unique kinase that phosphorylates Tyr-527 rather than auto- or transphosphorylation of Tyr-527 by the Src kinase itself (14-16). Recently, such a tyrosine kinase, C-terminal Src kinase (CSK) has been purified from rat brain (7). Expression of the corresponding cDNA in yeast demonstrated that the kinase specifically phosphorylates Tyr-527 of c-Src (17). Interestingly, the CSK tyrosine kinase has both src homology 2 and 3 (SH2 and SH3) domains (17) that may facilitate interaction with other cellular proteins (3). In an attempt to determine the control of c-Src phosphorylation by CSK in the chicken system, we have cloned
Abbreviations: SH2, src homology 2; SH3, src homology 3; CSK, C-terminal Src kinase; mAb, monoclonal antibody. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M85039).
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2190
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Proc. Natl. Acad. Sci. USA 89 (1992)
7.5/2.5 mM KCI/1 mM MgCl2/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/i mM Na3VO4/10 ,uM Na2MoO4), COS-7 cells were incubated for 10 min on ice in the same hypotonic buffer. Cells were then harvested, disrupted by Dounce homogenization, and centrifuged at 1000 x g for 2 min at 4°C. The supernatants were fractionated by centrifugation at 100,000 x g for 30 min at 4°C into pellet (P100) and supernatant (S100). RESULTS Isolation and Sequencing of Chicken csk DNA. Chicken genomic DNA was digested with EcoRI and hybridized at 65°C (1 M NaCl) to the rat csk probe after Southern transfer. Washing of the filter at 50°C in 0.1 x SSC/O.1% SDS led to the appearance of a single band on autoradiograms (data not shown). We therefore used these conditions to screen a chicken brain cDNA library with the rat csk probe. Five positive clones were isolated from 1 x 106 clones. Two of them, 2.4 and 1.9 kb, were analyzed by restriction enzyme mapping and subjected to sequence analysis. Fig. 1 shows the ATGTCAGGGATGCAGGCCGTTTGGCCATCCGGTACAGAATGTATCGCCAAGTACAACTTC M
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GACGGCCTCTGCTCGCGCCTCATCAAACCGAAGGTGATGGAGGGGACGGTGGCAGCTCAG D
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nucleotide sequence of the protein coding region of the 2.4-kb csk clone. The 2.4-kb clone also contains a poly(A) sequence (data not shown). As deduced from the sequencing data and restriction maps, the 1.9- and 2.4-kb clones start at the same 5' position but the shorter clone terminates in the middle of the 3' untranslated region. Despite several single nucleotide differences (data not shown), possibly due to the polymorphism in chickens, both of those chicken clones encode the same 450 amino acid residues, which are the same length as rat CSK. The deduced amino acid sequence of chicken CSK possesses 93.1% homology when compared to the rat sequence. Significant diversity is only noted in the N-terminal and C-terminal regions. The kinase domains, SH2 domains (100%o homology), and SH3 domains (98.1% homology) are highly conserved. The homology at the nucleotide level between chicken and rat csk is 82.9% within the coding region. Protein products of the cDNAs were confirmed by transient expression in COS-7 cells and detection by anti-rat CSK antibodies (Fig. 2). The endogenous CSK in COS cells was also detected with this antibody (lanes 1 and 3). The kinase activities of these proteins were confirmed in extracts of COS-7 cells using p60CSrC as a substrate (data not shown). Expression of csk mRNA. We then analyzed the expression of csk mRNA in a variety of chicken tissues (Fig. 3). All tissues tested, including cultured chicken embryo fibroblasts, expressed a 2.4-kb mRNA species, although spleen tissue appeared to contain the highest amounts. An additional 2.2-kb band was detected in spleen. Transformation of chicken embryo fibroblasts by a variety of oncogenic viruses containing v-src, v-mos, v-crk, v-myc, v-ras, or polyoma middle-sized tumor antigen gene did not change the levels of csk mRNA significantly (data not shown). A single molecular weight CSK protein was detected in cultured chicken embryo fibroblasts by Western blots with the polyclonal anti-rat CSK antibodies (data not shown). Does csk Belong to a Gene Family? To investigate whether several distinct CSK-like kinases exist (for example, one for each member of the Src family kinase), we performed Southern blot analysis of chicken genomic DNA (Fig. 4). Under stringent washing conditions, a single 18-kb EcoRI band was detected by hybridization with the chicken 2.4-kb cDNA fragment, which corresponds to the entire mRNA sequence (Fig. 4A). HindIII and BamHI digestion gave multiple bands of 12.5 and 1.8 kb and 12.5, 6.8, and 2.4 kb, respectively, with
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FIG. 1. Nucleotide and deduced amino acid sequences of chicken CSK and its comparison with rat CSK. Only those rat amino acid residues different from the chicken residues are shown below the chicken amino acids. The SH2, SH2', and SH3 domains are underlined (=, - - -, and -, respectively).
