Protein tyrosine phosphorylation in response to fertilization.

The sea urchin egg contains one or more protein tyrosine kinases which are active during the response of the egg to fertilization. In the present study, we have used an antibody specific for phosphotyrosine to determine which egg proteins are phosphorylated on tyrosine in response to fertilization. Analysis of immunoblots prepared from fertilized and unfertilized eggs revealed that fertilization results in a major increase in the phosphotyrosine content of a 350-kDa egg protein. Increased phosphorylation of this protein was detected as early as 1 min after fertilization, at which time it represented the most prominent phosphotyrosine containing protein in the egg. Tyrosine phosphorylation of this protein was transient however, and after 5 min post-insemination, the protein was dephosphorylated or otherwise degraded. Egg membrane proteins of approximately 40, 75, and 145 kDa were also found to act as substrates for protein tyrosine kinases in vitro, but did not exhibit significant changes in phosphotyrosine content during egg activation.

Fertilization results in the execution of a series of preprogrammed biochemical events which serve to stimulate egg metabolism, activate biosynthetic pathways, and initiate cell division. Studies on the role of protein phosphorylation in egg activation have demonstrated that a variety of protein kinases are active during this period of development (1)(2)(3). Of particular interest is the observation that the unfertilized egg contains one or more protein tyrosine kinases which are activated as a consequence of fertilization (4-7). Tyrosine-specific protein kinase activity is an intrinsic property of several oncogene-encoded proteins as well as of cell surface receptors for many growth factors or polypeptide hormones (8). Functional studies in a number of systems suggest that protein tyrosine kinases may play an important role in transducing a signal to induce cell proliferation or in otherwise regulating cell growth (9-12). Our objective has been to identify the protein tyrosine kinases which are activated at fertilization and study their role in egg activation.
To understand the specific enzymatic or cellular functions that are regulated by tyrosine phosphorylation, it is essential to identify the substrate proteins which are phosphorylated on tyrosine during egg activation. Earlier studies detected several egg proteins which act as substrates for endogenous * This work was supported by National Institutes of Health Grant HD-14846 and Research Career Development Award HD-00620 (to W. H. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed Dept. of Anatomy and Cell Biology, University of Miami School of Medicine, 1600 N.W. 10th Ave., Miami, FL 33101. protein tyrosine kinases in vitro (4,5). Specific changes were found in the tyrosine phosphorylation of egg membrane proteins prepared from embryos at different stages of development, suggesting that developmental changes occur in the activity of certain kinases and/or in the availability of substrate proteins (4).
The objective of the present study is to determine which egg proteins are phosphorylated in vivo by protein tyrosine kinases in response to fertilization. We have used an antibody specific for phosphotyrosine to detect phosphotyrosine-containing proteins in immunoblots prepared from eggs at different points during the egg activation process. These experiments revealed that a 350-kDa egg protein is transiently phosphorylated on tyrosine during the first few minutes after fertilization. During this period, the 350-kDa phosphoprotein is the predominant phosphotyrosine-containing protein detected in eggs by the Western immunoblot technique, suggesting that it may play an important role in egg activation.

EXPERIMENTAL PROCEDURES
Antibody Production-New Zealand White rabbits were immunized with keyhole limpet hemocyanin (Behring Diagnostics) which had been derivatized with the synthetic phosphotyrosine analogp-azobenzyl phosphonate (13). For the initial injections, 850 pg of protein (1.4 pmol of POJmg of protein) emulsified in Freund's complete adjuvant was given intradermally. Later injections of 500 pg were in incomplete adjuvant. Anti-phosphotyrosine activity in serum was monitored by a solid phase enzyme-linked immunoassay in which bovine serum albumin coupled to phosphotyrosine by the carbodiimide procedure (14) was used to coat wells of a microtiter plate and IgG binding was detected with biotinylated goat anti-rabbit IgG followed by avidincoupled alkaline phosphatase (Vector Laboratories). To purify the antibodies specific for phosphotyrosine, an affinity resin was prepared by derivatizing cyanogen bromide-activated Sepharose (Pharmacia LKB Biotechnology Inc.) with L-phosphotyrosine (Sigma) (15). Antibodies with a high affinity for phosphotyrosine were allowed to bind to this phosphotyrosine-Sepharose column which was then washed, and the antibodies were eluted with phenyl phosphate (16).
