MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 2855-2862 0270-7306/90/062855-08$02.00/0 Copyright © 1990, American Society for Microbiology
Activation of the Proto-Oncogene p60c-src by Point Mutations in the SH2 Domain
MELANIE C. O'BRIEN, YASUHISA FUKUI, AND HIDESABURO HANAFUSA* The Rockefeller University, New York, New York 10021-6399
Received 27 December 1989/Accepted 14 March 1990
To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60&csrc (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60(CSFC variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60CSrc variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G1701) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. Mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G1701 was elevated twofold. Expression of p60W14R (or, to a lesser extent, of p6OGl7OI) increased the number of proteins phosphorylated on tyrosine in infected cells. All of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60V-Sr(. Immunoprecipitated p60W14R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine- specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus.
The proto-oncogene p60c-src, like its viral counterpart p60v-src, is a membrane-bound tyrosine-specific protein ki- nase of molecular weight 60,000 (for a review, see reference 20). However, p6c-src is not transforming even when over- expressed (19, 41, 51). Differences in the coding sequence of p60v-src must therefore be responsible for the oncogenicity of this protein. p60c-src can be converted to a transforming protein by various amino acid substitutions (27, 34) or by replacement or truncation of the carboxyl terminus (re- viewed in references 16 and 18). In p6v src, the last 19 amino acids of p60csrc have been replaced by 12 entirely different amino acids. In addition, there are 10 other amino acid substitutions scattered throughout the protein (for a review, see reference 16).
Several important functional regions have been defined in p60src. The carboxy-terminal half of the molecule constitutes the catalytic domain, as suggested by the extent of homology with other protein kinases (including serine- and threonine- specific protein kinases) and demonstrated by proteolysis experiments (1, 31). It is also believed that the first seven amino-terminal residues of p60C-rc are required for myristy- lation and consequent attachment to the cell membrane (26; reviewed in reference 20). The function of the remaining amino-terminal sequence is not yet well understood, al- though various experiments suggest a regulatory capacity for this region (reviewed in reference 42). One clue in the search for amino-terminal function has been the discovery of two blocks of sequence similarity among many tyrosine-specific protein kinases, called src homology 2 (SH2) and src homol- ogy 3 (SH3). (The catalytic domain is considered to be src homology 1.) In p60csrc, SH2 includes residues 137 through 241, and SH3 extends from residue 84 to residue 114. SH2 was first described as a stretch of conserved amino
acids found in all cytoplasmic tyrosine kinases (49) but is now recognized in a diverse group of otherwise unrelated
* Corresponding author.
proteins. A newly described oncogene, crk, has sequence similarities to tyrosine-specific protein kinases in the SH2 and SH3 regions but has no catalytic domain (38). Transfor- mation by this oncogene does increase the level ofphosphor- ylation on tyrosine within the cell, presumably indirectly. One isozyme of phospholipase C (phospholipase C--y) has two copies of SH2 and one of SH3 (54). In addition, GTPase-activating protein (GAP), which activates GTPase in normal but not oncogenic p2lras, has two copies of SH2 (57). GAP has been shown to have increased phosphoryla- tion on tyrosine in cells transformed by v-src or other tyrosine kinases (11).
Experiments with deletion mutants of p60vsrc indicate that the SH2 region is important for transformation (2, 6, 28, 44, 58, 60). The mutants are transformation defective, incom- pletely transforming, or temperature sensitive. Various linker insertions and deletions in the SH2 region of fps produce host-dependent phenotypes or impaired kinase ac- tivity (9, 29, 49). Recently, mutations of SH2 in p60v-src have been found that also give a host-dependent transformation- defective phenotype (8, 56). It has been suggested that the SH2 region interacts with a host cell protein (42). We were interested to see whether disrupting the SH2
region by changing the most conserved residues would affect the transforming ability of p60csrc. We therefore carried out oligonucleotide-directed mutagenesis of four well-conserved residues in SH2 and characterized the resulting mutants. Several of these are activated, as measured by morphologi- cal transformation, anchorage independence, and elevated kinase activities in vivo and in vitro.
MATERIALS AND METHODS
Plasmid constructions. The template used for mutagenesis was a modification of p5H in which the MluI site down- stream of the c-src coding region was removed for conve- nient manipulation (gift of R. Jove; 22). This plasmid con- tains the complete coding sequence of chicken c-src and
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Cells and viruses. Chicken embryo fibroblasts (CEF) were prepared from 11-day-old embryos and cultured as described previously (15). Cultures were passaged at least once after infection to allow virus spread.
