The lethality of p60^v-src in Saccharomyces cerevisiae and the activation of p34^CDC28 kinase are dependent on the integrity of the SH2 domain

The Src family of protein tyrosine kinases has, in addition to a highly conserved catalytic domain, two regions, termed the Src homology 2 and 3 domains (SH2 and SH3, respectively), which are found in a variety of other protein families (Koch et al., 1991). The SH2 domains act as sites for interaction with proteins phosphorylated on tyrosine (Matsuda et al., 1991; Mayer et al., 1991, 1992; Overduin et al., 1992; Roussel et al., 1991), whereas the SH3 domain is present in many cytoskeletal proteins (Koch et al., 1991). Together, these domains are believed to direct the formation of protein complexes that function in signal transduction. The SH2 domain of v-Src extends from amino acid residue 140 to 240 in this 526 amino acid protein (Koch et al., 1991). Mutations in this SH2 domain of v-Src affect transforming activity, the specificity of cellular protein phosphorylation and the intracellular localization of Src protein, while alterations in the SH2 domain of c-Src protein can activate its transforming potential (Bryant and Parsons, 1984; Cross et al., 1985; Fukui et al., 1991; Hirai and Varmus, 1990; Kanner et al., 1991; Kaplan et al., 1990; Nemeth et al., 1989; O’Brien et al., 1990; Wang and Parsons, 1989; Wendler and Boschelli, 1989). The SH2 domains of various cytoplasmic protein tyrosine kinases are capable of binding to phosphotyrosine-containing peptides, and any particular SH2 domain exhibits a specificity for some phosphotyrosine-containing peptides over others (Matsuda et al., 1991; Mayer et al., 1991, 1992; Overduin et al., 1992; Roussel et al., 1991). In the recently described crystal and solution structure of SH2 domains, a distinct binding site for phosphotyrosine peptides was identified (Overduin et al., 1992; Waksman et al., 1992). The SH2 domain therefore appears to be the determinant that defines the cellular substrate proteins for cytoplasmic protein tyrosine kinases.

The transforming gene of Rous sarcoma virus, v-src, but not its cellular cognate gene, c-src, is toxic to the budding yeast Saccharomyces cerevisiae (Brugge et al., 1987; Kornbluth et al., 1987). We have found that native v-Src protein perturbs the formation of the mitotic spindle in yeast, but the ability of cells to arrest in G₂ in response to spindle dysfunction is lost. Cells pass through START and the cellular DNA content increases beyond the completely replicated 2N level (F. Boschelli, unpublished). This behavior is also seen in yeast strains with mutations that allow DNA replication to proceed in the absence of mitosis (Hoyt et al., 1991; Li and Murray, 1991). These mutations interfere with genes whose protein products function as regulators of the p34^CDC28 kinase, the S. cerevisiae homolog of the p34^cdc2 kinase present in all eukaroyotes (Forsburg and Nurse, 1991). As is the case with these endogenous yeast mutations, v-Src also abrogates regulation of Cdc28 kinase, and the histone H1-associated Cdc28 kinase activity increases when v-Src is expressed (F. Boschelli, unpublished). The cell cycle defects apparent when v-Src is expressed in yeast strongly suggest that the lethality of v-Src in yeast involves phosphorylation of yeast proteins with important cell cycle functions.

In this paper, further evidence to support this conclusion is presented. As shown here, the SH2 domain of v-Src is functionally significant in v-Src-mediated cell death in S. cerevisiae, since mutations in the SH2 domain can negate the lethal effects of v-Src expression. Catalytically active v-Src proteins with deletions in the SH2 domain, which are weakly transforming in vertebrate cells, have no effects on yeast viability, in spite of the fact that these altered Src proteins can phosphorylate yeast proteins extensively. In addition, instead of the large increase in Cdc28 kinase activity occurring when native v-Src is expressed, Cdc28 kinase activity is unaffected when the c-Src protein or an altered v-Src protein is expressed. These results suggest that a functional SH2 domain is required for phosphorylation of yeast proteins with a physiological role in the lethal activity of v-Src in yeast. These phosphorylation events must in some manner result in activation of p34^CDC28 kinase and loss of cell cycle control.

Standard methods were employed for yeast manipulations (Sherman et al., 1986). Strains BWG1-7A (Mataura3-52 leu2-3,112 ade2-100 his4-519) and PSY142 (Mata. ura3-52 leu2-3,112 lys2-801) were obtained from L. Guarente’s laboratory (MIT, Cambridge, MA). Diploid strain BP1 was made by crossing these two haploid strains. W303 (Mata ura3-1 leu 2-3,112 ade2-1 trp1-1 his3-11,15) was obtained from S. Ackerman (WSU).

