Purification and characterization of a Src-related p57 protein-tyrosine kinase from Xenopus oocytes. Isolation of an inactive form of the enzyme and its activation and translocation upon fertilization.

In the previous study (Fukami, Y., Sato, K.-I., Ikeda, K., Kamisango, K., Koizumi, K., and Matsuno, T. (1993) J. Biol. Chem. 268, 1132-1140), we found that an antibody termed anti-pepY antibody causes a severalfold activation of bovine brain c-Src. The anti-pepY antibody was raised against a synthetic peptide corresponding to residues 410-428 of chicken c-Src, one of the most conserved regions among the Src family protein-tyrosine kinases. In this study, we have used this antibody as an in vitro activator and purified a c-Src-related protein-tyrosine kinase from the particulate fraction of Xenopus laevis oocytes. A synthetic peptide corresponding to residues 7-26 of fission yeast Cdc2 was used as substrate. Immunoreactivity toward the antibody was also monitored during the purification. The purified kinase displayed a single polypeptide of 57 kDa on SDS-gel electrophoresis and showed a specific activity of 2.37 and 20.1 nmol/min/mg protein in the absence and the presence of the anti-pepY antibody, respectively. The purified enzyme underwent autophosphorylation and phosphorylated actin and the Cdc2 peptide exclusively on tyrosine residues. Specific antibodies against c-Src, Fyn, c-Yes, c-Fgr, Lck, Lyn, Hck, and Blk proteins did not recognize the p57 Xenopus tyrosine kinase. The kinase activity of the Xenopus enzyme was not affected by oocyte maturation but was found to be elevated severalfold upon fertilization. Fertilization also caused a translocation of the activated enzyme from the particulate fraction to the cytosolic fraction. The activation and translocation was observed within 1 min after fertilization. These results suggest a possible involvement of the p57 Xenopus tyrosine kinase in the signal transduction of fertilization.

The protein-tyrosine kinase activity was initially found to be associated with v-Src, the product of Rous sarcoma virus oncogene, and then with the products of several retroviral oncogenes and their cellular counterparts, proto-oncogenes (1). Soon after the discovery, the same activity was demonstrated for some growth factor receptors (2). It is now established that the protein-tyrosine kinase activity plays an important role not only in the cell transformation but also in the processes of normal cell growth and differentiation. c-Src, the 60-kDa normal cellular homologue of v-Src, is a prototype of the cellular members of the Src family protein-tyrosine kinases and is associated with the inner surface of the plasma membrane by virtue of its myristoylated amino terminus (3)(4)(5)(6). Like other members of the Src family, c-Src shares a related structural feature including unique amino-terminal region, Src homology 3 and Src homology 2 domains, and the catalytic domain (also referred to as Src homology 1 domain), as well as the noncatalytic regulatory sequence in the carboxyl terminus (5). c-Src is found in virtually all higher eukaryotes and many cell types, and its predominant expression has been found in cells such as differentiating neurons, platelets, and peripheral blood lymphocytes (4 -6). However, its mode of regulation and physiological function in the cell remain unclear.
In the present study, we have used oocytes of Xenopus laevis in order to investigate the physiological function of the members of the Src family protein-tyrosine kinases. To date, it has been suggested that the Src family kinases may be involved in the signal transduction mechanisms of Xenopus oocytes. For example, microinjection of v-Src or activated c-Src causes an acceleration of the rate of progesterone-induced oocyte maturation (7,8). Identification of Src-like tyrosine kinase activity in the frog has been done initially by Schartl and Barnekow (9). Steele et al. reported cDNA cloning of three members of the Src family kinases in oocytes, c-Src (10), Fyn (11), and c-Yes (12). However, the advancement of knowledge regarding the function and regulation of the Src family protein-tyrosine kinases in oocytes has been limited by the lack of purification of native enzymes and their detailed characterization.
In the previous study, we proposed a model in which the c-Src activity is negatively regulated by the intramolecular interaction between the amino terminus of Src homology 2 domain and the region containing the autophosphorylation site, named the inter-(Asp-Phe-Gly)-(Ala-Pro-Glu) (IDA) 1 region (13). Furthermore, we found that an antibody, termed anti-pepY antibody, raised against a synthetic peptide corresponding to residues 410 -428 in the IDA region of chicken c-Src causes a severalfold activation of bovine brain c-Src (13). The amino acid sequence in the IDA region is almost completely conserved among the Src family members. Thus, we thought that the anti-pepY antibody might be useful as a tool for the identification of Xenopus Src family kinases. As a standard protein-tyrosine kinase substrate, we used a synthetic Cdc2 peptide that corresponds to the tyrosine phosphorylation site of the fission yeast cdc2 gene product (residues [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. This peptide and related peptides have been used as phosphate acceptors for several protein-tyrosine kinases, such as v-Src (13,14), c-Src from bovine brain (13), a c-Src-related tyrosine kinase from bovine spleen (15), and a cytosolic tyrosine kinase from HeLa cells (16).