Plasmid DNA from the pSH constructs was ligated to a second plasmid containing viral coding sequences and trans- fected into secondary CEF, using the calcium phosphate precipitation method as described previously (7) except that the second plasmid was pBH-REP (23) instead of pSR-REP. After two more passages, replication-competent virus was harvested from the supernatant of confluent cultures, and these virus stocks were used in all experiments. The v-src- containing virus used here was harvested from similar trans- fections using pSR-XD2 and pBH-REP (7) and is derived from the Schmidt-Ruppin subgroup A (SRA) strain of Rous sarcoma virus (RSV) (10). Colony formation. Anchorage-independent growth was
assayed by seeding cells in Dulbecco modified Eagle medium containing 0.4% agar, 11% calf serum, 1% chick serum, 10% tryptose phosphate broth, and antibiotics. Secondary CEF were infected for 3 to 5 h at a multiplicity of infection of 10' and seeded at 1.6 x 106 cells in a 100-mm-diameter dish. The plates were incubated at 40°C and scored after approxi- mately 2 weeks.
Cell lysates and immunoprecipitation. Cells were lysed in RIPA buffer containing 150 mM NaCl, 10 mM Tris hydro- chloride (pH 7.4), 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 ,uM leupeptin, 1 ,uM antipain, and 0.1 puM aprotinin. Mono- clonal antibody (MAb) 327 (gift of J. Brugge; 36) was used in all experiments. Lysates were reacted with an excess of antibody and kept on ice for 1 h. A 50-plI sample of a 1:3 slurry of protein A-Sepharose CL-4B (Pharmacia) incubated with rabbit anti-mouse immunoglobulin G (Accurate) was added, and the lysates were rotated at 40C for 30 min. Immunoprecipitates were washed three times with RIPA buffer containing 300 mM NaCl, three times with RIPA buffer containing 150 mM NaCl, and twice with 40 mM Tris hydrochloride (pH 7.4).
In vitro kinase assays. Washed immunoprecipitates were suspended in 10 mM Tris hydrochloride (pH 7.4) and split into two equal volumes. One portion was boiled in sample buffer and electrophoresed on a 10% SDS-polyacrylamide gel. The gel was then transferred to a nitrocellulose filter, using a transfer buffer containing 20 mM Tris hydrochloride, 150 mM glycine, and 20% methanol. The second portion was suspended in 30 pul of kinase buffer containing 20 mM Tris hydrochloride (pH 7.2), 5 mM magnesium chloride, 5 puCi of [y-32P]ATP (10 mCi/ml; Amersham Corp.), and 5 p.g of acid-denatured enolase as an exogenous substrate. The reaction mixtures were left at room temperature for 10 min and then washed twice with RIPA buffer containing 10 mM NaCl. The samples were electrophoresed as described above, and then the gel was fixed in 10% ethanol-10% acetic acid for 30 min, dried, and exposed with an intensifying
screen at -70°C. The bands were excised from the dried gel for quantitation.
Immunoblotting. Nitrocellulose filters that were probed with-MAb 327 were first blocked for 60 min in phosphate- buffered saline (PBS) containing 0.05% Tween 20 and 2.5% normal sheep serum (Accurate). A 1:250 dilution ofMAb 327 was added, and the filters were agitated at room temperature overnight. The filters were then washed with PBS containing 0.05% Tween 20, blocked again for 30 to 60 min, and incubated with a 1:250 dilution of 1251I-labeled sheep anti- mouse immunoglobulins (100 ,uCi/ml; Amersham) in block- ing buffer for 2 h. Final washes were also in PBS with 0.05% Tween 20. The filters were dried and exposed as described above. For detection of phosphotyrosine-containing proteins, fro-
zen cell pellets were thawed on ice and suspended in 150 pul of Sol buffer (10 mM Tris hydrochloride [pH 7.4], 1% SDS, 1 mM sodium orthovanadate, 0.1 mM sodium molybdate, and 1 mM phenylmethylsulfonyl fluoride), sheared with a 25-gauge needle, and immediately heated in boiling water for 3 min. Samples were normalized for protein concentration and electrophoresed on a 10% gel as described above. The proteins were then transferred to nitrocellulose in a buffer containing 25 mM Tris, 92 mM glycine, and 20% methanol. The filter was blocked in buffer (10 mM Tris hydrochloride [pH 7.4], 0.1% Triton X-100, 0.9% NaCl, 0.02% sodium azide) containing 1% ovalbumin (Sigma Chemical Co.) and then incubated overnight with antibody to phosphotyrosine (prepared according to Wang [59]; gift of D. Sternberg). The filter was washed in buffer as described above, blocked again for 30 min, and incubated with 2 ,uCi of affinity-purified 125I-protein A (100 ,uCi/ml; Amersham) in a volume of 20 ml for 1 to 2 h. After final washes, the filter was dried and exposed as described above. Cyanogen bromide cleavage. Confluent, fully infected
plates of CEF were metabolically labeled for 4 h with 32Pi (1 mCi per plate; 25 mCi/ml; Dupont, NEN Research Prod- ucts). Cells were lysed in 1 ml of RIPA buffer containing 150 mM NaCl, 1 mM sodium orthovanadate, and 0.1 mM sodium molybdate. The lysates were immunoprecipitated with MAb 327 and electrophoresed on a 10% SDS-polyacrylamide gel as described above. Cleavage with cyanogen bromide was performed as described previously (22). Briefly, the bands containing p6Osrc were excised, washed in 10% methanol, and lyophilized. The bands were then incubated at room temperature for 30 min in 0.5 ml of 70% formic acid containing 50 mg of cyanogen bromide (Sigma) per ml. The bands were washed, dried, and loaded onto a 18.75% SDS- polyacrylamide gel exactly as described previously (21). After electrophoresis was complete (usually 24 h at 70 V), the gel was washed four times in 10% methanol-10% acetic acid-10% glycerol for several hours and then dried and exposed.