Eggs and Embryos-Gametes were collected from the sea urchin Lytechinus uariegatus or Strongylocentrotus purpuratus and the eggs were washed at pH 5.5 to remove the jelly coat. For controlled development, eggs were suspended with constant stirring (1% v/v) in sterile sea water buffered at pH 8.3 with 5 mM TAPS' (25 "C for L. uariegatus, 7-8 "C for S. purpuratw). Samples to be used in immunoblot experiments were removed before and at various times after fertilization and the eggs were rapidly pelleted in a hand centrifuge. The egg pellet was immediately extracted with 40 volumes of chloroform/methanol (2:l v/v) to arrest development and extract egg lipids. The protein residue was dried and solubilized in SDS gel sample buffer (17). Subcellular Fractionation-Unfertilized or fertilized eggs were washed three times in 10 volumes of calcium-and magnesium-free seawater (0.5 M NaC1,lO mM KCL, 10 mM NaH2C03, 25 mM EGTA (18)). The eggs were then suspended in 10 volumes of a homogeni- zation buffer consisting of calcium-and magnesium-free seawater to which was added 10 mM Na,P20,, 10 mM NaF, 10 p M Na3V04, 10 pg/ ml aprotinin (Sigma), and 10 pg/ml soybean trypsin inhibitor (Sigma). The egg suspension was homogenized in a Potter-Elvehjem homogenizer and layered over a discontinuous sucrose gradient consisting of 10 ml of 30%, 10 ml of 40%, and 5 ml of 78% sucrose dissolved in the above homogenization buffer. The gradients were centrifuged in a DuPont AH 627 rotor at 25,000 rpm for 4 h at 4 "C. Material collected at the 0/30%, 30/40%, and 40/78% interfaces was suspended in homogenization buffer and pelleted a t 25,000 rpm for 1 h. The pellets were solubilized in SDS gel sample buffer and immediately heated at 95 "C for 5 min. In some experiments, the cell surface complex fraction consisting of large sheets of plasma membrane with attached cortical vesicles was purified by a modification of the original method (18). The egg homogenate prepared as above was centrifuged at 1,000 X g for 1 min. The pellet was resuspended in homogenization buffer and centrifuge twice more (18), and then centrifuged through a sucrose step gradient as above. The purified cell surface complex was recovered from the 40/78% interface.
Immunoprecipitation and Immunoblot Analysis-For immunoprecipitation experiments, membrane fractions were phosphorylated in vitro by incubation in a phosphorylation buffer containing 10 mM HEPES, 10 mM MnC12, 10 p~ Na3V04, 10 pg/ml aprotinin, and 0.15% Nonidet P-40. The reaction was started by addition of [y"P] ATP (435 Ci/mmol) to a final concentration of 3.0 p~ and the samples were incubated a t 25 "C for 2 min. The 32P-labeled proteins were solubilized in an immunoprecipitation buffer containing 150 mM NaCI, 50 mM Tris, 10 mM EDTA, 10 mM N&P& 100 mM NaF, 10 p~ Na3V04, 5 mM phenylmethylsulfonyl fluoride, 1.0% Triton X-100, and 0.1 mg/ml aprotinin (Sigma) and centrifuged at 100,000 X g for 30 min at 4 "C. The solubilized proteins were then incubated with affinity-purified anti-phosphotyrosine antibody (0.3 pg/ml) at 4 "C for 4 h, after which 25 pl of protein A-Sepharose (Pharmacia) was added for each 0.1 pg of antibody. After 1 h, the immune complexes were collected by centrifugation, washed twice with immunoprecipitation buffer, once with 50 mM Tris, pH 7.5, solubilized in SDS gel sample buffer, and heated at 90 "C for 5 min. Immunoprecipitates to be analyzed for phosphoamino acid content were washed once more with distilled water and were solubilized in 6 N HCI containing 50 pg each of phosphoserine, phosphothreonine, and phosphotyrosine as standards. Acid hydrolysis and paper electrophoresis were done as described elsewhere (5); some analyses were performed by two-dimensional thin-layer electrophoresis (19).
Samples for Western immunoblot analysis were electrophoresed on SDS polyacrylamide gels and electrophoretically transferred to 0.45 p~ pore nitrocellulose sheets (Schleicher & Schuell). The nitrocellulose sheets were then blocked in 150 mM NaCI, 50 mM Tris (pH 7.5), 0.2% Nonidet P-40,1% gelatin, and 0.1% bovine serum albumin for 12 h, after which they were incubated in blocking solution containing the affinity-purified antibody (0.3 pg/ml) for 4 h. After several washed in blocking buffer, the blots were incubated with 56 pCi/pg '"I-protein (ICN) at a concentration of 1 pCi/ml for 1 h. The blots were then washed in blocking buffer and dried.