The pGal vector B656 was obtained from G. Fink’s laboratory (Whitehead Institute, Cambridge, MA). This 2 μm vector, pB656, has a URA3 gene and a Gal1 promoter upstream of a BamHI cloning site. PB656 was modified to change the BamHI cloning site to a HindIII-BamHI segment by digesting the plasmid with BamHI, filling in the overhangs with Klenow enzyme and ligating a 12 base pair HindIII linker (New England Biolabs) into this site. BamHI sites were regenerated upstream and downstream from the HindIII site, so the resultant plasmid was subjected to a partial BamHI digest and filled in with Klenow enzyme to yield the plasmid pB700 with the HindIII upstream from the BamHI site (with respect to the Gal1 promoter). The wt and mutant v-Src genes described by Wendler and Boschelli (1989) were modified for this work by insertion of a HindIII linker at the NcoI site at the beginning of the v-src gene (Schwartz et al., 1983). The wt and mutant genes were cloned into the HindIII-BamHI sites of the Gal vector to give pBsrc. The Gal vector pL5 was made by removing the part of the URA3 gene in pB656 and replacing it with the LEU2 gene from YEp13. PBsrc was digested with EcoRV (cuts in the URA3 gene) and a 12mer XhoI linker was inserted. This plasmid was digested with SalI and XhoI and the SalI-XhoI-flanked LEU2 gene from YEp13 was inserted. PLv-Src was con-structed by cloning the EcoRI-SalI Gal promoter-v-Src segment into the same region of pL5. Mutant genes were inserted by cloning the HindIII-SalI flanked src genes from pBsrc into the corresponding sites of pLv-Src. The HindIII-BamHI-flanked chicken c-src gene from pc-SrcHis (Wendler and Boschelli, 1989) was cloned into the corresponding region of the pBv-Src and pLv-Src clones to give pBc-Src or pLc-Src. Construction of the SH2 domain deletions in v-src has been described (Wendler and Boschelli, 1989).

src genes with deletions in the catalytic domain were constructed by M13-splint mutagenesis as described (Wendler and Boschelli, 1989). The D15Src gene has amino acid residues 438-464 deleted. This in-frame deletion was created by digesting a hybrid of the wild-type (wt) v-src gene in single-stranded M13 DNA and a complementary subclone of residues 8419-8581 (in the Schwartz et al. sequence of Prague C Rous sarcoma virus) also in M13 single-stranded DNA with HaeIII (Schwartz et al., 1983). The digested hybrid was ligated and transformed into Escherichia coli. Clones with the deletion were identified by restriction mapping and confirmed by sequencing. The D12Src gene was made by digesting a hybrid of intact v-src and a complementary subclone of residues 7916-8219 with RsaI. There are three RsaI sites in the double-stranded segment formed in this hybrid, and four different src genes were isolated from the clones after transformation of E. coli with the ligated digestion mix. One of these genes, the D12Src gene, has a deletion of nucleotides 8105-8197 and an insertion of one of the RsaI fragments (nucleotides 8146-8105) in the nonsense orientation. This insertion introduces a four amino acid (SVTM) sequence followed by a stop codon after residue 326 in the v-src gene, yielding a 330 amino acid Src protein. These two genes are non-transforming and catalytically inactive when expressed in Rat 1 fibroblasts (unpublished data, this laboratory). They were cloned into the 2μm expression pGal vector B656 via 5′ HindIII and 3′ BamHI sites distal to the GAL1 promoter. Haploid strain W303 was transformed with these plasmids, along with the control plasmid pB656 and the wild-type v-Src plasmid pBSrc.

Cells used for plate assays were grown in minimal raffinose liquid medium for 24 h. Liquid cultures were spotted on Gal plates. Growth was scored in two to three days. Growth was also scored by streaking for single colonies on solid galactose medium.

For viability assays, strains were grown overnight at 30°C in minimal medium, 0.67% yeast nitrogen base, 0.1% Casamino acids and 2% raffinose medium lacking uracil (YNBRaf). The overnight cultures were diluted to an absorbance at 600 nm of 0.1 into fresh medium, grown for 1.5 h and then galactose was added to 2%. Aliquots were taken at various times, briefly sonicated and plated onto solid minimal medium with glucose as carbon source. Cell number was determined with a hemocytometer.

Fluorescence microscopy was performed with a Zeiss Axiophot microscope. Cells were grown for 5 h in YNBRaf/Gal(2%/2%) after overnight growth in YNBRaf. Cells were formalin fixed for microtubule staining and treated with Zymolyase prior to incubation with YOL1-34 monoclonal antibody to α-tubulin (ICN) and FITC-conjugated goat anti-rat antibody (ICN) (Adams and Pringle, 1984). Nuclei were stained with DAPI (4,6-diamidino-2-phenylindole).

