Protein Tyrosine Phosphorylation during Meiotic Divisions of Starfish Oocytes’ GERARD

PEAUCELLIER,*+~

ANN C. ANDERSEN,*

AND

WILLIAM

H. KINsEYt

We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vitlo labeled Murthastrriusglacinlis oocgtes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproleins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35.kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias ruhww oocytes. 11, vitro phosphorylation of corticcs showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68.kDa protein. The proteins phosphorylatcd on tyrosine iu 19itro were almost similar in extracts from oocytes treated or not with the hormone. 6’ 1990 Aeadcmie Press. Inc

INTRODIJCTION

Protein phosphorylation at tyrosine plays an important role in cellular growth control and in the malignant transformation of vertebrate cells (Hunter and Cooper, 1985). The activation of protein tyrosine kinases is one of the earliest cellular responses to the stimulation of cell proliferation by polypeptide growth factors, and tyrosine kinase activity is essential for the action of insulin and growth factors (Chen et al., 1987; Chou et al., 1987). Moreover, many oncogene products also act as tyrosine kinases and share extensive homology with several polypeptide growth factor receptors (Heldin and Westermark, 1984; Yarden and Ullrich, 1988). Numerous protein substrates for tyrosine kinases have been identified in normal and malignant cells but their physiological roles are still hypothetical. Protein tyrosine kinase activity has been demonstrated in sea urchin egg membranes and found to increase following fertilization (Dasgrupta and Garbers, 1983; Ribot et &., 1984; Kinsey, 1984; Satoh and Garbers, 1985). A tyrosine kinase immunologically related to src has been described but its level was found to decrease during fertilization and early development, implying the existence of other tyrosine kinases (Kamel et al., 1986). All these experiments relied on in vitro phos-

phorylation of endogenous or exogenous substrates but in viva phosphorylations have also been demonstrated in eggs from several sea urchin species (Peaucellier et al., 198813).As for the other cell systems, the significance of these events is still unknown. Starfish oocytes may provide a better approach for studying the role of tyrosine phosphorylation in early development. Full-grown oocytes are arrested at first meiotic prophase, at which stage they readily take up exogenous phosphate, in contrast to sea urchin eggs which show a very low uptake (Chambers and White, 1954). Thus it is possible to load these oocytes with high activities of [““PIphosphate to study in viva phosphorylations. When oocytes are treated with l-methyladenine, the relay hormone produced by follicle cells under the influence of a peptide hormone of neural origin, they resume complete meiotic maturation, including the emission of two polar bodies and t,he formation of a haploid nucleus (see Masui and Clarke, 1979; Meijer and Guerrier, 1984, for review). Protein phosphorylation has been shown to increase considerably following lMeAde” addition and to decrease at first polar body emission, the same process occurring at the second meiotic division (Dorke et al., 1983; Capony et al., 1986). Recently, these phosphorylation cycles have been re-

’ This work was supported by CNRS/NSF cooperative research program, Association pour la Recherche contre le Cancer grant (to G.P.), National Institutes of Health Grant HD-14846, and Research Career Development Award HD-00620 (to W.H.K.). ‘To whom corrrspondcncc should bc addrcsscd.

” 1-MeAde, l-methyladenine; BCA, bicinchoninic acid; EGTA, ethylenebis(oxyethylenenitrilo)tetraacetic acid; GVBD, germinal vesicle breakdown; Hepes, 4-(2-hydroxyethyl).1-piperazineethanesulfonic acid; pha, posthormone addition; PMSF, phenylmethylsulPonyl fluoride; SBTI, soybean trypsin inhibitor; SDS, sodium dodecyl sulfate; TCA, trichloroacctic acid.

392

DEVELOPMENTAL

BIOLOGY

lated to the activation of an M phase-specific histone kinase, homolog of the yeast cdc2+ protein kinase (Labbi! et al., 1988). Little is known however on the sequence of events leading from the binding of 1-MeAde with its still unisolated membrane receptor (Yoshikuni et al., 1988a; Yoshikuni et al., 1988b) to the activation of the cytoplasmic histone kinase. Phosphorylations on tyrosine at the plasma membrane level are good candidates for a role in this signal transduction. The objective of the present study is to determine which oocyte proteins are phosphorylated by protein tyrosine kinases in response to hormonal stimulation and during the meiotic divisions. This investigation was greatly facilitated by the availability of an antibody specific for phosphotyrosine (Peaucellier et ab, 1988b). EXPERIMENTAL