Detection of Egg Proteins Phosphorylated on Tyrosine in
Vitro-Previous work in this and other laboratories has used chemical methods to show that membrane fractions from eggs and early embryos contain protein tyrosine kinase activity capable of phosphorylating endogenous membrane proteins in vitro. To further characterize the proteins which act as substrates for these kinases and to test the specificity of the anti-phosphotyrosine antibody in the sea urchin system, we tested its ability to recognize phosphotyrosine-containingproteins from egg membrane fractions which had been phosphorylated in vitro. Egg homogenates prepared before and at different times after fertilization were fractionated over a step gradient prepared from sucrose in calcium-and magnesiumfree seawater. Previous studies have shown that the membrane fraction banding at the 30140% interface has the highest level of protein tyrosine kinase activity (4). When aliquots of this fraction were incubated with [y3'P]ATP, solubilized in immunoprecipitation buffer, and incubated with the antiphosphotyrosine antibody, a characteristic set of phosphopro-teins were immunoprecipitated upon addition of protein A-Sepharose. Analysis of the immunoprecipitates by SDS gel electrophoresis and autoradiography revealed that phosphoproteins of 40,57, 75, and 145 kDa were immunoprecipitated by the antibody (Fig. 1). This result correlates well with earlier in vitro studies in which proteins of 45, 57, 74, and 120 kDa were found to contain phosphotyrosine by chemical analysis (4,5). Samples immunoprecipitated from egg extracts prepared at various times between fertilization and the first cell division contained an identical pattern of phosphoproteins. However, there was a fertilization-dependent increase in the amount of radioactivity incorporated as would be expected from previous measurements of protein tyrosine kinase activity. As seen in Fig. 1, immunoprecipitation of these proteins was inhibited by the presence of phosphotyrosine indicating that the antibody binds through its specific interaction with phosphotyrosine. Antibody specificity was further tested by analysis of the 32P-labeled phosphoamino acids present in the membrane proteins immunoprecipitated by the antibody. Egg membrane proteins phosphorylated in vitro as in Fig. 1 contain phosphoserine and phosphothreonine as the predominant labeled amino acids with phosphotyrosine accounting for about 10% of the radiolabeled species (Fig. 2 A ) . However, in the proteins immunoprecipitated by the antibody, phosphotyrosine accounts for over 95% of the radiolabeled phosphoamino acids (Fig. 2 B ) . Thus  The membranes were solubilized and divided into two aliquots, and the phosphotyrosine-containing proteins were immunoprecipitated with the anti-phosphotyrosine antibody as described under "Experimental Procedures." The phosphoproteins were then analyzed on a 10% SDS polyacrylamide gel and detected by autoradiography. Each immunoprecipitation contained 150 pg of membrane protein and 0.3 pg/ml affinity-purified antibody ( l a n e A ) . In some samples, phosphotyrosine ( l a n e B ) was included a t 5 mM as a competitive inhibitor to demonstrate the specificity of the antibody reaction.  Fig. 1, were solubilized and the phosphotyrosine-containing proteins immunoprecipitated as described. After immunoprecipitation, the phosphoamino acid content of the detergent-soluble ( A ) and immunoprecipitated ( B ) proteins was determined by two-dimensional paper electrophoresis at pH 3.5 (bottom to top) and pH 1.9 (right to left) (5). P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine. has any low affinity, nonspecific interactions with phosphoserine or phosphothreonine.