In this paper, we describe the purification of a Src-related protein-tyrosine kinase from the particulate fraction of Xenopus oocytes. Kinase activity of the enzyme was elevated severalfold by the addition of anti-pepY antibody. A 57-kDa polypeptide was identified in the immunoblot analysis using anti-pepY antibody and found to be copurified with the kinase activity. Significant activation and translocation of the enzyme was observed as early as 1 min after fertilization of the eggs, suggesting a possible role of the enzyme in the fertilization events of Xenopus.
Anti-pepY antibody was raised against a synthetic peptide, termed pepY (13), which corresponds to residues 410 -428 of the chicken c-Src (18). Anti-PTK1C antibody was raised against the bacterially expressed fusion protein consisting of the amino-terminal 13 residues of ␤-galactosidase and the catalytic fragment of v-Src protein (residues 226 -526) (13). Anti-Src-CT antibody was raised against a synthetic peptide corresponding to the carboxyl terminus (residues 520 -533) of the chicken c-Src, whose sequence is highly conserved among c-Src, c-Yes, Fyn, and c-Fgr (5). Preparation of these rabbit antisera was carried out as described previously (13). Anti-pepY IgG was purified by ammonium sulfate precipitation and DEAE-cellulose column chromatography as described (13), and the IgG preparation was used in the kinase assay without further affinity purification. Mouse monoclonal anti-Src antibody mAb327 was purchased from Oncogene Science. Rabbit polyclonal antibodies against c-Src (residues 3-18 of the human c-Src), c-Yes (residues 3-19 of the human c-Yes), c-Fgr (residues 47-66 of the human c-Fgr), Lyn (residues 44 -63 of the human Lyn), Hck (residues 8 -37 of the human Hck), and Blk (residues 21-43 of the human Blk) were obtained from Santa Cruz Biotechnology. Rabbit anti-Fyn (residues 29 -48 of the human Fyn) antibody was from Oncogene Science, and rabbit anti-Lck (residues 22-51 of the human Lck) was from Upstate Biotechnology. Mouse monoclonal anti-phosphotyrosine antibody (PY20) was purchased from ICN.
Kinase Assay-Protein kinase assay was routinely carried out for 20 min at 30°C using Cdc2 peptide as substrate. The standard assay mixture (25 l) contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 2 M [␥-32 P]ATP (3.7 kBq/pmol), 1 mM dithiothreitol, 1 mM Cdc2 peptide, and 5 l of the enzyme fraction that had been preincubated with or without anti-pepY antibody for 1 h at 4°C. The final concentration of anti-pepY antibody in the assay mixture was 40 g/ml. Phosphorylation reaction was started by the addition of the enzyme fraction and terminated by spotting an 18-l aliquot of the reaction mixture onto a P81 phosphocellulose filter (1.5 ϫ 1.5 cm, Whatman). The filter was washed with 75 mM H 3 PO 4 , and the radioactivity remaining on the filter was determined by Cerenkov counting in a liquid scintillation counter (Beckman LS 6000IC). In some experiments, phosphorylation of Cdc2 peptide was analyzed by SDS-gel electrophoresis using 20% polyacrylamide gel. For this purpose, reaction was terminated by the addition of SDS-sample buffer (19) and boiling for 3 min. The sample was subjected to the SDS-gel electrophoresis (19), and the gel was stained with Coomassie Brilliant Blue. The radioactivity in the gel was visualized by autoradiography. The radioactive band was excised and quantified in the liquid scintillation counter. We also used 0.2 mg/ml of actin as a phosphate acceptor under the standard assay conditions. Phosphorylated actin was analyzed by SDS-gel electrophoresis using 12.5% polyacrylamide gel.
Immunoblot Analysis-Column fractions were examined for the immunoreactivity with anti-pepY antibody throughout the purification by immunoblot analysis which was carried out as described previously (13), using 100-fold diluted anti-pepY serum. Alkaline phosphataseconjugated goat anti-rabbit IgG (Cappel) was used as the second antibody. The purified enzyme fraction was also characterized by immunoblot analysis using various anti-Src family antibodies. When mouse monoclonal antibodies were used, rabbit anti-mouse IgG (Cappel) was added prior to the addition of the goat antibody as specified previously (13). In the analysis of immunoprecipitated samples (see Fig. 7), [ 125 I]protein A (50 kBq/ml) was used instead of the alkaline phosphatase-conjugated goat antibody as described previously (20).
Purification of c-Src and Fyn from Bovine Brain-c-Src was partially purified from the Triton X-100-solubilized crude membrane fraction of bovine brain by successive chromatographies on DEAE-cellulose and two tyrosine-Sepharose columns as described previously (13). Immunoblot analysis with anti-Fyn antibody revealed that the Fyn protein, a Src family protein-tyrosine kinase, was present in a side fraction of this partially purified c-Src preparation. We separated Fyn protein from this c-Src preparation by MonoQ chromatography and used as Fyn preparation in this study.