PROCEDURES

Handling of oocytes. Ripe females of Marthasterias and Asterias rubens were obtained from the Biological Station of Roscoff. Fully grown prophaseblocked oocytes were prepared free of follicle cells by washing them several times in artificial Ca’+-free seawater (Do&e and Guerrier, 1975). Antibody production. The polyclonal anti-phosphotyrosine antibody was prepared as previously described (Peaucellier et al., 1988b), according to the method of Ross et al. (1981). Briefly, New Zealand White rabbits were immunized with keyhole-limpet hemocyanin which had been derivatized with the synthetic phosphotyrosine analog p-azobenzyl phosphonate. Antibodies were purified by affinity chromatography on cyanogen bromide-activated Sepharose (Pharmacia LKB Biotechnologie) derivatized with L-phosphotyrosine. They were eluted with 40 mMphenylphosphate at 40°C. [“PjPhosphate in vivo labeling. A 12% suspension of prophase-blocked oocytes was incubated with gentle stirring in seawater containing 0.5 mCi/ml carrier-free [“2P]orthophosphate (Amersham). After 3 hr of incubation, oocytes were washed three times with Ca’+-free seawater. Meiosis reinitiation was initiated by addition of 1 mM 1-MeAde to a final concentration of 2 PLM. Preparation of cortices. Cortices were prepared according to the method of Detering et al. (1977). Briefly, 0.6-ml aliquots of oocytes were washed in ice-cold seawater C (0.5 MNaCl, 10 mMKCl,2 mMNaHCO,, 25 mM EGTA, pH 8.0) and homogenized by hand with a Teflon pestle in 10 ml of seawater C containing protease and phosphatase inhibitors (1 mg/ml SBTI, 1 mMPMSF, 50 mM NaF, 10 mM Na pyrophosphate, 10 mM cu-naphthyl phosphate, 10 mM phenyl phosphate, 0.5 mM ZnClz, 0.1 mM Na orthovanadate). Cortices were pelleted by low speed centrifugation (1 min at 1OOO.g) and washed three times with homogenization medium. Phenyl phosphate and oc-naphthyl phosphate, which may inhibit antibody binding, were omitted in the last washing. In some exglacialis

VOL~JME

138, 1990

periments, cortical granules were removed according to Kinsey et al. (1980), by three washes in isotonic sucrose (1 M sucrose, 0.1 mg/ml SBTI) separated by centrifugation at 5000g for 20 min. When not used immediately (in vitro experiments only), cortices and membranes were frozen in liquid nitrogen and stored at -30°C. Immunoprecipitations. All operations were performed at 4°C. Each cortex sample was suspended in 3 ml of immunoprecipitation buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 0.1 mg/ml SBTI, 1 mM PMSF, 50 mM NaF, 10 mM Na pyrophosphate, 0.5 mM ZnCl,, 0.1 mM Na orthovanadate), frozen in liquid nitrogen, and stored at -30°C. After thawing in a cooled water bath at 4”C, and sonication for 5 set at 100 V, samples were centrifuged for 30 min at 105g.Two 1.3-ml aliquots were taken from each supernatant and incubated with anti-phosphotyrosine antibody (10 r.lg/ml) for 2 hr; one aliquot used as control also contained 5 mM L-phosphotyrosine. Protein A-Sepharose (Pharmacia) was added (25 PUsample) and, after 1 hr, the immune complexes were collected by centrifugation, washed four times with immunoprecipitation buffer and twice with 50 mM Tris, pH 7.5, solubilized in SDS sample buffer, and heated at 100°C for 4 min. Gel electrophoresis and autoradiography. For SDSpolyacrylamide slab gel electrophoresis, linear gradients (7.5-20% acrylamide, 0.28-0.05% bisacrylamide) were used with the discontinuous buffer system of Laemmli (1970). Molecular weight markers were purchased from Pharmacia. Gels were stained with Coomassie blue R-250, dried under vacuum, and processed for autoradiography using X-Omat AR films (Kodak). Densitometric analysis of autoradiograms was performed with a Shimadzu CS-930 densitometer. Some gels were incubated with alkali (2 hr in 1 N KOH at 56°C) after glutaraldehyde treatment (30 min in 10% glutaraldehyde) according to Bourassa et al. (1988). [“-?P]Ph,osphateincorporation. During the preparation of cortices and immunoprecipitation from in vivo labeled oocytes, aliquots of whole cell homogenates and 105g supernatants of cortices’ Triton extracts (0.5 and 0.8 ml, respectively) were precipitated with TCA (10%). Samples from total homogenates were processed as previously described (Doree et al, 1983). Triton extracts were washed five times with 10% TCA and four times with acetone. Pellets were dried, dissolved with 0.1 N NaOH, and separated in two aliquots. One was used for protein measurement with BCA reagent (Pierce) (Smith et ah, 1985). The other was neutralized with HCl and dissolved in ACS (Amersham) for liquid scintillation counting. Analysis of labeled phosphoamino acids. Labeled phosphoamino acid analysis was carried out as described by Cooper et al. (1983). Briefly, samples were precipitated with 10% TCA, washed with acetone,

dried, and hydrolyzed in 6 N HCl for 2 hr at 110°C. After addition of unlabeled phosphoserine, phosphothreonine, and phosphotyrosine, they were analyzed by two-dimensional thin-layer electrophoresis on lOO-pm-thick cellulose plates (Sigma) with migration at pH 1.9 (1500 V for 25 min) in the first dimension and at pH 3.5 (1500 V for 25 min) in the second dimension. The position of standards was determined by ninhydrin staining and radioactivity was scored by autoradiography or by scintillation counting of cellulose scraped from the spots. They were performed as In vitro phosphorylations. described previously (Peaucellier et al., 1988b). Briefly, cortices were incubated in a phosphorylation buffer containing 10 mM Hepes pH 7.5, 10 mM MnCl,, 10 pM Na3V04, 0.1 mg/ml leupeptin (Bachem), 0.15% Nonidet-P40 (BDH). The reaction was started by addition of [y-““P]ATP (Amersham) to a final concentration of 425 @Z/ml (about 0.1 PMATP); after a 5-min incubation at 20°C unlabeled ATP was added to a concentration of 25 PLMand samples were incubated for another 5-min period. Ten volumes of ice-cold immunoprecipitation buffer were added and samples were frozen in liquid nitrogen. Immunoprecipitation was performed as described for in viva experiments. RESIJLTS