FIG. 2. Expression of chicken csk cDNA in COS-7 cells. COS-7 cells (1 x 106) were transfected with 10 uig of pcDNA-I plasmid DNAs containing the 2.4-kb chicken csk cDNA (lane 2), the antisense orientation of the 2.4-kb cDNA (lane 3), the 1.9-kb chicken csk cDNA (lane 4), and vector alone (lane 1). Cells were harvested 72 hr after transfection and lysed in a Nonidet P-40 buffer. Solubilized proteins (40 /ug) were electrophoresed through a SDS/10%0 polyacrylamide gel. Proteins were then transferred to an Immobilon-P filter (Millipore) and CSK proteins were detected with polyclonal anti-rat CSK antibodies and 125I-labeled protein A. Molecular size markers (prestained; Amersham) are indicated in kDa.
Biochemistry: Sabe et al.
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bility that c-Src anchors CSK to cell membranes, we coexpressed csk and c-src in COS-7 cells. Although high levels of c-Src expression were achieved by transfection in COS-7 cells (Fig. SB), the amount of CSK in membrane fractions of cells transfected with csk and c-src remained unchanged compared to cells transfected with csk alone, suggesting that CSK may bind to cell membranes independent of c-Src. c-Src and CSK Do Not Form a Complex. To investigate the association of CSK and c-Src proteins in vivo, cDNAs of csk and c-src were cotransfected in COS-7 cells and expressed proteins were immunoprecipitated with mAb2-17. Both proteins were transiently expressed to high amounts in these cells as demonstrated by Western blotting of whole cell lysates with the corresponding antibodies (Fig. 6 A and B, lanes 1-3). Despite overexpression of both proteins, no CSK protein was detectable in anti-Src immunoprecipitates (Fig. 6A, lane 4), suggesting that under these conditions CSK does not form a complex with c-Src. CSK contains an SH2 domain, which could potentially bind to tyrosine residues on the Src protein. c-Src isolated from cells is almost entirely phosphorylated on Tyr-527 and hence is inactive (6, 14). During the cell cycle, c-Src becomes activated and autophosphorylates on Tyr416 (30). In addition, overexpression of c-Src may result in an active, Tyr416-phosphorylated c-Src population (31). Tyr-416 is also phosphorylated on v-Src. Although the lack of association between CSK and c-Src suggests that the CSK SH2 domain does not bind stably to Tyr-527 of c-Src, it may still complex with Tyr416 of the Src protein. We therefore used autophosphorylated v-Src protein to test the interactions with CSK. Purified v-Src and CSK proteins were incubated for 1 hr and immunoprecipitated with mAb2-17. After SDS/PAGE and Western blotting, membranes were probed with an anti-CSK antibody (Fig. 6C, lanes 3-6). No CSK protein was detected in the v-Src immunoprecipitates. In addition, CSK did not associate with unphosphorylated v-Src protein (lane 7).