PI kinase activity. Frozen cell pellets were thawed, lysed in NP40 buffer (20 mM Tris hydrochloride [pH 7.5], 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride) and immunoprecipitated with MAb 327 coupled to protein A- Sepharose beads (17). The immunoprecipitates were washed and reacted with micelles of phosphatidylinositol (PI) in the presence of [y-32P]ATP as described previously (12). The reaction products were separated on thin-layer silica gel plates (12) and visualized by autoradiography.
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POINT MUTATIONS IN SH2 ACTIVATE c-src 2857
c-src v- fps/fes v-abl v-crk PLC (N) PLC (C) GAP (N) GAP (C)
148 820 248 248 550 668 178 348
WYFGKITRRESERLLLNPENPRGTFLVRESETTKGAYCLSVSDFDNAKGLNVKHYKI WYHGAIPRSEVNELLKYS....GDFLVRESQG.KQEYVLSVL.WDGQPR....HFII WYHGPVSRNAAEYLLSSGIN..GSFLVRESESSPGQRSISLR... YEGR.VYHYRI WYWGRLSRGDAVSLLQGQRH .. GTFLVRDSGSIPGDFVLSVSESSRVS..... HYIV
A A A A
FIG. 1. Conserved residues in the SH2 domain. The amino acid sequences of the following gene products were aligned by eye: c-src (55); feline sarcoma virus v-fpslfes (53); Abelson murine leukemia virus gag-abl (45); CT10 v-crk (38); GAP, amino (N)- and carboxy (C)-terminal repeats of SH2 (57); and phospholipase C-y (PLC), N- and C-terminal repeats (54; nomenclature from reference 48). Numbers indicate the position of the first residue shown. c-src and RSV v-src (SRA) code for identical amino acids between residues 124 and 338. Asterisks mark residues conserved in at least six of the eight sequences shown. Arrowheads mark the residues selected for oligonucleotide-directed mutagenesis in c-src. Only a portion of the SH2 domain is shown.
RESULTS
Construction of point mutations and expression of mutant proteins. To investigate the function of the SH2 region in c-src, four highly conserved residues were chosen as the sites of nonconservative point mutations (Fig. 1). Trp-148 was changed to arginine (W148R), Arg-155 was changed to serine (R155S), Gly-170 was changed to isoleucine (G1701), and His-201 was changed to leucine (H201L). Oligonucle- otide-directed mutagenesis of the c-src gene was performed on a uracil-substituted vector (30). Replication-competent viruses were harvested after transfection of parental (c-src) and mutant plasmids into CEF, and were used in all exper- iments. CEF infected with the mutant viruses were collected, and
the lysates were analyzed for the presence of p6Osrc proteins by Western immunoblotting with MAb 327 (Fig. 2). Roughly equal amounts of p6Osrc were detected in each lane, indicat- ing that although the transforming abilities of the mutant proteins varied (see below), they were expressed at compa- rable levels in the infected cells. All the proteins, including parental p60c-src are highly overexpressed in this system, and the level of endogenous p60c-src is negligible in compar- ison. Morphology of infected cells and colony formation. Photo-
graphs of CEF infected with the mutant viruses are shown in Fig. 3. Cells expressing p60W148R appeared disorganized as well as more rounded and refractile than cells expressing the parental c-src protein. This was also true, to a lesser extent, for G1701 and R155S. Cells expressing p60H201L did not look different from the parental cells. We tested the mutant viruses for temperature sensitivity but did not detect any difference in the phenotypes at 41°C.
.sc+8jAbbc55\1O\ eWz
pp60-
FIG. 2. Expression of the altered proteins in CEF. Confluent, fully infected cultures of CEF infected with parental (c-src) or mutant viruses were collected and lysed as described in Materials and Methods. The p6Osrc in 100 ,ug of whole-cell lysates was detected by transfer to nitrocellulose and binding to MAb 327, followed by 1251I-labeled sheep anti-mouse serum.
The ability of CEF infected with the mutant viruses to grow in soft agar was tested (Table 1). In order of number of colonies induced, the viruses ranked W148R G1701 > R155S >> H201L. None of the viruses were as effective as RSV (expressing p60vsrc) at inducing anchorage-indepen- dent growth of infected cells. Colonies induced by the mutant viruses were compact, somewhat variable in size, and smaller on average than colonies induced by RSV.