Detection of membrane proteins phosphorylated vim Prophase-arrested oocytes of M. glacialis

in

were preloaded with 32P,then 1-MeAde was added after elimination of external radioactivity. As previously described (Do&e et al., 1983), incorporation of “P into proteins increased rapidly until GVBD, which occurred 18 min posthormone addition (pha) and then more slowly as oocytes proceeded to metaphase I (60-70 min pha). No significantly labeled protein could be immunoprecipitated, with anti-phosphotyrosine antibodies, from whole oocyte extracts at any stage (results not shown). Since most protein tyrosine kinases are known to be associated with the plasma membranes, cortices were prepared from preloaded oocytes, in the presence of protease and phosphatase inhibitors, and extracted with the nonionic detergent Triton X-100. The 1o”g supernatants of the extracts were used for immunoprecipitation with the anti-phosphotyrosine antibody. The immunoprecipitates were analyzed by SDS-gel electrophoresis and autoradiography. As shown in Fig. 1, several labeled proteins, ranging from 195 to 35 kDa were selectively bound by the antibody. The labeling was hardly significant in prophase-blocked oocytes, but increased rapidly upon reinitiation of meiosis, reaching a maximum after GVBD (40 min pha). Immunoprecipitation of these proteins was inhibited by the presence of 5 mM phosphotyrosine, indicating that the antibody was bound through its specific interaction with phospho-

I.

4 195 4 155 . 100 * 85 *r 43 .

. *

45

4 35 30 *

* FIG. 1. Immunoprecipitation of phosphotyrosine-containing proteins from itc ~ico labeled cortices. Corticcs were prepared from equal aliquots of a batch of ‘r2P-preloaded M glacialis oocytes and extracted with Triton. The high speed supernatant of each extract was divided in two equal aliquots which were incubated with anti-phosphotyrosine antibodies in either the absence (A) or the presence (B) of 5 mM phosphotyrosine. Immunoprecipitates were analyzed by SDS electrophoresis, on the same gel, and autoradiographg during 4 days. The times (in minutes post-hormone addition) at which the samples were taken are indicated at the top of each lane. The migrations and molecular weights (X10 ‘!) of marker proteins are indicated on the left,

tyrosine. It was previously shown, with the same batch of antibody, that preincubation with the same concentration of either phosphoserine or phosphothreonine could not prevent the binding of tyrosine-phosphorylated proteins (Peaucellier et al., 1988b). To further characterize the site of “P incorporation, an immunoprecipitate, prepared from oocytes at 20 min pha, and a 10”~ pellet fraction, used as control, were run in duplicates on a gel, half of which was treated with alkali before autoradiography. The label was found to be alkali resistant in all cases for the immunoprecipitate, while in controls the labeling of the prominent Sl-kDa protein almost disappeared (Fig. 2). This 31-kDa protein was probably the S6 ribosomal protein, which has been shown to be highly phosphorylated on serine only (Peaucellier et al., 1988a). This indicated that the immunoprecipitated proteins had not incorporated significant amounts of “‘P in serine residues but did not preclude some phosphorylation in threonine, which is also alkali resistant. Starfish oocyte proteins are known to undergo partial dephosphorylation upon completion of meiotic divisions (Doree et al., 1983), i.e., emission of first and second polar bodies, occurring under our experimental conditions at 85 and 120 min pha, respectively. In unfertilized eggs, development stops after formation of the female

394

DEVELOPMENTALBIOLOGY

195 .

155 c 100 * 85.

45 c 35 * -

31

FIG. 2. Alkali-resistance of in vine labeled phosphotyrosine-containing proteins. An immunoprecipitate from cortices of oocytes taken 20 min pha (1) and a 105g pellet fraction used as control (2) were divided in equal aliquots and submitted to SDS-gel electrophoresis. The gel was cut in two halves, which were treated (B) or not (A) with 1 N KOH (2 hr at 56°C) before autoradiography during 4 days (A) or 25 days (B).

pronucleus (180 min pha). Thus we investigated the changes in protein tyrosine phosphorylation during completion of meiotic divisions. M. glacialis oocytes had to be preloaded with 32P in prophase I, since the uptake of exogenous phosphate drops following treatment with 1-MeAde. Cortices were prepared from oocytes at various times and used for immunoprecipitation as in the previous experiment. The autoradiogram shown in Fig. 3 demonstrated a decrease in the 32P content of immunoprecipitated proteins upon completion of meiotic divisions. A detailed analysis of these experiments was done from densitometric scanning of the autoradiograms and complementary measurements of 32P incorporation into whole oocyte proteins and Triton-extracted proteins from cortices. During sample treatment for immunoprecipitation, aliquots of whole cell homogenates and 105g supernatants of cortices were precipitated with 10% TCA and prepared for measurement of protein content and incorporated radioactivity as described under Experimental Procedures. As shown in Fig. 4, the were very time courses of changes in 32P incorporation similar in total homogenates, cortices Triton extracts, and anti-phosphotyrosine immunoprecipitates. Quantitatively, the specific activities in cortices’ extracts were about twice those of the corresponding total homogenates, indicating that phosphorylations were specially important in the cortices. The actual incorporation into anti-phosphotyrosine immunoprecipitates was too low