Ovulation was induced in females by administration of human chorionic gonadotropin (400 -600 IU) via the dorsal lymph sac, and the frogs were left in water containing 0.1 M NaCl at 21°C. Ovulation began 8 -10 h after the hormone administration. The ovulated eggs were collected and washed extensively with 1 ϫ DeBoer's solution (DB) (110 mM NaCl, 1.3 mM KCl, 0.44 mM CaCl 2 , adjusted to pH 7.2 with the addition of NaHCO 3 ) (22) and two times with 0.5 ϫ DB. By this procedure, 200 -400 eggs were obtained per one animal.
Fertilization of Eggs-Sperm suspension was prepared by macerating testes removed surgically from males in 0.5 ϫ DB. Estimation of sperm concentration was made by counting in a hemocytometer, and the average value of 1.2 ϫ 10 7 sperm cells/ml was obtained by the maceration of one pair of testes in 5 ml of 0.5 ϫ DB. The sperm suspension was kept at 4°C until use. Insemination (fertilization) was conducted in 100-mm culture dishes at 21°C by adding 1 ml of sperm suspension/5 ml packed volume of unfertilized eggs in 0.05 ϫ DB. The insemination mixture was agitated manually. Insemination was stopped by washing the eggs with ice-cold 0.5 ϫ DB at the specified time, and the eggs were immediately frozen in liquid nitrogen and kept at Ϫ80°C until use. Aliquots of the inseminated eggs were incubated further at 21°C in 0.5 ϫ DB, they started "rotation" 12-25 min after insemination and began first cleavage 60 -90 min after the rotation. Under these conditions, 0, 65, 70, 70, 85, and 85% of the eggs underwent rotation after 0, 1, 2, 5, 10, and 20 min of the insemination procedure, respectively.
Purification of p57 Xenopus Tyrosine Kinase from the Particulate Fraction of Oocytes-All procedures were carried out at 0 -4°C. Oocytes (30 ml in a packed volume) were mixed with 90 ml of buffer A (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 10 mM ␤-mercaptoethanol, 10 g/ml leupeptin, and 100 M APMSF). Excess buffer was discarded immediately, and oocytes were disrupted in a Teflon-glass homogenizer (10 strokes). The homogenate (ϳ35 ml) was centrifuged at 15,000 ϫ g for 20 min, and the resulting supernatant (ϳ25 ml) was clarified by further centrifugation at 150,000 ϫ g for 1 h. After centrifugation, the clear supernatant was set aside as the cytosolic fraction. The pellet and fluffy part of the pellet were then carefully taken, diluted with buffer A, and recentrifuged at 150,000 ϫ g for 30 min. The pellet fraction obtained was solubilized by homogenization with 25 ml of buffer A containing 1% Triton X-100 (buffer B) in a Teflon-glass homogenizer (10 strokes). The homogenate was centrifuged at 150,000 ϫ g for 30 min, and the resulting supernatant was used as the Triton X-100-solubilized particulate fraction.
Purification of p57 Xenopus protein-tyrosine kinase from the solubilized particulate fraction was carried out according to the method applied for the purification of v-Src (23) and c-Src (13) with some modifications. The Triton X-100-solubilized particulate fraction (75 mg of protein in 25 ml) was applied onto a DEAE-cellulose column (1 ϫ 13 cm) equilibrated with buffer B. Unbound materials were washed out with 10-column volume of the same buffer. Proteins bound to the column were then eluted with a 100-ml linear gradient of NaCl (0 -0.6 M) in buffer B. Fractions of 2 ml were collected and assayed for the Cdc2 peptide kinase activity in the presence or the absence of anti-pepY antibody. Fractions were also subjected to the immunoblot analysis with anti-pepY antibody. The peak fractions were pooled, adjusted to 0.8 M (NH 4 ) 2 SO 4 by the addition of solid (NH 4 ) 2 SO 4 , and applied onto a tyrosine-Sepharose column (tyrosine-Sepharose I, 1.8 ϫ 6 cm) equilibrated with buffer B containing 0.8 M (NH 4 ) 2 SO 4 . After washing the column with a 10-column volume of the buffer, the tyrosine kinase was eluted with a 120-ml decreasing concentration gradient of (NH 4 ) 2 SO 4 (0.8 -0 M) in buffer B. Fractions of 4 ml were collected and assayed as above. Peak fractions were pooled, dialyzed against buffer B, and applied onto another tyrosine-Sepharose column (tyrosine-Sepharose II, 1.8 ϫ 6 cm) equilibrated with buffer B. Unbound materials were washed out with 10-column volume of the buffer, and the tyrosine kinase was eluted with a 120-ml linear gradient of NaCl (0 -0.8 M) in buffer B. Fractions of 4 ml were collected and assayed. Peak fractions were pooled, dialyzed against buffer B, and applied onto a MonoQ column (0.5 ϫ 5 cm) equilibrated with buffer B. After washing the column with 10-column volume of the buffer, the enzyme was eluted with a 40-ml linear gradient of NaCl (0 -1 M) in buffer B. Fractions of 1 ml were collected and assayed for the Cdc2 peptide kinase activity and immunoreactivity with anti-pepY antibody. Peak fractions were pooled, dialyzed against buffer B containing 20% glycerol, and stored at Ϫ80°C until use.