Tyrosine kinase activity of mutant c-src proteins. To deter- mine the specific kinase activities of the mutant p6Osrc proteins, we immunoprecipitated them from RIPA lysates with MAb 327. The immunoprecipitates were split; one half was used to measure protein, and the other was tested for kinase activity. The levels of protein detected after immu- noprecipitation and Western blotting varied (Fig. 4), in contrast to the results shown in Fig. 2 (comparable expres- sion of all mutants), but no particular p60 was consistently low between experiments. The specific kinase activities for all experiments were quantitated by counting the bands containing 1251 or 32P and dividing the phosphorylation counts by the protein counts. The ratio of the specific activity of each mutant to the specific activity of p60csrc was then calculated. Figure 5 shows these ratios, as the average of four separate experiments, for both autophosphorylation and enolase. p60W148R and p6OG170I had specific activities that were elevated about twofold over the level of the parental c-src protein. By contrast, the p60v-src protein has been found to have a specific kinase activity at least 10 times that of p60c-src (19). We wanted to see whether these elevated kinase activities
were reflected in the in vivo level of phosphorylation in infected CEF. Equal amounts of protein in lysates of in- fected cells were electrophoresed through a 10% SDS- polyacrylamide gel and then transferred to nitrocellulose. The blot was then probed with antibody to phosphotyrosine
TABLE 1. Colony formation
Virus No. of colonies>0.2 mm'
None ........................................ 0 SH (c-src) ............ ........................... 8 W148R ........................................ 42 R15SS ........................................ 20 G1701 ........................................ 46 H201L ........................................ 5 SRA (v-src) ....................................... 70
a Colony formation was assayed in 100-mm-diameter dishes as described in Materials and Methods.
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FIG. 3. Photographs of infected cells in monolayers. CEF were infected with parental (c-src) or mutant viruses, passaged twice to allow virus spread, and then overlaid with soft agar. (a) Mock-infected CEF; (b to f) CEF infected with c-src (b), W148R (c), R155S (d), G1701 (e), H201L (f).
(Fig. 6). The band detected at about 60 kilodaltons (kDa) in all lanes is most likely the src protein itself. In cells express-
ing p60csrc, this is the major tyrosine-phosphorylated pro-
tein. In cells infected with RSV (v-src lane), the number of proteins phosphorylated on tyrosine was greatly increased. Most known partially transforming mutants of c-src have tyrosine phosphorylation patterns intermediate between those of c-src and v-src (21, 40). CEF infected with W148R had increased levels of phosphorylation on tyrosine (Fig. 6). Prominent bands were seen at 36, 60, 67, and 120 to 140 kDa. Longer exposures showed bands at 80, 90, 170, and 200 kDa. The same proteins were also seen to have slightly increased levels of tyrosine phosphorylation upon infection with G170I
pp6O
(Fig. 6). The 36-kDa protein is most likely the calpactin I heavy chain, a well-known substrate of p60vsrc and trans- forming variants of p60csrc (32). The identities of the other proteins are unknown, although some match the molecular weights of proteins previously seen in cells transformed by various tyrosine-specific kinases (24, 35, 47, 59).
In vivo phosphorylation sites of mutant c-src proteins. p60c-src is normally found to be phosphorylated on tyrosine 527 in vivo. In many transforming variants of p60csrc, the in vivo phosphorylation site shifts to Tyr-416, the residue that is phosphorylated in p60v-src (20). To determine how the point mutations in SH2 affected the site of in vivo phosphor- ylation, we performed cyanogen bromide cleavage on src
B c (°C5
enolase
FIG. 4. Specific kinase activities of parental and mutant src proteins. Lysates of infected cells were immunoprecipitated with MAb 327 and divided in half. One half of each immunoprecipitate was used to quantitate the levels of src protein by Western blotting and detection by MAb 327, followed by 1251I-labeled sheep anti-mouse serum (A); the other half was used for an in vitro kinase assay with acid-denatured enolase as an exogenous substrate (B), and the products were separated on a 10% SDS-polyacrylamide gel.
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FIG. 5. Increase in the specific kinase activities of mutant pro- teins. Bands detected by autoradiography of gels such as those in Fig. 4 were excised and counted. The 32P counts were divided by the 125I counts for each sample. The ratio of these values to the value for c-src was determined for each mutant. The graph shows these ratios (for autophosphorylation and phosphorylation of the exogenous substrate enolase) as the average of four experiments, with the standard deviations given as error bars.
proteins metabolically labeled with 32Pi. Such cleavage can separate phosphopeptides containing tyrosines 416 and 527 (22). All four mutant p60s, as well as overexpressed p60csrc, yielded a cleavage product of 4 kDa that is known to contain Tyr-527 (Fig. 7). Only p6fv-src produced the phosphopeptide of 10 kDa that contains Tyr-416.
Coprecipitation of mutant c-src proteins with PI kinase activity. It has been shown that cells stimulated with platelet- derived growth factor or transformed with polyomavirus middle T antigen have increased PI kinase activity (4, 25). This activity converts PI into phosphatidylinositol 3-phos- phate (12, 61). PI kinase activity coprecipitates with various
kDa cf 10 *c
200-
92.5-
69-
46-
30-
FIG. 6. Detection of phosphotyrosine-containing proteins in cells expressing the mutant proteins. Lysates from cells infected with parental (c-src), mutant, or SRA (v-src) virus were normalized for protein content, and 100 ,ug of each sample was electrophoresed on a l1o SDS-polyacrylamide gel and transferred to nitrocellulose. Incubation with antibody to phosphotyrosine and detection with 125I-protein A was performed as described in Materials and Meth- ods.