V0~~~~138.1990

to be directly measured and, in any case, it accounted for less than 1% of the radioactivity of the Triton extract. Incorporation into the immunoprecipitates increased significantly faster after hormone addition than in the cortices’ extracts or whole cell proteins. Examination of the autoradiograms shows quite different kinetics of incorporation into individual immunoprecipitated proteins. Some of these changes have been quantitated by densitometry (Fig. 5). The labeling of a 155-kDa protein increased very quickly following hormone addition; it was clearly distinguishable as soon as 2 min pha, reached a maximum at 20 min pha, which corresponded to GVBD, and was decreased to its basal level at 40 min pha. In contrast, a 85-kDa protein showed only minor labeling at the beginning but increased steadily during the meiotic divisions and did not decrease, becoming one of the most prominent upon completion of meiosis. A group of proteins around 45kDa showed a regular increase in radioactivity, reaching a maximum just before first polar body emission and decreasing lately. A different temporal pattern was seen in the case of the 35-kDa protein where no significant label could be seen until 20 min pha, then it increased quickly, and disappeared when meiosis was completed. Shifts in the apparent molecular weight of this protein were also noticeable, after GVBD (20 min

A 195 b 155 b 100

b

85 b

B

40 75 95 180

40

75 95 180

4200

4

94

4

67

4

30

4

20

4

14

45 b 35 )

FIG. 3. Immunoprecipitation of phosphotyrosine-containing proteins from irk viva labeled cortices. Cortices were prepared from “Ppreloaded M. glacialis oocytes, extracted with Triton and incubated with anti-phosphotyrosine antibodies in the absence (A) or presence (B) of 5 mMphosphotyrosine, as in Fig. 1, but with a different batch of oocytes. Immunoprecipitates were analyzed by SDS-gel electrophoresis and autoradiography during 4 days. The times (in minutes posthormone addition) at which the samples were taken are indicated at the top of each lane. The migrations and molecular weights (X10m3) of marker proteins are indicated on the right.

P~XTTCEIMER,

ANDERSEN,

AND

395

KINSEY

P-Tyr

0

2

5 1020407595150

0

2

t min

5 102040759515O

0

t min

2

5 10 20 40 7595150 t mir

FIG. 4. Analysis of “‘P-incorporation during meiotic divisions. ““P-preloaded M. glacialis oocytes were treated with 1-MeAde for the indicated times. “‘P-incorporation was measured in total proteins (Tot), cortices Triton extracts (Mb), and anti-phosphotyrosine immunoprecipitates (I’-Tyr). In the first two graphs, incorporation is expressed as specific activities, measured by liquid scintillation counting, per fig of protein. In the last graph, incorporation was measured by densitometry, using the autoradiograms depicted in Figs. 1 and 3 (Time 40 min pha was used for adjustment between the two experiments).

pha in Fig. 1) and just before second polar body emission at 120 min pha (Fig. 6). This suggested that this protein became hyperphosphorylated during meiosis, which is known to reduce protein mobility in SDS electrophoresis. These results were obtained from two batches of oocytes from two different females; the similar labeling pattern of the common time point (40 min pha), which was also obtained in a separate experiment, is an indication of the reproducibility of these results. However, experiments performed at the end of the breeding period, with oocytes which did not emit perfectly both polar bodies, were unsuccessful due to much lower “P incorporation.

155

K

200

100

0

300

To investigate the generality of these results, similar experiments were performed on another starfish species: A. rubens. Figure 7 shows the autoradiogram obtained by immunoprecipitation with anti-phosphotyrosine antibodies of cortices’ detergent extracts. The pattern and variations of labeling in immunoprecipitated proteins were very similar to those obtained with M. glacialis oocytes. Prophase-blocked oocytes yielded no significantly labeled band, while “P incorporation was clearly seen in 200-, 160-, and lOO-kDa proteins at 10 min pha, before GVBD. Before first polar body emission (40 min pha), the label was increased in the 200-kDa protein, while it was decreased in the 160- and lOO-kDa ones and a 35-kDa protein could now be seen. After completion of both meiotic divisions (180 min pha), the 200- and 35-kDa bands were no longer visible, while the 160- and lOO-kDa were reduced. Thus, the differences between the two species are surprisingly small, and rather quantitative than qualitative, e.g., the lower incorporation at the 85- and 45-kDa levels in Asterias. Previous works had shown In vitro phosphorylations. that membrane fractions from sea urchin eggs contained protein tyrosine kinase activity capable of phosphorylating endogenous membrane proteins in vitro. Thus it seemed interesting to perform in vitro experi95

110 120

130

200

100

a FIG. 5. Changes in 32P-labeling of phosphotyrosine-containing proteins from in vi?:o labeled cortices. Incorporation was measured by densitometry, using the autoradiograms depicted in Figs. 1 and 3, in individual proteins of 155, 85, 45, and 35 kDa, from samples taken at the indicated times (in minutes pha). Time 40 min pha was used for adjustment between the two experiments.

FIG. 6. Mobility changes in the 35.kDa protein during second meiotic division. Cortices from oocytes taken at various times (in minutes, across the top), were immunoprecipitated as in Figs. 1 and 3. The 30- to 45-kDa region of the autoradiogram is shown, together with the migrations and molecular weights (X10 “) of marker proteins on the left.