Immunoprecipitation and Autophosphorylation-Samples were immunoprecipitated with 5 g of anti-pepY IgG for 2 h at 4°C, and immune complexes were collected by adsorption onto 10 l of protein A-Sepharose and washed three times with RIPA buffer (50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 10 g/ml leupeptin, and 100 M APMSF) (20) containing 150 mM NaCl. Washed immune complexes were then autophosphorylated under the standard assay conditions. Autophosphorylated samples were separated by SDS-gel electrophoresis and visualized by BAS2000 Bioimaging Analyzer (Fuji film, Tokyo). In the experiment shown in Fig. 7, anti-pepY IgG immobilized onto protein A-Sepharose (anti-pepY beads) was prepared according to the mannual of Harlow and Lane (24), and protein samples were immunoprecipitated with 20 l of the beads (containing 10 g of anti-pepY IgG) under the same wash conditions.
Other Methods-Silver staining of protein samples after SDS-gel electrophoresis was carried out according to the method of Oakley et al. (25). To estimate the protein in the gel, various amounts of bovine serum albumin were run in the same slab gel and quantified by a densitometer (Shimadzu, Kyoto) after silver staining. Protein concentrations of tyrosine-Sepharose II and MonoQ fractions were estimated as above. Proteins in crude fractions were estimated by the dye-binding method of Bradford (26) using the Bio-Rad Protein Assay Mix. Phosphoamino acid analysis was carried out according to the method of Hunter and Sefton (27) with a minor modification (28).

Identification and Purification of a 57-kDa Protein
Kinase from Xenopus Oocytes-The purification protocol is summarized in Table I. Throughout the purification, protein kinase activity was measured by the phosphorylation of Cdc2 peptide in the presence or the absence of anti-pepY antibody, a kinaseactivating antibody that was raised against a synthetic peptide corresponding to residues 410 -428 of the chicken c-Src (13). Immunoblot analysis with anti-pepY antibody was also carried out throughout the purification steps. At the first step of DEAE-cellulose column chromatography (Fig. 1A), a high and broad peak of anti-pepY antibody-independent protein kinase activity was observed at 0.2-0.4 M NaCl. This activity was ascribed to the endogenous protein phosphorylation of the fractions, because the activity was observed in the absence of Cdc2 peptide. On the other hand, a minor anti-pepY antibody-dependent Cdc2 peptide kinase activity was observed at 0.05-0.1 M NaCl (Fig. 1A, indicated by a bracket). Immunoblot analysis of the fractions with anti-pepY antibody revealed a 57-kDa immunoreactive protein that coeluted with the Cdc2 peptide kinase activity (Fig. 1B). Although a major immunoreactive protein of 30 kDa and some other minor proteins were detected in the fractions of higher NaCl concentrations, the c-Src protein of 60 kDa was not evidently detected by the immunoblot analysis (Fig. 1B). To our surprise, we could not detect c-Src even when Src-specific mAb327 monoclonal antibody was used for the immunoblot analysis, nor we could observe the autophosphorylation activity of c-Src in any DEAE-cellulose fraction after immunoprecipitation with mAb327 antibody (data not shown). Identification of c-Src is described later (see "Discussion"). It is also important to note that we could not observe the 57-kDa protein or anti-pepY IgG-dependent activity in a similar DEAE-cellulose preparation obtained from the cytosolic fraction of oocytes. Thus, we used only particulate fraction as a source of the enzyme.