FIG. 7. Phosphorylation state of mutant src proteins. Cells in- fected with parental (c-src), mutant, or SRA (v-src) virus were metabolically labeled with 32Pi, lysed, and immunoprecipitated with MAb 327. The bands as located by autoradiography were excised and digested with CNBr and then separated on an 18.75% SDS- polyacrylamide gel containing 6 M urea. The 10-kDa phosphopep- tide contains Tyr-416, and the 4 kDa phosphopeptide contains Tyr-527 (22).
transforming oncogene products, including activated vari- ants of p60-src , and seems to be necessary but not sufficient for such transformation (13). The mutant proteins were tested for PI kinase activity (Fig. 8). The activity in immu- noprecipitates of overexpressed p6fc-src (lane 2) was compa- rable to that found in mock-infected CEF (lane 1). In contrast, the phosphatidylinositol 3-phosphate spot pro- duced by immunoprecipitates of p60vsrc (lane 3) was much larger. Both p60W148R and p60G17OI showed levels of PI kinase activity greater than that of p60csrc (compare lanes 4 and 6 with lane 2). Neither p60R155S nor p60H2O1L showed elevated PI kinase activity (lanes 5 and 7).
DISCUSSION
We have shown that certain point mutations in the SH2 region of c-src lead to partial activation of the transforming ability of this proto-oncogene. Cells infected with viruses
l 2 3 4 5 6 7
99... -PIP
O0 U) C LONL °EEO FIG. 8. PI kinase activity associated with mutant src proteins.
The PI kinase activity brought down with MAb 327 immunoprecip- itates of src proteins was assayed by incubation with PI and [y-32P]ATP. The products were analyzed by thin-layer chromatog- raphy on a silica plate. Lanes: 1, mock-infected CEF; 2, parental (c-src)-infected CEF; 3, SRA (v-src)-infected CEF; 4 to 7, CEF infected with mutant viruses as shown. PIP, Phosphatidylinositol 3-phosphate.
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2860 O'BRIEN ET AL.
expressing the altered c-src genes W148R, R155S, and G1701 had a morphology different from that of cells overexpressing c-src; they also grew colonies in soft agar. The morpholog- ical changes and colony induction were not, however, as strong as those seen on infection with virus expressing p60vsrc. Many previous mutations of c-src have produced partially transforming phenotypes (27, 40; reviewed in refer- ence 16). However, such activation by point mutations in the SH2 region has not been previously reported.
It could be argued that the transforming properties of cells infected with these mutants are due to spontaneous viral mutations that arise during serial passage of CEF. There is precedent for such spontaneous transforming mutants (19, 34). We do not think that this is the case, because the transformed phenotypes described here are always seen when the cells are passaged just once after infection. Fur- thermore, transforming characteristics were never observed in the cultures infected with H201L or virus expressing c-src.
In addition to the altered morphology and formation of colonies, several biochemical properties of the mutant pro- teins indicated partial transformation. (i) Two of the mu- tants, p60w148R and p60G17OI, had elevated specific kinase activities in vitro. (ii) The same two mutants produced an increase in the number of proteins phosphorylated on ty- rosine in vivo, although p60G170I had only a slight effect. (iii) The two mutant proteins, p60W148R and p60G17OI, were shown to associate with a PI kinase activity. Taken together, these observations indicate that point mutations at positions 148 and, to a lesser extent, 170, are capable of activating the transforming ability of p60csrc. The mutants were all shown to retain the major phosphor-
ylation site of p60`src, Tyr-527. Even long exposures of gels such as that in Fig. 7 showed no evidence of any phosphor- ylation on Tyr-416. Other partially activated c-src proteins have been found to be phosphorylated on both Tyr-416 and Tyr-527 in varying proportions (21, 50). There is some indication that these constitute separate populations and that the molecules phosphorylated on Tyr-416 are responsible for the transforming phenotype. This could be the case here if the population of molecules phosphorylated on Tyr-416 was so small as to be undetectable. However, there are other examples of activated forms of p60c-src that do not demon- strate a shift to phosphorylation on Tyr-416: neuronal p60c src+, which contains a six-amino-acid insert in the SH3 domain (33, 37), mutants containing various substitutions and small deletions around amino acids 90 to 95 (43), and p60c-src phosphorylated by p34cdc2 (3, 39, 52) or by platelet- derived growth factor (14). It is notable that many of these activations involve small increases in specific kinase activ- ity, comparable to the increases measured here. The effect of specific amino acid substitutions in the SH2