396

DEVELOPMENTAL

0

10 40

BIOLOGY

180

VOLUME

138, 1990

vivo experiments. In all cases, a 68kDa protein was the

most heavily labeled. The more significant changes occurred late in meiosis with the appearance of two bands of 85 and 80 kDa. Cortices, as obtained by our low speed washes procedure, are complex structures consisting of the plasma membrane, with associated peripheral proteins, the vitelline layer, and cortical granules. As described for sea urchin eggs, further washes with isotonic sucrose allow the elimination of most of these granules, which represent nearly three-fourths of total proteins (Kinsey et al., 1980). Such purified membranes from iI4 glacialis oocytes were used for in vitro phosphorylation and immunoprecipitation. The pattern of radiolabeled proteins was very similar to that of cortices (Fig. 9, lane S), the 68-kDa proteins being the most phosphorylated. This indicates that cortical granule material was not a significant source of enzyme or protein substrate for tyrosine phosphorylations. FIG. 7. Immunoprecipitation of phosphotyrosine-containing proteins from in eivo labeled cortices of A. rubens oocytes. Cortices prepared from “‘P-preloaded A. rubens oocgtes were used for immunoprecipitation as in Figs. 1 and 3. The times (in minutes posthormone addition) at which the samples were taken are indicated at the top of each lane. The migrations and molecular weights (X10-“) of marker proteins are indicated on the left.

DISCUSSION

The meiotic divisions of oocytes from various phyla have been extensively used for cell biology studies (see Masui and Clarke, 1979; Meijer and Guerrier, 1984, for review) since they are well suited for the analysis of hormone interaction with isolated cells and provide cell populations dividing with high synchrony. Prophasements with starfish oocyte membranes and see if there blocked starfish oocytes can be loaded with high specould be some relationship with the in vivo processes. cific activities of [““PIphosphate, part of which is rapCortices were prepared from M. glacialis oocytes, before or at various stages after 1-MeAde addition, incubated A with [T-~P]ATP, and extracted with Triton X-100. Aliquots of the 105gsupernatants, as used for immunopreP-Ser cipitation, were precipitated with TCA and hydrolyzed in 6 N HCl at 110” for 2 hr before separation of phosP-Thr phoamino acid by thin layer electrophoresis. The autoradiogram in Fig. 8A shows that similar amounts of 3zP P-Tyr were incorporated in phosphotyrosine and phosphoserine, while phosphothreonine accounted for only 15% of total incorporation. Similar ratios of incorporation were found in extracts from oocytes blocked in prophase B I or after completion of meiosis (results not shown). By comparison, in the detergent extract of cortices from in P-Ser vivo labeled oocytes, taken 20 min pha, 32P incorporation in phosphotyrosine was below the detection level P-Thr (less than 1% of total), while phosphoserine and phosphothreonine accounted for 80 and 20% respectively (Fig. 8B). Detergent extracts from in vitro phosphorylated corFIG. 8. Phosphoamino acid analysis of in viva and in 7&-o labeled tices were also used for immunoprecipitation with proteins. Cortices of M ylacialis oocytes, taken 20 min pha, were anti-phosphotyrosine antibody followed by SDS-gel labeled in vitro (A) or in viva (B) and extracted with Triton. Proteins electrophoresis and autoradiography. As shown in Fig. were hydrolyzed with 6 N HCl for 2 hr at 110°C and phosphoamino 9, the differences, in total incorporation and the pattern acids were separated by two-dimensional thin layer electrophoresis at of radiolabeled proteins, in samples from different pH 3.5 (bottom to top) and pH 1.9 (left to right). P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine. stages of meiosis were far less important than for in

0

10 40 180

S

200 b

94 e 67 . 43 e

30 .

20 . 14 . PIG. 9. Immunoprecipitation of phosphotyrosine-containing proteins from in c,i~v phosphorylated cortices. Corticcs (left panel) prepared from M. gltrricrlis oocytes taken at various times (in minutes, across the top), from a single hatch, were phosphorglated irr r,ifro with [y-‘“P]ATP and extracted with Triton. Phosphotyrosine-containing proteins were immunoprecipitated and analyzed by SDS-gel electrophoresis and autoradiography. Lane S shows the result of a similar experiment performed with sucrose-washed memhranes from prophase-blocked oocgtes. The migrations and molecular weights (XlC”) of marker prot,eins are indicated on the left.

idly incorporated into ATP. We have previously shown that the specific activity in the ATP pool remains constant, after removal of external radioactivity, for several hours in hormone-treated and in control oocytes (Do&e et ul., 1983). In this report, we have used such preloaded oocytes to detect the proteins which are phosphorylated on tyrosine in vivo, by immunoprecipitation with an antibody specific for phosphotyrosine. The specificity of the antibody used in this study was previously checked both in ELISA assays and in immunoprecipitation experiments with an excess of free phosphoamino acids. The reliability of such antibodies, which are purified by affinity chromatography on a column of phosphotyrosine-Sepharose, has been reviewed recently (Wang, 1988). Phosphorylated proteins could be immunoprecipitated only from membrane fractions of hormonetreated oocytes. This is consistent with the previous in vitro studies on sea urchin eggs which showed that tyrosine kinase activity is concentrated at the plasma membrane level (Dasgrupta and Garbers, 1983; Ribot et al., 1984), as in most other cell systems. The absence of significantly labeled proteins in immunoprecipitates from prophase-blocked oocytes and their appearance a few minutes after hormone addition imply a strong stimulation of phosphorylations, since preincubation