FIG. 3. Expression of csk mRNA in chicken tissues. Poly(A)+ RNA (2 ,ug) from chicken medulla (lane 1), cerebellum (lane 2), intestine (lane 3), spleen (lane 4), telencephalon (lane 5), liver (lane 6), and cultured chicken embryo fibroblasts, which were prepared from 11-day embryos (lane 7), was electrophoresed on a 1% formamide/agarose gel, transferred to a Zetabind filter (Cuno), and hybridized with a 32P-labeled chicken csk cDNA probe. The relative positions of the 28S and 18S rRNAs are indicated by arrows.
the same probe (Fig. 4A). To analyze the existence of related genes in the chicken genome, genomic DNA was hybridized with a 1.2-kb Bal I/Sma I fragment (nucleotides 25-1192) of the coding region. A 0.96-kb HincII/Bgl I fragment of the Rous sarcoma virus v-src gene corresponding to exons 4-10 of chicken c-src, which encompass the 3' half of the SH3 domain, the whole SH2 domain, and the 5' half of the kinase domain (SH1 domain) (27), was used as a comparison. As shown in Fig. 4 B-D, the src probe cross-hybridized with many other genes under lower stringency conditions as expected (28). Under the same conditions, the chicken csk probe revealed one predominant band. Even under the mildest conditions (lx SSC/0.1% SDS; 50°C), only two additional very faint bands were detected with the csk probe. Under these hybridization conditions, a gene with a homology of =69% can be detected (29). CSK and c-Src Lcalize to Membranes. CSK was first isolated from a rat membrane fraction (7). The sequence, however, does not reveal any hydrophobic domain or lipid attachment site. Fig. 5A shows the subcellular distribution of CSK expressed in COS-7 cells by cDNA transfection. Although the majority of CSK is present in the soluble fraction, a substantial amount associates with cell membranes. A similar distribution of endogenous CSK is found in COS-7 cells (Fig. SA, vector). In contrast, c-Src is almost entirely found in the membrane fraction (Fig. 5B). To test the possi-
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DISCUSSION
Sequence analysis of chicken CSK demonstrates that the amino acid sequence of CSK is highly conserved between rat and chicken (93.1% in amino acid and 82.9o in nucleotide sequence). This evolutionary preservation of CSK supports an important functional role for this enzyme. Since p60c-src is also highly conserved (94.2% of amino acids between human and chicken) (32), Tyr-527 phosphorylation by CSK may be an ancient regulatory mechanism. Chicken and rat CSK share unusual features for nonreceptor tyrosine kinases; they do not contain a C-terminal regulatory tyrosine residue and they lack autophosphorylation sites. The regulatory mechanisms of CSK therefore differ from other tyrosine kinases.
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Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 4. Southern blot analysis of chicken genomic DNA with csk cDNA probe. (A) Genomic DNAs digested with HindIII (lane 1), BamHI (lane 2), or EcoRI (lane 3) were electrophoresed on 0.7% agarose, transferred to a nitrocellulose filter (Millipore), and hybridized with a 32P-labeled 2.4-kb EcoRI fragment of csk cDNA. Washing conditions were 0.2x SSC/0.1% SDS at 65°C. (B-D) Genomic DNAs digested with EcoRI (lanes 1 and 3) or HindIII (lanes 2 and 4) were processed as described above and hybridized with 32P-labeled csk (lanes 1 and 2) or src (lanes 3 and 4). cDNA probes each consisted of their coding regions (see text). Washing conditions were 0.2x SSC/0.1% SDS at 650C (B), O.1x SSC/0.1% SDS at 50°C (C), and 1x SSC/0.1% SDS at 500C (D). Molecular sizes in kb are indicated by arrows.
Biochemistry: Sabe et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
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FIG. 5. Subcellular localization of CSK and c-Src. COS-7 cells (1 x 106) transfected with 5 j.g each of pcDNA-I (vector), csk cDNA (CSK), c-src cDNA (c-Src), or 5 A&g each of csk cDNA and c-src cDNA (CSK & c-Src) were fractionated to S100 (lanes S) and P100 (lanes P) before Western blot analysis with polyclonal anti-rat CSK antibody (A) or mAb327 (B) as described. Samples applied on lanes S and P represent 10%o of the total recovered S100 fractions and all of the recovered P100 fraction, respectively.