To purify the anti-pepY antibody-dependent kinase activity further, the peak fractions indicated by the bracket in Fig.  1A were pooled and subjected to two successive tyrosine-Sepharose column chromatography (Table I). The first tyrosine-Sepharose chromatography was carried out by starting at high ionic strength using 0.8 M (NH 4 ) 2 SO 4 , which facilitates hydrophobic interactions, and the second chromatography was performed at low ionic strength, which permits affinity binding (23). The Cdc2 peptide kinase activity was eluted at 0.55-0.45 M (NH 4 ) 2 SO 4 in the first tyrosine-Sepharose chromatography and at 0.15-0.25 M NaCl in the second chromatography (data not shown). The peak fraction was further purified by MonoQ column chromatography, and the anti-pepY antibody-dependent enzyme activity was recovered at 0.15-0.2 M NaCl. The 57-kDa immunoreactive protein was found to coelute with the kinase activity throughout these chromatographic steps (not shown). SDS-gel electrophoresis and silver staining patterns of the last three chromatography fractions are shown in Fig. 2. The final MonoQ fraction contained a single polypeptide migrating at a molecular size of 57 kDa, coinciding with the size of the immunoreactive protein. The basal specific activity of the purified enzyme was 2.37 nmol/min/mg in the absence of anti-pepY antibody under the standard assay conditions (Table I). However, in the presence of the antibody (40 g/ml), the enzyme was activated and showed a specific activity of 20.1 nmol/min/mg under the same assay conditions. Purified p57 Xenopus Enzyme Is a Protein-tyrosine Kinase That Undergoes Autophosphorylation-During the above purification, enzyme activity toward Cdc2 peptide was monitored by the filter assay. To alternatively measure the kinase activity and to confirm the effect of anti-pepY antibody, phosphorylated Cdc2 peptide was analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 3, lanes 1 and 2). We also used actin as an exogenous substrate (lanes 3 and 4). As evident in the figure, the phosphorylation of peptide and actin was significantly stimulated by the antibody. Quantitation of the radioactivity in the excised gels revealed that stimulation of the phosphorylation of Cdc2 peptide and actin was 8-and 4-fold, respectively. In the presence of anti-pepY antibody, phosphorylation of a 57-kDa protein was also observed (lanes 2 and 4). The same phosphoprotein was detected in the absence of exogenous substrate (lane 6), suggesting that the p57 Xenopus enzyme is activated and undergoes autophosphorylation in the presence of anti-pepY antibody. Phosphoamino acid analysis revealed that the phosphorylation of Cdc2 peptide, actin, and 57-kDa protein occurred exclusively on tyrosine residues (data not shown). Thus, we will hereafter refer to this enzyme as p57 Xenopus tyrosine kinase.
Kinetic Properties of the p57 Xenopus Tyrosine Kinase-Us-ing Cdc2 peptide as substrate, we examined kinetic properties of the purified Xenopus tyrosine kinase. Because the optimal kinase activity was obtained at 10 -20 mM MgCl 2 (data not shown), phosphorylation reactions were carried out in the assay mixtures containing 20 mM MgCl 2 for 10 min at 30°C. Phosphorylation of 1 mM Cdc2 peptide was linear up to 1 h at 100 M ATP (not shown). Table II shows the kinetic constants for the phosphorylation of Cdc2 peptide in the presence or the absence of anti-pepY antibody. The anti-pepY antibody little affected K m values for Cdc2 peptide and ATP. However, V max values were found to be significantly increased (up to 7-fold) by the presence of anti-pepY antibody. Immunochemical Properties of the p57 Xenopus Tyrosine Kinase-It has been reported that the mRNAs of c-Src, Fyn, and c-Yes genes are present in Xenopus oocytes (10 -12). In order to examine the structural similarity between the p57 Xenopus tyrosine kinase and the known Src family proteins, immuno-  3 and 4), and autophosphorylation (lanes 5 and 6) was examined under the standard assay conditions using the second tyrosine-Sepharose fraction (7.8 ng protein/assay). Phosphorylated peptides and proteins were subjected to SDS-gel electrophoresis with 20% (for lanes 1 and 2) or 12.5% (for lanes 3-6) polyacrylamide gels and then visualized by autoradiography. The arrowhead indicates the position of the phosphorylated 57-kDa protein.
blot analysis was performed with several Src subfamily-specific antibodies. In Fig. 4, partially purified c-Src (lane 1) and Fyn (lane 2) preparations from bovine brain were included as representatives of the Src family proteins, because the amino acid sequences of these mammalian proteins are essentially conserved in amphibians (10 -12). c-Src and Fyn were recognized by both anti-pepY antibody (Fig. 4, top panel) and the corresponding specific antibody, anti-Src antibody mAb327 (middle panel) or anti-Fyn antibody (bottom panel). On the other hand, the p57 Xenopus tyrosine kinase (lane 3) was recognized only by anti-pepY antibody but not by mAb327 or anti-Fyn antibody. We found two more antibodies, anti-PTK1C and anti-Src-CT, that recognize the p57 Xenopus tyrosine kinase (data not shown). The anti-PTK1C antibody was raised against the catalytic fragment of v-Src protein (13), and thus it may be widely reactive with other Src family members. The anti-Src-CT antibody was raised against the carboxyl-terminal region (residues 520 -533) of chicken c-Src. This antibody may also be cross-reactive, because the sequence of the antigen is well conserved among other Src family members such as c-Yes, Fyn, and c-Fgr (5). We also tested other commercially available polyclonal antibodies (anti-c-Src, anti-c-Yes, anti-c-Fgr, anti-Lck, anti-Lyn, anti-Hck, and anti-Blk), but none of them recognized the p57 Xenopus tyrosine kinase (data not shown). These results suggest that the p57 Xenopus tyrosine kinase is a member of the Src family, but its identity is still not clear.