Cells were grown overnight in YNBRaf. These cells were diluted to an A₆₀₀ of 0.2-0.3 in fresh YNBRaf and grown for 1.5 h. Galac tose was added to a final concentration of 2% (w/v) and cells were grown for a further 5 h. Cells were centrifuged and washed in 0.2 M Tris-HCl (pH 7.5) and then resuspended in this buffer. Ethanol was added to the cells to give a final concentration of 70%. After a 30 min fixation period, cells were washed with 0.2 M Tris-HCl buffer and resuspended in 0.5 ml of 50 mM Tris-HCl, 50 mM EDTA (pH 7.5). Cells were treated with 1 mg of RNase A for 2 h at 37°C, and then treated with pepsin (2.5 mg/ml) for 5 min on the bench, followed by washing with 0.2 M Tris-HCl buffer. Cells were then incubated overnight with propidium iodide (5 mg/ml). Free propidium iodide was removed by successive washes with 0.2 M Tris-HCl (pH 7.5). Cells were analyzed with a Becton Dickinson FACSCAN analyzer.

YNBGal media was inoculated to an A₆₀₀ of 0.3 with cells grown in raffinose. Cells were harvested by centrifugation about 8 h after addition of galactose and washed once in RIPA buffer (10 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1 mM EDTA, 0.1% sodium deoxycholate and 1% NP-40). Cells were vortexed for 10 min with cold, acid-washed, 0.45 mm glass beads in RIPA buffer with PMSF (1 mM), and clarified extracts were prepared by centrifugation in a microcentrifuge for 20 min. Immunoprecipitations with mAb327 (a generous gift from J. Brugge, University of PA) and enolase kinase assays were performed as described (Wendler and Boschelli, 1989), except that reactions were performed in the cold (4°C) for 20 min.

Direct detection of Src proteins in crude extracts was performed by chemiluminescence detection on western blots. Overnight cultures in YNB-raffinose medium were shifted to YP galactose (1% yeast extract, 2% peptone, 2% galactose) medium at an A₆₀₀ of 0.3. The cells were grown at 30°C for 6 h and then harvested by centrifugation. The harvested cells were washed in Tris-buffered saline (20 mM Tris-HCl, pH 7.8, 0.15 M NaCl, 0.1 mM EDTA; TBS) and frozen in liquid nitrogen until used.

Extracts were prepared by breaking cells with 0.45 mm glass beads in TBS with protease inhibitors. The broken cells were incubated with RIPA buffer (TBS with 10% glycerol, 1% NP-40 and 0.1% sodium deoxycholate) for 20 min and then clarified by centrifugation in a SM-24 rotor in a Sorvall superspeed centrifuge at 12,000 r.p.m. Protein concentration in the clarified extracts was determined with the Bio-Rad protein assay reagent. Equal amounts of protein (100-200 μg) were resolved on a 9% SDS-polyacrylamide gel and blotted to PVDF membranes. Monoclonal antibody to residues 2-17 of Src protein (Microbiological Associates) was used as primary antibody. To detect the Src protein, the Amersham ECL chemiluminescence kit was used. Control experiments with v-Src protein from Rat 1 fibroblasts transformed by v-src were performed to show that the yeast and mammalian Src proteins co-migrated.

Cells grown overnight in YNBraffinose were diluted into fresh YNB-raffinose medium to an A₆₀₀ of 0.3 and grown for 1.5 h prior to the addition of galactose to 2% (w/v). After 3 h at 30°C, sodium orthovanadate was added to 1 mM and cells were shaken for a further 15 min. Cells were then centrifuged and resuspended in 20% trichloroacetic acid. The cells were pelleted again and neutralized with 2 M Tris-HCl (pH 8), and then resuspended in 50 mM Tris-HCl (pH 7.5), 10% glycerol. Extracts in 50 mM Tris-HCl (pH 7.5), 10% glycerol and 10 mM β-mercaptoethanol were prepared by breaking with 0.45 mm glass beads, and then were clarified by centrifugation for 20 min in a microcentrifuge. The insoluble pellets were then further extracted by incubation in 50 mM Tris-HCl (pH 7.5), 7 M urea and 10 mM β-mercaptoethanol, and clarified by centrifugation for 20 min in a microcentrifuge. Relative protein concentrations were estimated by analyzing aliquots of cell extracts on an SDS-polyacrylamide gel and staining with Coomassie blue. For western blots, samples with approximately equal amounts of protein were loaded on polyacrylamide gels and blotted onto PVDF membranes (Millipore). The membranes were probed with affinity-purified monoclonal antibody IgG2bk to phosphotyrosine (Upstate Biotechnologies). Blots were blocked with 3% gelatin (Bio-Rad) prior to incubation with anti-body. Non-specific reaction was determined by incubating the antibody with 5 mM phosphotyrosine prior to probing the membrane.