with “‘P (3 hr) was far much longer, and the specific activity of ATP did not change. The simplest interpretation is that tyrosine phosphorylation was initiated by hormone treatment. However, these experiments give no information on the actual amounts of immunoprecipitated proteins, since they were too small to be seen on the Coomassie blue-stained gels. The radioactivity in immunoprecipitates was also too small to allow direct analysis of phosphoamino acids, and phosphotyrosine labeling was even below the detection level in hydrolysates of Triton-extracted cortices. Thus, the proportion of labeled threonine vs tyrosine in immunoprecipitates remains unknown; however, the alkali resistance of all bands precludes significant labeling on serine. Similarly, it was not feasible to measure the actual amounts of phosphate incorporated so that the observed increases in ‘izP incorporation may result from increased turnover due to phosphatase activation. Previous experiments, on whole oocytes, showed a net increase in phosphate incorporation following hormone addition (Dorire ef ul., 1983) and no change in phosphatase activity (Pondaven et Al., 1987), so that actual increases can also be expected in the present experiments. The shift in apparent molecular weight of the 35-kDa phosphoprotein is another clue for changes in actual phosphate content. The different kinetics observed for the immunoprecipitated proteins allow some speculations on the significance of these results. Incorporation in the 155-kDa protein is well in advance of the average of both whole oocyte and membrane phosphorylations. It is also abruptly dephosphorylated after GVBD. It has been shown that the presence of l-MeAde in the medium was required only during a well-defined minimum period of time (about 5 min under conditions similar to ours), called the “hormone-dependent period,” the duration of which could not be reduced by increasing hormone concentration (Dorke et ul., 1976; Guerrier and Doree, 1975). This temporal correlation suggests that phosphorylation of the 155-kDa protein is involved in transduction of the hormonal signal. Yet this could be associated with other effects of hormone-receptor interaction such as the various surface changes which have been described during the same time period (see Schroeder and Stricker, 1983, for review). The behavior of the 35-kDa protein is also intriguing since it is seen only during the period when the meiotic spindle forms and condenses. This may be related to preparation for cortical contraction at polar body emission. Most other major phosphoproteins have the same phosphorylation kinetics seen in total homogenates and cortices’ extracts, which gives no clue to their role in meiotic divisions. The results from i71 vitro experiments show little correlation with those of in vivo phosphorylations. Half of the incorporated 32Pis found in phosphotyrosine, which

398

DEVELOPMENTAL

B~o~ocu

indicates that tyrosine kinases are major intrinsic phosphorylating enzymes in cortices and that diffusible serine/threonine kinases are responsible for most of the in vivo phosphorylations of cortices proteins. The similarity of phosphorylation patterns in crude cortices and sucrose-washed ones, lacking cortical granules, support the idea that tyrosine kinases are localized in the plasma membrane or strongly associated with it. The extent of tyrosine phosphorylation is very similar in cortices from prophase-blocked oocytes and from hormone-treated ones. This implies that both tyrosine kinases and their protein substrates are already present in resting oocytes. Various mechanisms could be invoked to explain this inhibition and its release by hormone treatment. The simplest would be the existence of inhibitors which would dissociate from the enzyme under in vitro conditions or the inaccessibility of the substrates under in vivo conditions. The latter hypothesis would explain that the 68-kDa protein can be the major substrate in vitro without being significantly labeled in vivo. The importance of tyrosine phosphorylation in the stimulation of cell metabolism and division is well documented. A large increase in tyrosine phosphorylation has been shown to follow treatment of fibroblasts with serum (Morla and Wang, 1986) or purified growth factors (Kohono, 1985), as well as insulin stimulation of hepatocytes or adipocytes (White et al., 1985; Kasuga et al., 1982). This has led to the discovery of intrinsic tyrosine kinase activity in receptors for many growth factors and for insulin. However, most studies have been restricted to the Go/G1 transition. The present results indicate that tyrosine phosphorylation is also an important event in the special case of meiotic divisions. This is in agreement with results obtained on the homologous system of Xenopus oocytes, for which progesterone is the natural inducer of meiosis reinitiation. Microinjection of the oncogenic tyrosine kinase ~~60’.“‘” into prophase I-blocked oocytes has been shown to accelerate the rate of progesterone-induced maturation, through a protein synthesis-dependent pathway (Spivack et al., 1984). In these experiments ~~60”.“” alone did not induce maturation, but increased serine phosphorylation of ribosomal protein S6, which is an early event of meiosis reinitiation in both frog and starfish oocytes (Peaucellier et al., 1988a). Insulin, which mimics progesterone action, increases tyrosine kinase activity in the plasma membrane, one of the substrates being an insulin-stimulated serine kinase which is immunoprecipitable by anti-phosphotyrosine antibodies (Sakanoue et ab, 1988). One main issue remains the establishment of homologies between the tyrosine kinase substrates identified in this study with those found in other systems. Correlations based on molecular weight are probably of little