Activation of the p57 Xenopus Tyrosine Kinase upon Fertilization-In order to investigate whether the kinase activity of p57 Xenopus tyrosine kinase changes during the cell cycle, enzyme preparations from oocytes, unfertilized eggs, and fertilized eggs were compared at the step of DEAE-cellulose column chromatography. Fig. 5A shows that the kinase activity in oocytes was detected at 40 -90 mM NaCl and stimulated severalfold in the presence of anti-pepY antibody as already indicated in Fig. 1A. A similar pattern was obtained with the preparation from unfertilized eggs (Fig. 5B). However, in fertilized eggs (10 min after insemination), significant kinase activity was found to be independent of anti-pepY antibody (Fig. 5C). The kinase activity measured in the presence of anti-pepY antibody was virtually the same as other preparations (Fig. 5, A-C), suggesting that total amount of the kinase remained almost constant. Therefore, the increased antibodyindependent activity is likely due to an increase in the specific activity of the p57 Xenopus tyrosine kinase upon fertilization.
Translocation and Tyrosine Phosphorylation of the p57 Xenopus Tyrosine Kinase upon Fertilization-Further evidence for the possible involvement of p57 Xenopus tyrosine kinase in fertilization was obtained from the analysis of localization of the enzyme. In Fig. 6, cytosolic and particulate fractions from oocytes, unfertilized eggs, and fertilized eggs were prepared and subjected to DEAE-cellulose column chromatography, and the corresponding eluates were analyzed by immunoprecipitation with anti-pepY antibody followed by autophosphorylation. This method allowed us to detect the p57 Xenopus tyrosine kinase even in the partially purified preparations. As already  were subjected to SDS-gel electrophoresis with 12.5% polyacrylamide gel and analyzed by immunoblotting with anti-pepY antibody (100-fold diluted serum), 10 g/ml mAb327, and 10 g/ml anti-Fyn antibody, respectively.
FIG. 5. DEAE-cellulose column chromatography of the p57 Xenopus tyrosine kinase from oocytes, unfertilized eggs, and fertilized eggs. Oocytes (A), unfertilized eggs (B), and fertilized (10-min inseminated) eggs (C) (each ϳ5 ml in a packed volume) were prepared as described under "Experimental Procedures." Triton X-100-solubilized particulate fractions from these materials were separately subjected to DEAE-cellulose column (1 ϫ 6 cm) chromatography. Unbound materials were washed out with 45 ml of buffer B, and proteins bound to the column were eluted with a 30-ml linear gradient of NaCl (0 -0.35 M) in buffer B. Fractions of 0.5 ml were collected, and aliquots (10 l) were assayed for the Cdc2 peptide kinase activity in the presence (q) or the absence (E) of anti-pepY antibody. The solid lines indicate the NaCl gradients. mentioned, the DEAE-cellulose fraction prepared from the cytosol of oocytes did not contain the antibody-reactive kinase (lane 1). On the other hand, a major band of p57 protein was detected in the solubilized particulate fraction (lane 2). Unfertilized eggs showed a similar pattern (lanes 3 and 4). However, after a 10-min insemination, significant autophosphorylation of p57 protein was found in the cytosolic fraction (lane 5). The result suggests a translocation of the enzyme to the cytosol upon fertilization, although the activity that remained in the particulate fraction was still quite high (lane 6). The amount of enzyme found in the cytosol (as judged by its autophosphorylation activity) was estimated to be about 10% of that in the particulate fraction.
To confirm the translocation and activation of the p57 Xenopus tyrosine kinase further, preparations of the p57 protein from cytosolic and particulate fractions were monitored for their tyrosine phosphorylation during the course of fertilization. In Fig. 7A, the amount of p57 protein is monitored by blotting with anti-pepY antibody after immunoprecipitation with the same antibody. It is shown that a small amount (about 10%) of the p57 protein translocated within 1 min after insemination. The translocation did not progress further (lanes 2-4), and the amount of p57 protein in the particulate fraction remained almost constant (lanes 5-8). In Fig. 7B, on the other hand, tyrosine phosphorylation of the immunoprecipitated p57 protein is monitored by blotting with the monoclonal antiphosphotyrosine antibody PY20. The translocated cytosolic p57 protein was found to be highly phosphorylated (lanes 2-4). Tyrosine phosphorylation of p57 protein in the particulate fraction appeared to be slightly increased after insemination (lanes 6 -8), in comparison with the basal phosphorylation (lane 5). To estimate the enzyme activation, relative phosphotyrosine content of each preparation was calculated by quantifing the intensity of p57 bands. The values obtained for the particulate preparations at 1, 5, and 10 min were 2.4, 1.3, and 1.7, respectively, taking the phosphotyrosine content at 0 min as 1.0. In contrast, those for the cytosolic preparations at 1, 5, and 10 min were 11.5, 14.6, and 9.8, respectively. The result indicates that the p57 Xenopus tyrosine