Overnight cultures in YNB-raffinose were diluted to an A₆₀₀ of 0.3 and incubated at 30°C for 1 h prior to the addition of galactose. Cells were harvested at the indicated times, washed in EB (80 mM β-glycerophosphate, pH 7.3, 20 mM EGTA, 15 mM MgCl₂, 1 mM DTT), and frozen in liquid nitrogen. Cells were thawed in cold water and broken by vortexing three times for 45 s with 0.45 mm glass beads at 4°C in EB with 1 mM phenyl-methylsulfonyl fluoride (PMSF), 1 mM benzamidine, 1 mM phenanthroline. Broken cells were removed by centrifugation in a microcentrifuge for 20 min. Protein concentrations were determined with the Bio-Rad assay. For the kinase assays, duplicate tubes with 180 μg of extract protein was rotated end-over-end for 1.5 h at 4°C along with 1 mg BSA in EB and p13^suc1 Sepharose beads prepared as described by Brizuela et al. (1987). The beads were washed in EB three times and then two times in kinase buffer (50 mM Tris-HCl, pH 7.5, 15 mM MgCl₂₎. Reactions were performed at room temperature in 50 μl kinase buffer with 1 mg/ml of histone H1 (Boehringer Mannheim) and 50 mM cold ATP and 5 μCi of [γ-³²P]ATP (New England Nuclear) for the indicated times (5- or 10-min reaction periods) and terminated by addition of SDS-gel sample buffer and incubation at 95°C for 5 min. Control experiments were performed to show that the 5- and 10-min reaction times fall in the linear range of the reaction.

The wt v-src, c-src, and v-src genes with deletions in the SH2 domain coding regions, were cloned into URA3 2μm expression plasmids and transformed into haploid strain BWG1-7A by the lithium acetate procedure (Itoh et al., 1983). The Src proteins with SH2 domain deletions used here are active kinases in murine fibroblasts (Wendler and Boschelli, 1989), and these proteins, when expressed in yeast, also possess kinase activity in vitro. Src kinase activity in the BWG1-7A (pBsrc) strains was measured by the enolase kinase assay (Fig. 1) (Wendler and Boschelli, 1989). Extracts from the control strain with vector alone (pB656) have no activity whereas all of the Src-expressing strains have immunoprecipitable activity that phosphorylates acid-denatured enolase. Proteins with the expected molecular masses for the Src proteins are also labeled in the assay. These strains were also pulse-labeled in vivo with [³⁵S]methionine (Reid, 1983). Src proteins were immuno-precipitated from all of these labeled extracts with monoclonal antibody mAb327 (Lipsich et al., 1983). In addition, immunoblot analysis of Src immunoprecipitates also showed that Src proteins were present (data not shown). All of the Src proteins were detected, but either method yielded results suggesting that the level of wt and altered v-Src protein expression is lower than that of c-Src, in agreement with Kornbluth et al. (1987).

The ability of the various Src proteins to inhibit yeast growth was determined by plating approximately 10² cells from mid log cultures in minimal raffinose media of BWG1-7A strains with the pBSrc plasmids onto a minimal galactose or raffinose plate as shown in Fig. 2. All of the strains grew on the raffinose plate, and on the galactose plate, all strains grew except the strain with the wt v-Src plasmid. Cell viability time-course experiments (Fig. 3) were performed with wt v-Src, c-Src, and D17Src, which has approximately 10% of wt v-Src transforming activity in Rat 1 fibroblasts and about 50% of the specific kinase activity of wt v-Src in the enolase kinase assay (Wendler and Boschelli, 1989). The strain with the wt v-Src plasmid died rapidly whereas all of the other strains grew well. These results indicate that the tyrosine kinase activity of Src proteins is not intrinsically toxic to yeast.

The BWG1-7A (pBSrc) strains were then mated to PSY142 strains that had been transformed with pLSrc plasmids. The diploids were selected on medium lacking both uracil and leucine to ensure that both plasmids were present. When these strains were first incubated on raffinose liquid medium and then shifted to solid medium with galactose as the carbon source, no dual combination of Src proteins blocked growth except where v-Src was one of the pair. Therefore, none of the Src proteins with deletions in the SH2 domain can rescue the defect in the other altered Src proteins, or cause c-Src protein to become toxic when expressed in the same cell.