In addition to the 40-, 57-, 75-, and 145-kDa proteins described above, a high molecular mass protein of approximately 350 kDa was found to be phosphorylated in the cell surface complex fraction collected from the 40/78% interface of the sucrose gradient. This membrane fraction contains sheets of plasma membrane which have retained their association with the cortical secretory vesicles, cytoskeletal elements, and vitelline layer proteins. Detergent-insoluble protein aggregates in this material reduced the specificity of immunoprecipitation; however, when phosphorylated membranes were analyzed directly by SDS gel electrophoresis and treated with alkali prior to autoradiography, the 350-kDa protein became apparent (Fig. 3A). Phosphoamino acid analysis of the 350-kDa band cut from blots of preparative gels demonstrated that this protein is phosphorylated on tyrosine and on threonine residues (Fig. 3B). Western immunoblots of egg samples taken before and at various times after insemination. This method is particularly well suited for the fertilization system, in which metabolic labeling studies are complicated by the fact that the mature, unfertilized egg exhibits a very low rate of phosphate uptake, approximately 1% of the rate in the fertilized egg (20, reviewed in Ref. 21). In addition, the fact that the fertilization-dependent increase in phosphate transport does not begin until 20-30 min after fertilization (22) makes it difficult to study protein phosphorylation during the first few minutes after sperm egg fusion. In Fig. 4, samples of S. purpuratus eggs were taken before insemination and a t different times between fertilization and the first cell division. After electrophoresis on an SDS polyacrylamide gel, the proteins were transferred to nitrocellulose and treated with the anti-phosphotyrosine  imum by 5 min post-fertilization. At points later than 10 min post-insemination, the amount of phosphotyrosine was greatly reduced, suggesting that the protein had been dephosphorylated or otherwise degraded. Densitometric scanning was performed on the autoradiographs from several such experiments to estimate the relative change in phosphotyrosine content of the 350-kDa protein. As seen in Fig. 5, fertilization resulted in a 4-fold increase in the phosphotyrosine detectable in this protein by antibody binding.

Detection of Proteins Phosphorylated on Tyrosine in Vivo-
The 350-kDa protein was found to be present in quantities too low to detect by protein staining techniques; therefore, we were unable to determine whether the increase in phosphotyrosine content represents an increase in the stoichiometry of phosphorylation of a constant level of 350-kDa protein, or an increase in the amount of P-350 which remains phosphorylated at a constant stoichiometry.
As an additional control to insure that binding of the antibody to the 350-kDa protein was specific for phosphotyrosine, blots of egg samples prepared at 5 min after fertilization were incubated with antibody solutions containing either phosphoserine, phosphothreonine, or phosphotyrosine as competitive inhibitors of antibody binding. As seen in Fig. 6, phosphoserine and phosphothreonine had little effect on binding of the antibody to the 350-kDa egg protein, whereas 5 mM phosphotyrosine almost completely inhibited antibody binding. This demonstrates that antibody binding was specific for phosphotyrosine.
Western blot analysis of eggs and embryos from two other marine invertebrates revealed that a similar 350-kDa phosphotyrosine-containing protein is detectable in L. uariegatus  in eggs of the sea biscuit Clypeaster rosacea (Fig. 7). In L. variegatus, the time course of phosphorylation of P-350 was similar to that in S. purpuratus; however, the level of phosphotyrosine in P-350 from unfertilized L. variegatus eggs was barely detectable, and consequently the relative increase after fertilization was larger than that in S. purpuratus and varied more from experiment to experiment. Unfertilized eggs from C. rosacea consistently had a high level of phosphotyrosine in the 350-kDa protein. Fertilization resulted in a modest (approximately 2-fold) increase in phosphotyrosine content which declined to low levels by 15 min post-insemination. These studies in different species seem to indicate that, although the phosphotyrosine content of P-350 in the'unfertilized egg may vary from species to species, fertilization results in the phosphorylation of this protein to some maximal level which is maintained until about 10-15 min post-insemination. As development proceeds, the protein is either dephosphorylated or otherwise degraded in each species. Subcellular Localization-To determine the subcellular localization of the phosphotyrosine-containing form of the 350-kDa protein, eggs were homogenized in the presence of phosphatase inhibitors and fractionated on a sucrose gradient also containing these inhibitors. The fractions were then subjected to SDS gel electrophoresis and Western blot analysis using the anti-phosphotyrosine antibody. As seen in Fig. 8, antibody binding to the 350-kDa protein was detected primarily in the cell surface complex membrane fraction banding at the 40/ 78% interface. Similar results were seen in fractions prepared from fertilized eggs (not shown).