kinase is activated upon fertilization (within 1 min after insemination), and a part of the activated enzyme immediately translocates from the particulate fraction to the cytosol of eggs. DISCUSSION In Xenopus oocytes, the presence of mRNAs of three Src family kinases, c-Src, Fyn, and c-Yes, has been demonstrated (10 -12). cDNAs of receptor/tyrosine kinases for fibroblast growth factor (31) and platelet-derived growth factor (32) have also been obtained. In addition, more than 30 clones representing partial sequences of tyrosine kinase family members have been identified by the polymerase chain reaction method (33). To our knowledge, however, the biochemical identification and characterization of protein-tyrosine kinase activity in oocytes has been limited to the studies on the insulin and insulin-like growth factor receptor/kinase done by Hainaut et al. (34). Therefore, the p57 Xenopus tyrosine kinase purified in this study is the first Src-related kinase biochemically characterized in oocytes. The enzyme was found exclusively in the particulate fraction of oocytes and identified as a Cdc2 peptide kinase activity that was stimulated in the presence of anti-pepY antibody (Fig. 1A). The antibody was originally raised against a synthetic peptide corresponding to residues 410 -428 in the IDA region of chicken c-Src and has been found to activate the bovine c-Src (13). Because the IDA region is one of the most conserved regions among the Src family kinases, we thought that the anti-pepY antibody might be useful for isolation of the Src family kinases, providing not only immunochemical detection but also highly specific in vitro activation of the enzymes. In fact, the p57 Xenopus tyrosine kinase was purified to near homogeneity by using this activating antibody and found to have a limited activity in the absence of the FIG. 6. Translocation of the p57 Xenopus tyrosine kinase upon fertilization. Cytosolic (C) and particulate (P) fractions from each ϳ5 ml of packed volume of oocytes, unfertilized eggs, and fertilized (10-min inseminated) eggs were prepared as described under "Experimental Procedures." Samples were subjected to the DEAE-cellulose column (1 ϫ 6 cm) chromatography as in Fig. 5, except that proteins bound to the column were eluted with 12 ml of buffer B containing 0.2 M NaCl. The first 2 ml was discarded, and the next 10 ml was collected as the eluate. Amount of proteins subjected to the immunoprecipitation was normalized so that each sample was derived from the equal amount (packed volume, 1.5 ml) of oocytes or eggs. Consequently, aliquots of the eluates containing 3 mg of proteins from cytosolic fractions and 0.3 mg of proteins from particulate fractions were immunoprecipitated with anti-pepY IgG and analyzed by autophosphorylation as described under "Experimental Procedures." Phosphoproteins were separated by SDSgel electrophoresis with 12.5% polyacrylamide gel and visualized by a BAS2000 Bioimaging Analyzer (Fuji film). Autophosphorylated p57 protein is indicated by an arrowhead.

FIG. 7. Tyrosine phosphorylation and translocation of the p57
Xenopus tyrosine kinase in the course of fertilization. Insemination was carried out with a 5-ml packed volume of eggs in each experiment, and samples (including unfertilized eggs) were once frozen as described under "Experimental Procedures." DEAE-cellulose fractions were prepared from the cytosolic (lanes 1-4) and particulate fractions (lanes 5-8) and immunoprecipitated with anti-pepY antibody as in Fig.  6, except that anti-pepY beads was used instead of free IgG. Immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis with 8% polyacrylamide gel and analyzed by immunoblotting with anti-pepY antibody (A) or with anti-phosphotyrosine antibody PY20 (B) as described under "Experimental Procedures." Immune complexes treated with [ 125 I]protein A were visualized by the Bioimaging Analyzer. The closed arrowhead indicates the position of p57 protein, and the open arrowhead shows IgG heavy chain leaked from the beads. antibody ( Fig. 3 and Table II). To our surprise, however, we could not identify the c-Src protein or its activity in oocytes. The c-Src protein(s), which may be translated from either of the two mRNAs present in oocytes (10), would have been detected in the first DEAE-cellulose chromatography fractions by anti-pepY antibody or by the anti-Src monoclonal antibody (mAb327). Actually, by using mAb327, we have been able to identify the Xenopus c-Src in the DEAE-cellulose chromatography fractions prepared from brain. 2 Therefore, we think expression level of c-Src in oocytes is very low and beyond the detection limit, in spite of the reported presence of mRNAs. The p57 Xenopus tyrosine kinase did not react with mAb327 antibody (Fig. 4) or with other Src family-specific antibodies tested. This result suggests that the p57 Xenopus tyrosine kinase is not c-Src itself or the product of its partial proteolysis but yet an unidentified Src family member in oocytes. Direct protein sequencing of the purified kinase and its cDNA cloning will allow us to define the molecular structure of the enzyme.