The Src tyrosine kinase family consists of eight structurally related kinases (5, 33). The expression of each of these kinases is strictly regulated during development and among different tissues. The activity of several of these kinases was shown to be controlled by phosphorylation of a C-terminal tyrosine residue, and amino acid sequences surrounding the critical C-terminal tyrosine residue are well conserved among the different Src-like kinases (5, 10). Moreover, a suppression of c-Src kinase activity was noted when residues 495-509 of Lck were substituted for the C-terminal peptide of c-Src, indicating a similarity in tertiary structures of these two C-terminal regions (34). To date, only CSK can phosphorylate the regulatory C-terminal tyrosine residue of c-Src. Since several tyrosine kinases are regulated by C-terminal phosphorylation, each kinase may be phosphorylated by a unique C-terminal kinase. Alternatively, CSK may be sufficient to phosphorylate all members of the Src tyrosine kinase family. Our data support the latter possibility. First, we have detected only a single csk gene on Southern blots under conditions in which cross-hybridization of Src family members is observed. Second, our data demonstrate that csk mRNA and protein are expressed in all tissues. Third, we have shown that purified CSK phosphorylates Src family proteins such as Lyn and Fyn at their C-terminal regulatory tyrosine residues (35). Fourth, we have detected large amounts of CSK protein in a mouse hematopoietic cell line, in which c-Src protein was not detected (H.S. and H.H., unpublished data), suggesting the existence of a potential substrate(s) other than c-Src. The expression of csk mRNA seems to correlate with the abundance of Src-like tyrosine kinases. The csk mRNA is particularly abundant in spleen. This organ is also rich in cytoplasmic tyrosine kinases since these are found in large amounts in lymphoid cells (36). Since unphosphorylated c-Src is fully active in the presence of Mg2+-ATP (ref. 15; H.S. and H.H., unpublished data), phosphorylation at Tyr-527 must occur rapidly to prevent cellular transformation. The structure and cellular distribution of CSK suggested a possibility of the formation of a complex between CSK and c-Src for the following reasons: first, both CSK and c-Src have been isolated from the membrane fraction of rat brain (7); second, CSK possesses an SH2 domain (17) and may therefore bind to phosphotyrosine residues (37)-in particular, CSK SH2 may recognize Tyr-416 of c-Src, which becomes phosphorylated in enzymatically active c-Src by autophosphorylation; third,
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i.
FIG. 6. CSK does not stably associate with Src. (A and B) COS-7 cells (1 x 106) were transfected with 5 ug each of csk and c-src cDNAs (lanes 1-4) or 5 ,ug csk cDNA alone (lanes 5-8). After solubilization with 1 ml of 1% Nonidet P-40 buffer, lysates were processed to SDS/polyacrylamide gel electrophoresis (lanes 1-3 and 5-7) or immunoprecipitated with mAb2-17 followed by protein G-Sepharose, and the recovered proteins were processed for gel electrophoresis after they were divided into a ratio of 10 (A, lane 4) to 1 (B, lane 4). Each gel was then subjected to Western blot analysis with polyclonal anti-rat CSK antibody (A) or with mAb327 (B), respectively. The amounts of each of the lysates applied in lanes 1 and 5, 2 and 6, and 3 and 7 were 20%6, 2%, and 0.2% of that used for immunoprecipitation, respectively. (C) Each 100 ng of purified CSK proteins was incubated with v-Src proteins, which had been autophosphorylated in vitro, in 100 Al of phosphate-buffered saline containing 0.1% Nonidet P-40 on ice for 1 hr in the presence of acetylated bovine serum albumin as a carrier and subjected to immunoprecipitation with mAb2-17 (lanes 3-7). The recovered proteins were processed for Western blot analysis with polyclonal anti-rat CSK antibody. The amounts of phosphorylated v-Src proteins used were none (lane 3), 0.25 ,ug (lane 4), 0.5 gg (lane 5), and 1 ,tg (lane 6); 1 Ag of unphosphorylated protein was also used (lane 7). Lanes 1 and 2, 6% of total supernatants of lane 5 and 6, respectively. Lanes 8-10, pure CSK proteins-0.35 ng (lane 8), 3.5 ng (lane 9), and 35 ng (lane 10).