DISCUSSION
Fertilization resembles many other mitogenic processes in that a resting cell (the egg) is stimulated to divide in response to an external stimulus (sperm fusion). A number of in vitro studies have shown that the protein tyrosine kinase activity in egg homogenates increases in response to fertilization (4)(5)(6)(7). In this report we have used an antibody specific for phosphotyrosine to identify the egg proteins which are phosphorylated in vivo during the response to fertilization. We have found that a 350-kDa protein is phosphorylated by one or more protein tyrosine kinases as early as 1 min after fertilization. By 15 min after fertilization, this protein is largely dephosphorylated or otherwise degraded. The 350-kDa protein could not be detected in blots by protein staining techniques such as colloidal gold (Aurodye, Pierce) or in gels by silver staining. Therefore, we were unable to determine whether the changes in the phosphotyrosine content of the protein represent an increase in the phosphorylation of a constant pool of 350-kDa protein, or a change in the amount of the protein which remains phosphorylated at a constant stoichiometry. The 350-kDa protein was by far the major phosphotyrosine-containing protein during the first few minutes after fertilization. Although the 350-kDa and other egg proteins of 40, 57, 75, and 145 kDa could be phosphorylated on tyrosine in vitro, the lower molecular weight proteins were difficult to detect by Western blot of whole eggs because the level of antibody binding to these proteins was similar to the background antibody binding in this region of the gel. Numerous attempts were made to detect phosphorylation of these proteins by immunoprecipitation of eggs metabolically labeled with '*PO4. However, the unfertilized egg has a very low rate of phosphate uptake which does not increase until 20-30 min after fertilization (22). Therefore, even prolonged labeling of large samples of unfertilized eggs (100 mg of egg protein for 48 h) did not produce enough incorporation to demonstrate phosphotyrosine chemically in individual proteins during the first few minutes after fertilization.
The rapid phosphorylation of the 350-kDa egg protein was surprising in view of our earlier in vitro studies which indicated that the protein tyrosine kinase activity in egg homogenates did not increase significantly until 20 min after fertilization (5). However, Satoh and Garbers (7), using a different peptide substrate, were able to detect a 2-fold increase in protein tyrosine kinase activity within the first 3 min postinsemination. Activation of a protein tyrosine kinase that early in the egg activation process would provide a potential mechanism for the increase in phosphorylation of the 350-kDa protein that we have identified here. To evaluate the possibility that the fertilizing sperm could supply the phosphorylated 350-kDa protein at fertilization, we have analyzed sea urchin sperm by Western blot technique and could find no evidence that the 350-kDa protein is phosphorylated in sperm. It is possible that the sperm may contain a kinase, not present in the egg, which may phosphorylate the 350-kDa protein. However, the fact that the 350-kDa protein can be phosphorylated in cell surface complex fractions prepared from unfertilized eggs as well as our preliminary observation that the parthenogenic agent A23187 can stimulate phosphorylation of the 350-kDa protein in vivo suggest that the sperm is not required.
It is useful to consider the above results in the context of the other biochemical changes which are induced by fertilization. Depolarization of the plasma membrane restingpotential occurs within the first few seconds after sperm-egg fusion' (23) and is followed at 15 s by the phosphorylation and turnover of polyphosphoinositides (24, 25). Accumulation of inositol 1,4,5-triphosphate is thought to trigger the "calcium transient," the temporary elevation of free calcium concentrations to 2-5 PM (26,27)  continuing until about 3 min post-fertilization. The calcium transient is accompanied by exocytosis of the cortical secretory vesicles and intense endocytotic activity (28,29). Tyrosine phosphorylation of the 350-kDa protein reported here is first detected at 60 s after fertilization at a time when intracellular calcium levels would be high.
Other responses to fertilization such as the stimulation of protein synthesis, beginning 5-10 min after fertilization (30), initiation of DNA synthesis at 25-30 min after fertilization (31), and the first cell division at 60 min, occur subsequent to the phosphorylation of the 350-kDa protein. Although protein tyrosine kinases, in general, do not require calcium and are not stimulated by calcium, the temporal correlation between the calcium transient and the phosphorylation of the 350-kDa egg protein suggests that these two events may be related. Future work with parthenogenic agents such as the calcium ionophore A23187 may clarify the role of the calcium transient in the phosphorylation of the 350-kDa egg protein.
The rapid, often transient phosphorylation of tyrosine residues in cellular proteins has been observed in several other cell types during their response to growth factors or polypeptide hormones. Treatment of fibroblasts with serum (32) or purified growth factors (33) as well as insulin stimulation of hepatocytes and adipocytes (34,35), resulted in a severalfold increase in the phosphotyrosine content of specific proteins. The receptors for many growth factors and for insulin possess intrinsic protein tyrosine kinase activity, which explains the rapid phosphorylation response (as early as 30 s (35)) in these systems. Interestingly, stimulation of the epidermal growth factor receptor kinase has been shown to result in the transient phosphorylation, on tyrosine, of several cytoskeletonassociated proteins in the 300-350-kDa size range (36). Fertilization is initiated by the cell surface interaction between the sperm and egg (37,38) and it is intriguing to speculate whether the binding of a sperm to the sperm receptor present on the egg surface (39) may activate a protein tyrosine kinase in the egg plasma membrane.