We have used the anti-pepY antiboby as an in vitro activator of the Xenopus tyrosine kinase. Similar anti-IDA peptide antibodies have been used to identify and analyze Src-related tyrosine kinases. Casnellie et al. (35) identified Lck, the 56-kDa Src family protein-tyrosine kinase in T cells, by using an antibody against the synthetic peptide corresponding to residues 409 -421 of v-Src. Litwin et al. (15) reported the purification of a Src-related tyrosine kinase from bovine spleen by using a similar antibody. The purified enzyme displayed three closely spaced protein bands of 50 -55 kDa on SDS-gel electrophoresis, implying that the enzyme is a member of the Src family proteins. As to the effect of the antibody on the kinase activity, Casnellie et al. (35) reported that the Lck activity was inhibited by their antibody. Recently, Peaucellier et al. (36) identified a 57-kDa protein-tyrosine kinase in sea urchin eggs. The enzyme was identified by an antibody against the synthetic peptide corresponding to residues Thr-Tyr-Thr-Ala-Gln-Ala-Gly-Ala-Lys-Asn-Pro-Ile-Lys-Trp (a part of the IDA region) of the sea urchin c-Abl. They also found that the binding of the antibody completely inhibited the kinase activity (36). These conflicting results concerning the effect of antibody on the kinase activity might be explained by the difference in the epitopes that are recognized by these anti-IDA antibodies. Our recent experiments showed that the binding of the antibody to the carboxylterminal half of the IDA region of c-Src results in the activation of c-Src, whereas the binding to the amino-terminal half results in the inhibition. 3 The major epitope for the anti-pepY antibody is shown to reside in the the carboxyl-terminal half of the IDA region of Src (13). The antibody used by Casnellie et al. (36) was raised against a peptide that lacks seven carboxyl-terminal amino acids when aligned with our pepY. Thus, the resulting antibody would not be able to bind to the carboxyl-terminal half of the IDA region of Lck. This might be a critical difference between the two anti-IDA antibodies that possess apparently opposite effects on the kinase activity.
Kinetic analysis of p57 Xenopus tyrosine kinase (Table II) revealed that K m for Cdc2 peptide of this enzyme (360 M) is comparable with those of the Src-related protein-tyrosine kinase from bovine spleen (380 M) (15) and the 94-kDa cytosolic tyrosine kinase from HeLa cells (270 M) (16). On the other hand, the K m value for ATP (71 M) indicated that the nucleotide affinity of the Xenopus tyrosine kinase seems quite low when compared with those of c-Src (2.2 M) (37) and v-Src (14.3 M) (38). It is lower than those reported for c-Yes (32 M) (39) and Lck (30 M) (40). Importantly, anti-pepY antibody did not have any significant effect on K m for either ATP or Cdc2 peptide, but it caused a significant increase in V max (Table II). This result is consistent with the idea that the antibody activates inactive form of the kinase and increases a population of active enzyme without affecting the affinity constants.
It is well known that protein kinase activity plays an important role in oocyte maturation (41). As to the role of Src family kinases, Spivack et al. (7) and Unger and Steele (8) demonstrated the acceleration of the rate of progesterone-induced oocyte maturation by the microinjection of v-Src protein and by the expression of activated c-Src, respectively. Furthermore, much attention has been focused on protein-tyrosine phosphorylation that occurs upon fertilization of sea urchin eggs (42)(43)(44). Accordingly, we have examined the activity of the p57 Xenopus tyrosine kinase in the course of oocyte maturation and fertilization. Although we could not detect any activation of the enzyme during the oocyte maturation, we found a significant activation and translocation of the enzyme after fertilization (Figs. 5-7). The time course analysis (Fig. 7) showed that the process is very fast and almost completed within 1 min. In sea urchin eggs, a quick increase in tyrosine phosphorylation of a subset of proteins has been observed after fertilization (42,43). It is of special interest that the 350-kDa sperm receptor protein is shown to be tyrosine phosphorylated within 5 s after fertilization (44). cDNA for the sperm receptor has been cloned (45), and it is evident that the sperm receptor itself is not a proteintyrosine kinase. Thus, it is likely that one or more proteintyrosine kinase(s) is activated upon fertilization and phosphorylates the sperm receptor. Such a signal transduction system including functional and physical interaction between the receptor proteins without kinase activity and nonreceptor tyrosine kinases has been well established for T cell receptor complex (46). Although the physiological role of tyrosine kinase activity in fertilization is still obscure, Moore and Kinsey (47) demonstrated that tyrosine kinase inhibitors did not block the early events of fertilization such as elevation of the fertilization envelope, but they blocked the later events such as pronuclear migration, DNA synthesis, and cell division in sea urchin eggs. Their observations indicate the importance of tyrosine phosphorylation for the ongoing of fertilization. As a candidate for the protein-tyrosine kinase being responsible for the increased tyrosine phosphorylation of proteins at fertilization, a 57-kDa tyrosine kinase (36) and an Abl-related 220-kDa tyrosine kinase (48) have been reported. Both kinases are localized to the egg cortex (36,48). Recently, Kinsey reported a transient kinase activation and time-dependent phosphorylation and dephosphorylation of the 57-kDa kinase on both tyrosine and threonine residues after fertilization (49). Based on the biological properties and its apparent molecular size, it may be supposed that the sea urchin 57-kDa tyrosine kinase is homologous to the p57 Xenopus tyrosine kinase.
The fertilization-dependent activation of p57 Xenopus tyrosine kinase suggests that this enzyme might be under the regulatory mechanism of early embryogenesis. Further studies on the mechanism by which p57 Xenopus tyrosine kinase is activated upon fertilization may contribute to the understanding of the role of protein-tyrosine phosphorylation in early embryogenesis of Xenopus.