region varied; we attempted to make nonconservative changes, but it is difficult to know what properties of a particular residue are crucial. The introduction of a positive charge at position 148 had a particularly strong effect, greater than the effect of eliminating a charge at position 155. The conserved glycine at position 170 could be in a type II reverse turn (5) or another structural element in which this smallest of side chains is strongly favored. Trp-148 is abso- lutely conserved in all known copies of SH2. Arg-155, Gly-170, and His-201 are not, however, because the C- terminal version of SH2 in GAP has lysine at position 155, cysteine at position 170, and arginine at position 201 (Fig. 1). It is possible that the two copies of SH2 in GAP serve different functions and are therefore subject to different
selection pressures. We attempted to disrupt p6f-fsrc at His-201 by substituting a nonpolar residue, leucine. This substitution appeared to have no effect. A different substi- tution, perhaps a larger side chain, might have had more effect. The host range mutants created by mutations of SH2 in
v-fps and v-src have led to speculation about a putative host-cell protein that interacts with the SH2 region. Rey- nolds et al. (46) have reported that two proteins, of 110 and 130 kDa, coprecipitate with p60v-src and transforming vari- ants of p60csrc. However, they found that the two proteins were not coprecipitated with p60d1155/527F. This transforma- tion-defective mutant has an activating substitution of phen- ylalanine for tyrosine at residue 527, and also a deletion of residues 155 to 157 within the SH2 region. This result implicates the SH2 region in the binding of cellular proteins that are correlated with transformation. Verderame et al. (56) have reported the isolation of a host-dependent mutant of v-src that has a deletion of three nucleotides resulting in the in-frame loss of the Phe-172 codon. Our results are surprising because previous alterations of
SH2 in v-fps and v-src have been shown to reduce kinase activity. The action of the SH2 region could differ between the cellular and activated viral versions of oncogenes. How- ever, Nemeth et al. (40) report that deletion of residues 112 to 225 in p60c-src suppressed the slight phenotypic alterations normally induced by this protein. Deletion of these residues also suppressed the transforming activity of the activated c-src variant F527, and the NP mutant (deletion of residues 15 to 225) was less transforming than c-srcNX (deletion of residues 15 to 89). The discrepancy with our results could be due to the different types of mutations reported; in general, large deletions are more likely to perturb the overall folding pattern of a protein. Small deletions and point mutations probably give only local disruption, which in this case could affect the binding of the putative cellular protein or interfere with a possible cooperation between the SH2 region and the kinase domain in substrate selection. The results reported here are consistent with the idea that interaction of p60csrc with crucial transforming substrates is regulated in the normal cell and that disruption of the SH2 region can deregulate this interaction.
ACKNOWLEDGMENTS
We thank Bruce Mayer for critically reading the manuscript. We also thank David Stemnberg, Kathy Barker, and Sally Kombluth for contributions to this work. We are grateful to Richard Jove for invaluable discussions.
This work was supported by Public Health Service grant CA44356 from the National Cancer Institute. M.C.O. was supported by Public Health Service training grant AI07233 from the National Institutes of Health.
LITERATURE CITED 1. Brugge, J. S., and D. Darrow. 1984. Analysis of the catalytic
domain of phosphotransferase activity of two avian sarcoma virus-transforming proteins. J. Biol. Chem. 259:4550-4557.
2. Bryant, D., and J. T. Parsons. 1982. Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation. J. Virol. 44:683-691.
3. Chackalaparampil, I., and D. Shalloway. 1988. Altered phos- phorylation and activation of pp60csrc during fibroblast mitosis. Cell 52:801-810.
4. Courtneidge, S. A., and A. Heber. 1987. An 81 kd protein complexed with middle T antigen and pp60csrc: a possible phosphatidylinositol kinase. Cell 50:1031-1037.
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5. Crawford, J. L., W. N. Lipscomb, and C. G. Scheliman. 1973. The reverse turn as a polypeptide conformation in globular proteins. Proc. Natl. Acad. Sci. USA 70:538-542.
6. Cross, F. R., E. A. Garber, and H. Hanafusa. 1985. N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene. Mol. Cell. Biol. 5:2789-2795.
7. Cross, F. R., and H. Hanafusa. 1983. Local mutagenesis of Rous sarcoma virus: the major sites of tyrosine and serine phosphor- ylation of p6O0' are dispensable for transformation. Cell 34: 597-607.
8. DeClue, J. E., and G. S. Martin. 1989. Linker insertion-deletion mutagenesis of the v-src gene: isolation of host- and tempera- ture-dependent mutants. J. Virol. 63:542-554.
9. DeClue, J. E., I. Sadowski, G. S. Martin, and T. Pawson. 1987. A conserved domain regulates interactions of the v-fps protein- tyrosine kinase with the host cell. Proc. Natl. Acad. Sci. USA 84:9064-9068.
10. DeLorbe, W. J., P. A. Luciw, H. M. Goodman, H. E. Varmus, and J. M. Bishop. 1980. Molecular cloning and characterization of avian sarcoma virus circular DNA molecules. J. Virol. 36:50-61.
11. Ellis, C., M. Moran, F. McCormick, and T. Pawson. 1990. Phosphorylation of GAP and GAP-associated proteins by trans- forming and mitogenic tyrosine kinases. Nature (London) 343: 377-381.