VOLUME

138, 1990

use, due to the phylogenetic distance between echinoderms and higher vertebrates, which have been used for most studies. Possible exceptions would be highly conserved regulatory proteins such as ~~60”‘” or the cdc2 protein kinase, which were found to have similar molecular weights in echinoderms and mammals (Kamel et al., 1986; Labbi! et ah, 1988). Both are susceptible to phosphorylation on tyrosine and their phosphorylation state is subject to cell-cycle regulation (Chackalaparampil and Shalloway, 1988; Draetta et ah, 1988). However, such proteins were not detected in the present experiments since no significant label was found at the 60- and 34-kDa levels. This is in agreement with our recent observation that the starfish cdc2 kinase is not labeled under similar conditions (Labbe et aZ., 1989), which still do not exclude the existence of phosphotyrosine with low turnover. Another kind of tyrosine kinase substrate which may be involved in starfish oocyte maturation are those of the lipocortin/calpactin family (see Crompton et ah, 1988, for review). They show calciumdependent association with membranes or cytoskeleton and inhibit the release of arachidonic acid from membranes by phospholipase AZ. In starfish, there is evidence for a release of arachidonic acid, mediated by phospholipase AZ, which has some implication in meiosis reinitiation (Meijer et ah, 1984). The 35-kDa protein which was shown to be transiently phosphorylated in this study may belong to this family, since most lipocortins have similar molecular weights. Future work on isolation and amino acid sequence analysis of the tyrosine kinase substrates identified in this study should provide insights on their role in meiosis reinitiation and cell division. We thank

Madame

A. Cueff

for her skillful

technical

help.

REFERENCES BOITKASSA, C., CIIAPDELAINE, A., ROBERTS, K. D., and CHEVALIER, S. (1988). Enhancement of the detection of alkali-resistant phosphoproteins in polyacrylamide gels. Anal. Biochem. 169.356-362. CAPONY, J.-P., PICARD, A., PEAIICFX,LIER, G., LABBI?, J.-C., and DORI?E, M. (1986). Changes in the activity of the maturation-promoting factor during meiotic maturation and following activation of amphibian and starfish oocytes: Their correlation with protein phosphorylation. Dn: Riol. 117, l-12. CIIACKALAPARAMPIL, I., and SHALLOWAY, D. (1988). Altered phosphorylation and activation of ~~60’~“” during fibroblast mitosis. Cell 52, 801-810. CHAMBERS, E. L., and WHITE, W. E. (1954). The accumulation of phosphate by fertilized sea urchin eggs. Biol. Bull. 106,297-307. CHEN, W. S., LAZAR, C. S., POENIE, M., TSIEN, R. Y., GILL, G. N., and ROSENFELD, M. G. (198’7). Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. Nature (London) 328, 820-823. CHOII, C. K., DTJLL, T. J., RUSSELL, D. S., GHERZI, R., LEBWOHL, D., ULRICH, U., and ROSEN, 0. M. (1987). Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J. Biol. Chem. 262.1842184’7.

COOPEK, J. A., SEFTON, B. M., and HUNTEX, T. (1983). Detection and quantification of phosphotgrosine in proteins. Ilr “Methods in Enzymology” (J. D. Corhin and J. G. Hardman, Eds.), Vol. 99, pp. 387-401. Academic Press, San Diego. CROMPTON, M. R., Moss, S. E., and CRIIMPTON, M. J. (1988). Diversity in the lipocortin/calpactin family. &[I 55, l-3. DASCGRIIPTA, J. D., and GAKUERS, D. L. (1983). Tyrosine protein kinasr activity during emhryogenesis. J. Bin/. (%cnc. 258, 6174-61’78. DETERINC;, N. K., DE(‘KXK, G. L., SCHMELI,, E. D., and LENNAKX, W. J. (1977). Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs. .J. Cdl Bid. 75, m-914. DOR&E, M., and GIIERRIEK, P. (1975). Site of action of I-methgladenine in inducing oocvte maturation in starfishes: Kinetical evidences for receptors localized on the cell surface. Exp. Cd/ Rvs. 91, 296-300. DOK~E, M., GTTEKRIPX, P., and LEONARI), N. J. (1976). Hormonal control of meiosis: Speciticity of the I-methgladenine receptors in starfish oocytcs. Proc. Not/. Accctl. Sci. ILSA 73, 16691673. D~~kfi:, M., PEAU(:ELI,IER, G., and PI(‘ARI), A. (1983). Activity of the maturation-promoting factor and the extent of protein phosphoqlation oscillate simultaneouslg during meiotir maturation of starlish oocytes. UC>?). Biol. 99, 489501. DRA~:TTA, G., PIWNI(‘A-WOKMS, H., MORKISON, L).. DKUKER, B., RotrERTS, T., and BF:A(‘H, D. (1988). Human cdc2 protein kinasr is a major cell-cycle regulated tyrosine kinasr substrate. A’of?~t~, (LOT/dor,l 336, 738-743. GITERRIF:K, P., and DO&E, M. (1975). Hormonal control of reinitiation of meiosis in starfish. The requirement of I-methgladeninc during nuclear maturation. L)pl: Biol. 47, 341-348. HXLI)IN, C. H., and WESTF:RMARK, B. (1984). Growth factors: Mechanism of action and relation to oncogones. Cc’// 37, 9-20. HUNTER,T., and COOPER, J. A. (19%). Protein-tgrosine kinases. AI/VU. Kc~c! Iliocllf~t?/. 54, X97-930. KAMF,L, C., VENO, I’. A., and KINSEY, W. H. (1986). Quantitation of a src-like tvrosine protein kinase during fertilization of the sea urchin egg. Biochrn/. Biol,h gs. Rtes. Con/ nt UK 138, 349-355. KASU~A, M., ZI(:K, Y., BLITH, 1). L., KARI~SONN, F. A., HARIN~, H. IT., and KAI~N, C. R. (1982). Insulin stimulation of phosphorylation of the f-subunit of the insulin receptor. J. Viol. C’!/cnr. 257, 989-9894. KINSEY, W. H. (1984). Regulation of tvrosine-specific kinase activity at fertilization. Ur)>. Rio/. 105, 137-143. KINSXY, W. H., DE:CKER, G. L., and L~;NNAR%, W. J. (1980). Isolation and partial characterization of the plasma memhrane of the sea urchin egg. J. &/I Biol. 87, 24X-254. KOHONO, M. (1985). Diverse mitogenic agents induce rapid phosphorglation of a common set of cellular proteins at tyrosine in quiescent mammalian cells. ,I. Rio/. Ulrn). 260, 1771~1779. LAHRI?, J.-C., LEE, M. G., NURSE, P., PICAKZ), A., and DOR~E, M. (1988). Activation at M-phase of a protein kinasc encoded by a starfish homologue of the cell cycle control gene cdc2’. Nofurc iLm2tfcn,) 335, 251-253. LAB&,, J.-C., PI~ARD, A., PEAII(‘EI,I,IER, G., LEE, M., NURSE, P., and ~hlR6X, M. (1989). Purifcation of MPF from starfish: Identification as the Hi histone kinase ~34’““~ and a possihle mechanism for its periodic activation. Cell 57, 253-263. LAEMMLI, IT. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nofurc? (Lo&or/l 227, 680-685. MASI~I, Y., and CLARKE:, H. J. (1979). Oocgte maturation. I?rt. Ret?. Cytol. 57, 185-223.