We have demonstrated that wt v-Src expressed in ura3::v-SRC strains causes accumulation of large budded cells, with 90% of the cell population having this phenotype after 5 h of growth in galactose medium (F. Boschelli, unpublished). These cells are blocked in nuclear division and defective in mitotic spindle formation. We therefore studied the phenotypic properties of the strains expressing Src proteins from 2μm plasmids. About 60% of cells expressing wt v-Src are large-budded, but neither c-Src nor D17Src causes an accumulation of large-budded cells. Similarly, the spindle morphology in the c-Src and D17Src strains is normal, whereas v-Src expression from the 2μm plasmids causes spindle defects similar to that seen in ura3::v-SRC strains (Fig. 4). Normal mitotic cells with extended intranuclear micro-tubules and extended nucleus are as well-represented in the c-Src (second row) and D17Src (third row) strains as in the control strain (top row). A pair of large-budded cells are shown for the v-Src sample (bottom row). In both of these cell pairs, the nucleus is not extended and a complex pattern of cytoplasmic microtubules is present. About 52% of the cells are large-budded cells in this Zymolyase-treated population, and of these, only 20% have extended intranu-clear microtubules. The remaining large-budded cells have short intranuclear microtubules, which are diverse in shape, and nuclear transfer across the bud neck is apparently blocked. A significant fraction (∼40%) of the v-Src-arrested cells are unbudded in these samples. Most of these cells have microtubule staining that colocalizes with the nucleus, and are probably cells in G₁. Less than 5% of the cells are aploid single cells, and the remaining cells are small-budded. Since in the aploid cells mitochondrial staining is easily visible, these cells represent single cells without a nucleus, probably resulting from cleavage of a large-budded cell blocked in nuclear transfer by Zymolyase. If this is the case, then some cells in the population undergo cytokinesis in spite of the nuclear division block. It is also possible that these cells arise from multiple budding of arrested cells. Although the proportion of cells with particular properties differs between ura3::v-Src strains and the 2μm v-Src expression strain described here, we can state that the expression of v-Src, but not c-Src and D17Src has a deleterious effect on nuclear division and spindle formation.

When v-Src is expressed in ura3::v-SRC strains, the DNA content of the cells increases beyond the 2N G₂ content of DNA. We performed FACS analysis of the 2μm plasmid strains to determine whether c-Src or D17Src affects DNA replication. The experiment shown in Fig. 5 demonstrates that while expression of v-Src causes an elevation in DNA content, neither c-Src nor D17Src has this effect. In the wt v-Src strain, the G₁ peak is similar to that present in the other strains. The DNA peak corresponding to the G₂ peak in the control, c-Src and D17Src strains, in the v-Src strain is significantly shifted to a higher fluorescence intensity, which indicates that the DNA content of these cells increases when wt v-Src is expressed. The appearance of the G₁ peak in the wt v-Src strain is unlike the situation in ura3::v-SRC strains. This peak may reflect a cell population that has lost the plasmid, although it is possible that some of these cells are arrested in G₁ in response to v-Src expression. Either possibility is consistent with the smaller fraction of large-budded cells in the strains with the 2μm plasmids compared to the ura3::v-SRC strains.

We have also observed an increase in genetic alterations in strains expressing v-Src but not c-Src or D17Src. A variably large number of survivors of galactose induction in the diploid ade2/ADE2 BP1 v-Src strains are auxotrophic for adenine (usually >1 in 10⁻³), with the red pigment characteristic of strains with deficiencies in either the ADE2 or ADE5 genes. In addition, many of these survivors also appear as small colonies. We have previously demonstrated that such small colonies appear frequently as a result of v-Src expression and are associated with both aneuploidy and sporulation defects (F. Boschelli unpublished). Adenine-deficient and slow-growing strains are not observed in strains after c-Src or D17Src are expressed (the detection limit is about 1 in 10⁻⁴).

Expression of wt v-Src in ura3::v-SRC strains increases the activity of p34^CDC28 kinase more than 30-fold after 5 h of incubation in galactose medium (F. Boschelli, unpublished). The increase in Cdc28 kinase activity parallels the loss in cell viability, the accumulation of large-budded cells, the nuclear division block and the ability to pass START in the absence of mitosis in those strains. The lack of phenotypic response in cells expressing either c-Src or D17Src suggests that these Src proteins do not influence Cdc28 kinase activity. This possibility was examined by measuring the histone H1-associated Cdc28 kinase activity after shift of the strains from raffinose to raffinose-galactose. Extracts prepared from these cells were incubated with p13^suc1 beads to affinity-purify p34^CDC28 protein (Brizuela et al., 1987). The beads were then incubated with histone H1 and [γ-³²P]ATP, after which aliquots were analyzed on a SDS-polyacrylamide gel. The results of such an experiment are shown in Fig. 6. Histone H1 phosphorylation in the 5 h wt v-Src sample is elevated 10-fold over the 1.5 h wt v-Src sample, whereas a much smaller increase in histone H1 kinase activity occurred in D17Src strain. The same result was obtained with c-Src and control plasmid strains (not shown). The increase in histone H1 phosphorylation between the 0 h and the 1.5 h time points occurs repeatedly in many different strains, and may be associated with carbon source shift. In other experiments, no increase was seen in the control (B656 plasmid), D17Src or c-Src strains after 8 h growth in medium containing galactose (not shown). Alkali-stable phosphorylation of enolase was observed in these c-Src, D17 and v-Src samples, but not in the control strain with the B656 plasmid (not shown). Therefore, all of the Src proteins were active in these extracts, but only wt v-Src has an effect on p34^CDC28 kinase activity.