12. Fukui, Y., and H. Hanafusa. 1989. Phosphatidylinositol kinase activity associates with viral p60src protein. Mol. Cell. Biol. 9:1651-1658.
13. Fukui, Y., S. Kornbluth, S.-M. Jong, L.-H. Wang, and H. Hanafusa. 1989. Phosphatidylinositol kinase type I activity associates with various oncogene products. Oncogene Res. 4:283-292.
14. Gould, K. L., and T. Hunter. 1988. Platelet-derived growth factor induces multisite phosphorylation of pp60c-rc and in- creases its protein-tyrosine kinase activity. Mol. Cell. Biol. 8:3345-3356.
15. Hanafusa, H. 1969. Rapid transformation of cells by Rous sarcoma virus. Proc. Natl. Acad. Sci. USA 63:318-325.
16. Hanafusa, H. 1986. Activation of the c-src gene, p. 100-105. In P. Kahn and T. Graf (ed.), Oncogenes and growth control. Springer-Verlag KG, Berlin.
17. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory man- ual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
18. Hunter, T. 1987. A tail of two src's: mutatis mutandis. Cell 49:1-4.
19. Iba, H., T. Takeya, F. R. Cross, T. Hanafusa, and H. Hanafusa. 1984. Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts. Proc. Natl. Acad. Sci. USA 81:4424 4428.
20. Jove, R., and H. Hanafusa. 1987. Cell transformation by the viral src oncogene. Annu. Rev. Cell Biol. 3:31-56.
21. Jove, R., T. Hanafusa, M. Hamaguchi, and H. Hanafusa. 1989. In vivo phosphorylation states and kinase activities of trans- forming p60csrc mutants. Oncogene Res. 5:49-60.
22. Jove, R., S. Kornbluth, and H. Hanafusa. 1987. Enzymatically inactive p60csrc mutant with altered ATP-binding site is fully phosphorylated in its carboxyl-terminal regulatory region. Cell 50:937-943.
23. Jove, R., B. J. Mayer, H. Iba, D. Laugier, F. Poirer, G. Calothy, T. Hanafusa, and H. Hanafusa. 1986. Genetic analysis of p60v-src domains involved in the induction of different cell transforma- tion parameters. J. Virol. 60:840-848.
24. Kamps, M. P., and B. M. Sefton. 1988. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2:305-315.
25. Kaplan, D. R., M. Whitman, B. Schaffhausen, D. C. Pallas, M. White, L. Cantley, and T. M. Roberts. 1987. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell 50:1021-1029.
26. Kaplan, J. M., G. Mardon, J. M. Bishop, and H. E. Varmus.
1988. The first seven amino acids encoded by the v-src onco- gene act as a myristylation signal: lysine 7 is a critical determi- nant. Mol. Cell. Biol. 8:2435-2441.
27. Kato, J.-Y., T. Takeya, C. Grandori, H. Iba, J. B. Levy, and H. Hanafusa. 1986. Amino acid substitutions sufficient to convert the nontransforming p60csr, protein to a transforming protein. Mol. Cell. Biol. 6:4155-4160.
28. Kitamura, N., and M. Yoshida. 1983. Small deletion in src of Rous sarcoma virus modifying transformation phenotypes: identification of 207-nucleotide deletion and its smaller product with protein kinase activity. J. Virol. 46:985-992.
29. Koch, C. A., M. Moran, I. Sadowski, and T. Pawson. 1989. The common src homology region 2 domain of cytoplasmic signaling proteins is a positive effector of v-fps tyrosine kinase function. Mol. Cell. Biol. 9:4131-4140.
30. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367-382.
31. Levinson, A. D., S. A. Courtneidge, and J. M. Bishop. 1981. Structural and functional domains of the Rous sarcoma virus transforming protein (pp6Osrc). Proc. Natl. Acad. Sci. USA 78:1624-1628.
32. Levy, J. B., and J. S. Brugge. 1989. Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts. Mol. Cell. Biol. 9:3332-3341.
33. Levy, J. B., T. Dorai, L.-H. Wang, and J. S. Brugge. 1987. The structurally distinct form of pp60csr' detected in neuronal cells is encoded by a unique c-src mRNA. Mol. Cell. Biol. 7: 4142-4145.
34. Levy, J. B., H. Iba, and H. Hanafusa. 1986. Activation of the transforming potential of p60csr' by a single amino acid change. Proc. Natl. Acad. Sci. USA 83:4228-4232.
35. Linder, M. E., and J. G. Burr. 1988. Immunological character- ization of proteins detected by phosphotyrosine antibodies in cells transformed by Rous sarcoma virus. J. Virol. 62:2665- 2673.
36. Lipsich, L. A., A. J. Lewis, and J. S. Brugge. 1983. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J. Virol. 48:352-360.
37. Martinez, R., B. Mathey-Prevot, A. Bernards, and D. Baltimore. 1987. Neuronal pp60csr' contains a six-amino acid insertion relative to its non-neuronal counterpart. Science 237:411-415.
38. Mayer, B. J., M. Hamaguchi, and H. Hanafusa. 1988. A novel viral oncogene with structural similarity to phospholipase C. Nature (London) 332:272-275.