MEIJEK, L., and GIIERRIER, P. (1984). Maturation starfish oocgtcs. Zr//. Rc’rl. Qfol. X6, 129196.

and fertilization

in

MEIJER, L.. GIIFKRIEI~, P., and MA(‘I,oI.P, J. (1984). Arachidonic acid, 12- and 15-hpdrosyeicosatetraenoic acids, eicosapentaenoic acid, and phospholipase A:! induct starfish oocyte maturation. I)rr. Biol. 106,368-378. MORI,A, A. O., and WAS (I,orrtlo)rj 294, 654-656. SAKANCXIE, Y., HASI~IMOTO, E., NAKAMIIRA, S-I., and YA~I~\MI:RA, H. (198X). Insulin-stimulated serine kinasc in X(yrco/~/r.s oocgte plasma membrane. Biocli(~),,. Kio/,l/ !/.s. Rw (‘on, ,t, II U. 150, 11761184. SATOH, N., and Gar~u~:r~s, D. L. (1985). Protein tvrosine kinasr activity of eggs of the sea urchin St~o~/!/!//oc,c~)/(rotcts (,),r,,cr or/ 11s: The regulation of its increase afttlr fertilization. L)f,c~. Rio/. 111, 515-519. S(‘HKOIX)E:R, T. E., and STKI(‘KER, S. A. (1983). Morphological changes during maturation of starfish oocytes: Surface ultrastructure and cortical actin. L)car,. Bio(. 98, 373-3384. SiwITf%, P. K., KKO~IN, R. I., HI~:KMANSON, G. T., MAI,I.I~Z, A. K., GAKTNEK, F. H., PROV~:N%ANO, M. I)., FI .IIIVIOTO, E. K., GOEKE:, N. M.. OLSON, B. J., and KUNK, D. C. (198.5). Measurement of protein using bicinchoninic acid. Arctr/. Bioch(j)rc. 150, 76-85. SI>I\ A-K, J. G., EKIESON, R. L., and MALLEn, J. L. (1984). Microinjcction of pp60”” into Xer/o/))ts oocytcs increases phosphorglation of ribosomal protein S6 and ac.celeratcs the rate of proyestcrone-induced mciotic maturation. ,\fo/. (‘(‘II. Viol. 4, 1631-1634. WAX;(:, J. Y. J. (1!)88). Antihodics for phosphot;vrosine: Analytical and preparative tool for t4’ros~l-l)hosl)horylatoti proteins. Awl. Biof//cw/. 172, 1-T. WHITE, M. F., MAI~ON, R., and KAHN, C. R. (1985). Insulin rapidly stimulates tgrosinr phosphorylation of a M,-1X5,000 protein in intact cells. ,v~:clf)rre il,or/clon) 318, 183-185. YAKI)E:N, Y., and IJI,LRI(‘I I, A. (1988). Growth factor receptor tyrosine kinases. II)))/)/. I{(,,,. Biocl~c~rr~. 57, 443-478. YOSHII(~;NI, M., ISH11i;\w.4, K., ISOBE, M., GOTO, T., and NA(;AIIAMA, Y. (1988a). Synergistic actions of disulfide-reducing agents on lmethq’ladenineinduced oocvte maturation in starfish. I)c)~. Biol. 128 ,’ 216-2‘39.1. YOST-IIK~INI, M., ISHIKA~A, K., Isone, M., GOTO, T., and NAGAHAMA, Y. (1988h). Characterization of l-methgladenine binding in starfish oocyte cortices. Pro