We explored the possibility that the increase in phosphorylation of histone H1 might be due to Src protein activity adhering to the p13^suc1 beads, either non-specifically or as part of a complex with p34^CDC28. The phosphorylated histone H1 bands were excised from the gel and analyzed for the presence of phosphotyrosine by two-dimensional high-voltage electrophoresis/thin-layer chromatography. All of the radioactivity migrated as phosphoserine or phosphothreonine (not shown). We conclude that phosphorylation of histone H1 in these assays is not due to phosphorylation by p60^v-src.

The inability of c-Src protein or the altered v-Src proteins to affect yeast growth or morphology, or Cdc28 kinase activity, might be explained by either a reduced level of yeast protein phosphorylation or an alteration in the target protein specficity by deletion of a region of the SH2 domain. To distinguish between these two extreme models, the phosphorylation of yeast proteins on tyrosine by the various Src proteins was examined by Western analysis of blots probed with antibody to phosphotyrosine. These extracts were prepared from cells exposed to sodium ortho-vanadate for 15 min prior to harvesting, and to eliminate phosphatase and protease activities the cells were resuspended in trichloroacetic acid (Kanik-Ennulat and Neff, 1990). Extracts were prepared by breaking neutralized cells in buffer followed by further extraction of the insoluble pellet with 7 M urea. These extracts were analyzed by SDS-PAGE and blotted to PVDF filters. Antibody to phosphotyrosine from Upstate Biotechnologies was used to probe the blots, as this antibody is highly specific. Representative blots of roughly equivalent amounts of protein from buffer-soluble and urea extracts of control cells with the pGal plasmid with no insert, and cells expressing v-Src, c-Src or D17Src are shown in Fig. 7. Very little reaction occurs in the control cell lane in either the buffer-soluble (Fig. 7, left) or urea-soluble fractions (Fig. 7, right), and all of the reaction is removed by incubation of the antibody with 5 mM phosphotyrosine during the probing of the filter (not shown). The greatest difference in the samples is in the buffer-soluble fraction of the wt v-Src lane, where antibody reaction is markedly greater than in any of the other buffer-soluble samples. In contrast, the v-Src and D17Src lanes have similar levels of antibody reaction in the urea fraction blot. The reaction of antibody with yeast proteins in the c-Src lane from either buffer-soluble or urea-soluble fractions is nearer to the control cell lane in intensity than the other Src protein lanes. This difference is not due to loading unequal amounts of protein because approximately the same amount of protein was loaded in each lane. Also, some bands react equally well with the antibody in all four lanes. Buffer-soluble proteins of 50-60 kDa and 85 kDa from the v-Src extracts, but not D17Src extracts, react with the antibody. In the blot shown, the 50-60 kDa proteins are strikingly apparent, whereas the 85 kDa band is less intense. The major proteins that interact with the phosphotyrosine antibody in the urea fraction of v-Src extracts but not D17Src extracts, are the 50-60 kDa proteins seen in the soluble fraction blot, and proteins of 120 and 130 kDa, which are faint bands in this blot. On lower-percentage acrylamide gels where more protein can be loaded, the 120-130 kDa bands become prominent. Another doublet of 150-160 kDa also appears under these conditions in the wt v-Src lane but not in the D17Src lane.

Most of the antibody reaction in the D17Src sample occurs in the urea fraction, and many proteins in the wt v-Src sample are also present in this fraction. However, since these extracts were prepared from trichloroacetic acid-treated samples, the proportions of cell protein in the buffer-soluble or the urea-soluble fractions may be artifactual. Also, the 50-60 kDa proteins remaining in the urea fraction probably represent incompletely extracted proteins, since more exhaustive washing of the buffer-insoluble pellet can remove most of these proteins. We have performed these experiments with cell extracts prepared without trichloroacetic acid treatment, and found that antibody reaction is reduced even when 1 mM sodium vanadate and 50 mM sodium fluoride are included in the breaking buffers.

We cannot rule out the possibility that some of the proteins appearing in the blots shown in Fig. 7 are interacting with the phosphotyrosine antibody for reasons other than that they contain phosphotyrosine. However, the proteins with the molecular mass noted above do not appear in lanes with the control pGal plasmid, or with chicken c-Src or D17Src proteins, i.e. these proteins react with the antibody in a v-Src-dependent manner. Also, the intensity of the antibody reaction with proteins in the D17Src urea fraction sample is much greater than in either the control pGal or the c-Src lanes, again indicating an Src-dependent antibody reaction. While some other modification of these proteins might conceivably account for their reaction with antibody, the most straightforward interpretation of such a reaction is that these proteins are phosphorylated on tyrosine. Those proteins reacting with the antibody only in the wt v-Src samples and not in the D17Src samples represent substrate proteins whose phosphorylation is dependent on the integrity of the SH2 domain.