since common tyrosine phosphorylation sites are not present in CSK, SH2 domain-mediated binding may be critical in directing CSK activity to its substrates, thereby localizing CSK kinase activity; fourth, the formation of a stable complex between c-Src and CSK could explain the observation that the high intracellular c-Src levels cause cellular transformation by an active Tyr-416-phosphorylated c-Src subpopulation since in this instance concentration of CSK may be limiting (31). Despite all these implications, we have not been able to detect a stable complex between CSK and c-Src by immunoprecipitation. Also, no significant association was seen in vitro between CSK and v-Src (the Src protein phosphorylated on Tyr-416). In these experiments, the two proteins either were expressed in COS-7 cells to concentrations much higher than those of physiological conditions or were incubated in vitro at concentrations even higher than those found in vivo. Furthermore, we demonstrated that the translocation of CSK to membrane fractions was not increased by coexpression of c-Src. From a Western blot analysis, we confirmed that amounts of overexpressed CSK protein are in excess of those of the endogenous c-Src protein. Therefore, a significant increase in the level of CSK is expected in the membrane fractions where overexpressed c-Src localizes if CSK stably associates with c-Src. The possibility still exists that our conditions (1% Nonidet P-40) disrupted such a complex, although binding between phosphorylated tyrosine residues and SH2 domains in other systems is preserved in RIPA buffer (1% sodium deoxycholate/1% Triton X-100) (38). Alternatively, such a complex
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may be stabilized by cellular proteins that are not present in COS-7 cells. More likely though, association of CSK and c-Src occurs only transiently. It is conceivable that phosphorylation of Tyr-527 of c-Src by CSK in turn leads to dissociation of the enzyme from its substrate. To further investigate complex formation, the binding between CSK and p60c-src mutants that may bypass such a negative control mechanism needs to be investigated. We thank Rosemary Williams for her technical assistance. We thank Joan Brugge for mAb327, Lu-Hai Wang for the chicken brain cDNA library, and Yasuhisa Fukui for v-Src protein. We are grateful to Tomohiro Kurosaki for his kind help in sequencing analysis and Raymond Birge for critical reading of the manuscript. We also thank Kathleen Baker, Yoshiaki Tagawa, and Naoki Sawada for contributions to this work. This work was supported by Grant CA44356 from the National Cancer Institute and Grant 2517 from the Council for Tobacco Research. H.S. was supported by the Human Frontier Science Program Organization. B.K. was supported by a fellowship from the Charles H. Revson Foundation 1. Bishop, J. M. (1991) Cell 64, 235-248. 2. Hunter, T. (1991) Cell 64, 249-270. 3. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R. & Soltoff, S. (1991) Cell 64,281-302. 4. Jove, R. & Hanafusa, H. (1987) Annu. Rev. Cell Biol. 3, 31-56. 5. Cooper, J. A. (1990) in Peptide and Protein Phosphorylation, ed. Kemp, B. (CRC, Boca Raton, FL), pp. 85-113. 6. Cooper, J. A., Gould, K. L., Cartwright, C. A. & Hunter, T. (1986) Science 231, 1431-1434. 7. Okada, M. & Nakagawa, H. (1989) J. Biol. Chem. 264, 2088620893. 8. Kmiecik, T. E. & Shalloway, D. (1987) Cell 49, 65-73. 9. Jove, R., Hanafusa, T., Hamaguchi, M. & Hanafusa, H. (1989) Oncogene Res. 5, 49-60. 10. MacAuley, A. & Cooper, J. A. (1990) New Biol. 2, 828-840. 11. Iba, H., Cross, F. R., Garber, E. A. & Hanafusa, H. (1985) Mol. Cell. Biol. 5, 1058-1066. 12. Marth, J. D., Cooper, J. A., King, C. S., Ziegler, S. F., Tinker, D. A., Overell, R. W., Krebs, E. G. & Perlmutter, R. M. (1988) Mol. Cell. Biol. 8, 540-550. 13. Amrein, K. E. & Sefton, B. M. (1988) Proc. Natl. Acad. Sci. USA 85, 4247-4251. 14. Jove, R., Kornbluth, S. & Hanafusa, H. (1987) Cell 50, 937-943. 15. Kombluth, S., Jove, R. & Hanafusa, H. (1987) Proc. Natl. Acad. Sci. USA 84, 4455-4459.
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