39. Morgan, D. O., J. M. Kaplan, J. M. Bishop, and H. E. Varmus. 1989. Mitosis-specific phosphorylation of p60csr' by p34cdc2- associated protein kinase. Cell 57:775-786.
40. Nemeth, S. P., L. G. Fox, M. DeMarco, and J. S. Brugge. 1989. Deletions within the amino-terminal half of the c-src gene product that alter the functional activity of the protein. Mol. Cell. Biol. 9:1109-1119.
41. Parker, R. C., H. E. Varmus, and J. M. Bishop. 1984. Expres- sion of v-src and chicken c-src in rat cells demonstrates quali- tative differences between pp60v-sr and pp60csrc. Cell 37: 131-139.
42. Pawson, T. 1988. Non-catalytic domains of cytoplasmic protein- tyrosine kinases: regulatory elements in signal transduction. Oncogene 3:491-495.
43. Potts, W. M., A. B. Reynolds, T. J. Lansing, and J. T. Parsons. 1988. Activation of pp60c-src transforming potential by muta- tions altering the structure of an amino terminal domain con- taining residues 90-95. Oncogene Res. 3:343-355.
44. Raymond, V. W., and J. T. Parsons. 1987. Identification of an amino terminal domain required for the transforming activity of the Rous sarcoma virus src protein. Virology 160:400-410.
45. Reddy, E. P., M. J. Smith, and A. Srinivasan. 1983. Nucleotide sequence of Abelson murine leukemia virus genome: structural similarity of its transforming gene product to other onc products with tyrosine-specific kinase activity. Proc. Natl. Acad. Sci. USA 80:3623-3627.
46. Reynolds, A. B., S. B. Kanner, H.-C. R. Wang, and J. T. Parsons. 1989. Stable association of activated pp605" with two
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.org/ D
tyrosine-phosphorylated cellular proteins. Mol. Cell. Biol. 9: 3951-3958.
47. Reynolds, A. B., D. J. Roesel, S. B. Kanner, and J. T. Parsons. 1989. Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene. Mol. Cell. Biol. 9: 629-638.
48. Rhee, S. G., P.-G. Suh, S.-H. Ryu, and S. Y. Lee. 1989. Studies of inositol phospholipid-specific phospholipase C. Science 244: 546-550.
49. Sadowski, I., J. C. Stone, and T. Pawson. 1986. A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fuji- nami sarcoma virus p130gag-fPs. Mol. Cell. Biol. 6:4396-4408.
50. Sato, M., J.-Y. Kato, and T. Takeya. 1989. Characterization of partially activated p60csrc in chicken embryo fibroblasts. J. Virol. 63:683-688.
51. Shalloway, D., P. M. Coussens, and P. Yaciuk. 1984. Overex- pression of the c-src protein does not induce transformation of NIH 3T3 cells. Proc. Natl. Acad. Sci. USA 81:7071-7075.
52. Shenoy, S., J.-K. Choi, S. Bagrodia, T. D. Copeland, J. L. Mailer, and D. Shalloway. 1989. Purified muturation promoting factor phosphorylates pp60c-src at the sites phosphorylated dur- ing fibroblast mitosis. Cell 57:763-774.
53. Shibuya, M., and H. Hanafusa. 1982. Nucleotide sequence of Fujinami sarcoma virus: evolutionary relationship of its trans- forming gene with transforming genes of other sarcoma viruses. Cell 30:787-795.
54. Stahl, M. L., C. R. Ferenz, K. L. Kelleher, R. W. Kriz, and J. L.
Knopf. 1988. Sequence similarity of phospholipase C with the non-catalytic region of src. Nature (London) 332:269-272.
55. Takeya, T., and H. Hanafusa. 1983. Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus. Cell 32: 881-890.
56. Verderame, M. F., J. M. Kaplan, and H. E. Varmus. 1989. A mutation in v-src that removes a single conserved residue in the SH-2 domain of pp6O-src restricts transformation in a host- dependent manner. J. Virol. 63:338-348.
57. Vogel, U. S., R. A. F. Dixon, M. D. Schaber, R. E. Diehl, M. S. Marshall, E. M. Scolnick, I. S. Sigal, and J. B. Gibbs. 1988. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature (London) 335:90-93.
58. Wang, H.-C. R., and J. T. Parsons. 1989. Deletions and inser- tions within an amino-terminal domain of pp6Ov-src inactivate transformation and modulate membrane stability. J. Virol. 63: 291-302.
59. Wang, J. Y. J. 1985. Isolation of antibodies for phosphotyrosine by immunization with a v-abl oncogene-encoded protein. Mol. Cell. Biol. 5:3640-3643.
60. Wendler, P. A., and F. Boschelli. 1989. Src homology 2 domain deletion mutants of p60v-src do not phosphorylate cellular pro- teins of 120-150 kDa. Oncogene 4:231-236.
61. Whitman, M., C. P. Downes, M. Keeler, T. Keller, and L. Cantley. 1988. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature (London) 332:644-646.
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.org/ D