Since the effects of v-Src on yeast viability might be ascribed to the ability of the SH2 domain in v-Src to bind to and consequently stabilize phosphotyrosine-containing proteins in yeast, we examined the effect of expressing two v-Src proteins with deletions in the catalytic domain; the D12Src protein, which has a four amino acid insertion after residue 326 followed by a stop codon, and D15Src, which has residues 438-464 deleted (see Materials and Methods). In Fig. 8 (top), the growth of strains expressing these altered Src proteins is compared with that of a strain with the control B656 plasmid and a strain expressing wt v-src. Only the strain expressing the wt v-src gene failed to grow on solid medium containing galactose. Colony size in the control strain and the strains expressing the altered Src proteins is indistinguishable, indicating that no significant growth inhibition occurs. Also, no changes in cell morphology were observed. The expression of these proteins was confirmed by Western analysis with monoclonal antibody to amino acid residues 2-17 of Src (Fig. 8, middle). Intact v-Src (lane 2) migrates just above a cross-reacting yeast protein evident in the control strain without Src (lane 1). D15Src co-migrates with this cross-reacting band as a diffuse band, whereas D12Src migrates as a doublet at 35 kDa (lanes 3 and 4).

We crossed these strains with a strain expressing v-Src from a stable single copy integrant (F. Boschelli, unpublished) and measured the growth of these strains on galactose-containing medium. In all cases, the diploid failed to grow on this medium. Therefore, the Src proteins with the deletions in the catalytic domain do not affect the growth-inhibitory activity of the wt v-Src protein. When the D12 and D15Src proteins were co-expressed with chicken c-Src or with D17Src, which has a SH2 domain deletion, no changes in growth or phenotype were observed. We conclude that an intact SH2 domain and a functional catalytic domain cannot act in trans, at least with the particular Src proteins studied.

Expression of catalytically defective D12 or D15Src proteins does not cause an increase in the level of phosphoty-rosine-containing proteins beyond that observed in control strains with the plasmid without a src gene (Fig. 8, bottom). The phosphotyrosine-containing proteins observed in blots of the wt v-Src extracts are therefore attributable to a combination of the catalytic activity of v-Src in conjunction with an intact SH2 domain. These results indicate that the phenotypic effects of v-Src expression are dependent on both the catalytic activity of the protein and the SH2 domain.

The results presented in this paper strongly suggest that the lethal effects of v-Src expression in yeast are related to the ability of v-Src to phosphorylate yeast proteins that have important roles in cell growth and division. Seven different Src proteins with deletions within the SH2 domain do not affect the viability of yeast, even though all of these proteins are catalytically active when assayed in immuno-precipitates in vitro. The ability of one of these altered Src proteins to phosphorylate many yeast proteins is not affected by the SH2 domain alteration. However, the altered Src protein cannot phosphorylate all of the proteins that are phosphorylated by the wt v-Src protein. The pattern of protein phosphorylation in the v-Src strain differs from that of the D17Src strain, even though the extent of phosphorylation is similar, at least in the urea-soluble fraction. Expression of v-Src leads to an accumulation of large-budded cells, an increase in the frequency of genetic alterations, spindle dysfunction, an increase in DNA content and an increase in the activity of p34^CDC28 kinase. These phenotypic alterations are completely absent when the altered v-Src proteins or the chicken fibroblast c-Src protein are expressed. They are also absent when catalytically inactive v-Src proteins are expressed. These results suggest that the ability of v-Src to kill yeast is a reflection of the ability of v-Src protein to alter the physiological activity of certain yeast proteins by phosphorylating them on tyrosine.

The importance of the SH2 domain for the transforming activity of v-Src has been extensively documented in vertebrate cells (Bryant and Parsons, 1984; Cross et al., 1985; Fukui et al., 1991; Hirai and Varmus, 1990; Kanner et al., 1991; Kaplan et al., 1990; Nemeth et al., 1989; O’Brien et al., 1990; Wang and Parsons, 1989; Wendler and Boschelli, 1989). Deletions and point mutations in the SH2 domain can result in a drastic reduction of transforming potential of src genes (Bryant and Parsons, 1984; Cross et al., 1985; Wendler and Boschelli, 1989). Also associated with alterations in the SH2 domain is an inability to phosphorylate proteins that are modified by the wt v-Src protein, in a manner that correlates with reduced transforming activity (Kanner et al., 1991; Nemeth et al., 1989). The SH2 domain is also involved with localization of Src proteins, perhaps because of its role as a recognition sequence for substrate proteins (Hamaguchi and Hanafusa, 1987; Kaplan et al., 1990). The results presented here demonstrate that the SH2 domain is critical for the ability of v-Src to kill yeast, and in conjunction with this altered biological activity, the SH2 domain affects target protein recognition in yeast.

Others have already demonstrated that the chicken c-Src protein expressed in fibroblasts has little effect on yeast growth even though the amount of c-Src protein expressed in yeast is much greater than seen for v-Src, such that c-Src kinase activity in vitro is nearly as great as that of v-Src protein produced in yeast (Cooper and Runge, 1987; Kornbluth et al., 1987). However, as shown here, the reaction of phosphotyrosine antibodies with blots of extracts from strains expressing c-Src is much reduced compared to the v-Src or D17Src samples. The in vivo activity of the c-Src protein towards yeast proteins must therefore be much lower than either the v-Src or the D17Src proteins.

This conclusion would suggest that there is some cooperation between the SH2 domain of c-Src and the carboxylterminal regulatory element of c-Src, which contains a tyrosine residue, Tyr527, that in vertebrate cells serves as the major regulatory switch for c-Src kinase (Bagrodia et al., 1991; Chackalaparampil and Shalloway, 1988; Piwnica-Worms et al., 1987). Phosphorylation of Tyr527 inactivates p60^c-src, whereas dephosphorylation of Tyr527 results in p60^c-src activation (Cooper et al., 1986; Cooper and King, 1986; Courtneidge, 1985; Piwnica-Worms et al., 1987). Since only a fraction of c-Src proteins are phosphorylated on Tyr527 in yeast cells the reason for the relatively low level of yeast protein phosphorylation on tyrosine compared to the wt or mutant v-Src might seem obscure (Kornbluth et al., 1987). The carboxyl terminus of c-Src protein phosphorylated on Tyr527 is believed to physically bind to the SH2 domain and block the SH2 domain from interacting with other proteins, whereas the dephosphorylated form of the carboxyl terminus does not (Roussel et al., 1991). Perhaps the unphosphorylated form of the carboxyl terminus of c-Src is still capable of blocking the SH2 domain such that c-Src protein cannot interact with yeast proteins to the same extent as does v-Src. The uncovering of the SH2 domain of c-Src in vertebrate cells in response to dephos-phorylation of Tyr527 might therefore be due to some active mechanism, perhaps physical association of the dephosphorylated carboxyl terminus of c-Src with some other protein. The carboxyl terminus of c-Src might also interfere with the intracellular localization of c-Src protein and render certain protein targets related to cell death inaccessible. These two possibilities need not be mutually exclusive.

At present, we do not know whether the proteins detected on the Western blots as being phosphorylated by the wt v-Src protein and not by D17Src represent physiologically important events. As is the case with vertebrate cells transformed by v-src, a large number of proteins phosphorylated on tyrosine are present in yeast as a result of v-Src expression. The differences in the phosphorylation pattern between the D17Src and wt v-Src may reflect events that are not physiologically relevant, and the true mediators of v-Src-dependent lethality may be relatively minor bands on such blots. Nonetheless, since a large number of phosphorylated proteins are present as a result of D17Src expression, it must at least be concluded that the general elevation of tyrosine phosphorylation of yeast proteins has little effect on yeast viability, and that the identity of the phosphorylated yeast proteins is the critical determinant in the lethal effect of v-Src expression. In support of this conclusion, we note that there are no intermediate phenotypic effects when D17Src is expressed, which might be expected if the phenotype was due to a general increase in the level of intracellular phosphotyrosine.

Isolation of proteins phosphorylated by v-Src from yeast extracts may prove to be as technically difficult as is the case in vertebrate cells. Fortunately, it is possible to use a genetic approach to determine the molecular basis for v-Src lethality in S. cerevisiae, Such an approach has shown that a mutation in the START gene CDC37 suppresses the ability of v-Src to kill yeast (F. Boschelli, unpublished results).

This work was supported by an Institutional American Cancer Society Award (IN-63) and awards from the Meyer L. Prentis Comprehensive Cancer Center to F.B.; S.M.U. was supported by a Howard Hughes Fellowship for Undergraduate Research. Some of these experiments were performed at MIT in J. M. Buchanan’s laboratory and were supported by NIH grant RO1CA36928-03 to J.M.B.; F.B. gratefully acknowledges the generous support from Jack Buchanan during that period. Jim Oleson, Phil Wendler, Kim Arndt and Peter Drain aided in starting this work at MIT. Thanks are also due to Vincent Chau and Richard Needleman at WSU for use of laboratory facilities. The authors are grateful to Andy Laudano for doing the phosphoamino acid analysis of the histone H1 samples. Flow cytometry was performed at the Wayne State University Flow Cytometry Core Facility.