- Email: [email protected]
Polyphoshoinositide synthesis and protein phosphorylation in the plasma membrane from full-grown Bufo arenarum oocytes
Comp. Biochem. Physiol.Vol. 102B, No. 3, pp. 585-590, 1992 Printed in Great Britain
0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
POLYPHOSPHOINOSITIDE SYNTHESIS AND PROTEIN PHOSPHORYLATION IN THE PLASMA MEMBRANE FROM FULL-GROWN BUFO A R E N A R U M OOCYTES TELMA S. ALONSO*, IDA C. BONINI DE ROMANELLI,ANA M. ROCCAMODE FERNANDEZ and FRANCISCOJ. BARRANTES Instituto de Investigaciones Bioquimicas, Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET) and Universidad Nacional del Sur, C. C. 857, 8000 Bahia Blanca, Argentina (Tel.: 09 131342; Fax: 054 91 27876)
(Received 4 November 1991) Abstract--1. Polyphosphoinositide content and phosphorylation of lipids and proteins were analyzed in oocytes of the toad Bufo arenarum Hensel. 2. Plasma membrane-enriched fractions obtained from full-grown, prophase-arrested oocytes incorporated 32p into both phospholipids and proteins after incubation with [?-32p]ATP in an Mg2+-containing medium. Phosphatidylinositol 4-phosphate (PIP), phosphatidate (PA) and phosphatidylinositol-4,5-bisphosphate (PIPa) were the only labelled lipids. The 32p incorporation depended on incubation time, the amount of protein, and the ATP concentration. 3. Autoradiography of polyacrylamide gel electropherograms and scintillation counting showed that the radioactivity was mainly associated with a group of membrane proteins having an M r of 87,000. 4. This paper provides evidence for the capacity of prophase-arrested oocytes from Bufo arenarum to synthesize polyphosphoinositides and to phosphorylate distinct membrane proteins.
INTRODUCTION Although polyphosphoinositides have been found to be only minor constituents of the animal tissues examined to date, increased attention is being given to their metabolism and function in a variety of membranes (Abdel-Latif, 1989). There is general agreement now that activation of some receptors leads to the phosphodiesteratic breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2), through the action of phospholipase C, into myo-inositol 1,4,5triphosphate and 1,2-diacylglycerol, which may serve as second messengers in a number of calciumdependent functions (Abdel-Latif, 1986). In amphibian oocytes it has been suggested that polyphosphoinositides play a possible role in the maturation process. Thus, the oocyte maturation induced by progesterone is promoted by cholinergic muscarinic stimulation (Dascal et al., 1984) which in Xenopus laevis has been shown to induce the hydrolysis of PIP 2 (Oron et al., 1985). Ras-injected oocytes have shown changes in inositol lipid metabolites in response to induction of maturation (Lacal et al., 1987). D a t a on inositol lipid content and metabolism in full-grown oocytes capable of undergoing maturation are scarce. This paper presents evidence for the presence of an active biosynthesis of polyphosphoinositides in a plasma membrane-enriched fraction from full-grown, prophase-arrested Bufo arenarum
oocytes. Phosphorylation of proteins is also demonstrated.
distinct
membrane
MATERIALS AND METHODS
Preparation of full-grown oocytes The ovarian tissue was removed from adult Bufo arenarum Hensel female toads, placed in a Ca2+-free Barth's medium, rinsed, and cut into small pieces. Full-grown oocytes (1.7-1.8 mm in diameter) in stage V were selected under a stereo-microscope following the criteria of Valdez Toledo and Pisano (1980). They were treated with 0.25% (w/v) collagenase (Sigma type I) in a Ca2+-free Barth's medium with agitation for 1.5 hr at 23°C. After this treatment, oocytes free from follicular cells were rinsed several times with Barth's medium, now containing Ca 2+ (1 mM). Isolation of the plasma membrane -enriched fractions The procedure described by Blondeau and Baulieu (1985) was followed with some additional steps. Oocytes and eggs were rinsed three times and homogenized in 10 mM HEPES buffer, pH 7.9, containing 83 mM NaCI and 1 mM MgCI2. Homogenates were centrifuged at 1000g for 10min. The supernatants were centrifuged at 10,000g for 20 min. The pellets containing the plasma membrane were rehomogenized in the same buffer and centrifuged at 1000g for 10min. The supernatants were centrifuged at 10,000g for another 20 rain. The resulting pellets were resuspended in the phosphorylation buffer and used immediately. The activity of 5'-nucleotidase was used as an enzyme marker for membrane enrichment, following the procedure of Michell and Hawthorne (1965). Phosphorylation of plasma membrane-enriched fractions The standard medium used for lipid phosphorylation was 7.5mM HEPES, pH 6.8 10mM MgCI2 62mM NaC1. Membrane samples (about 0.3 mg of protein) were preincubated in this medium for 5 min at 30°C after 2rain of
*To whom all correspondence should be addressed. Abbreviations--PA, phosphatidic acid; PI, phosphatidylinositol; PC, phosphatidylcholine; PE phosphatidylethanolamine; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5 bisphosphate. 585
586
TELMAS. ALONSOet al.
sonication. The phosphorylation assay was initiated by the addition of 50 ,aM (7-32P)ATP (spec. act. 0,67 mCi//~mol). Incubation was carried out in a final volume of 250 #1 and was ended by addition of 2.25 ml ice-cold distilled water followed by 2.50 ml ice-cold 20% trichloroacetic acid. The precipitated material was centrifuged in a benchtop centrifuge. The supernatant was removed and the pellet extracted with chloroform-methanol-concentrated HCI (400 : 200 : 3,
with H202 at 70°C overnight, and counting their radioactivity in a Beckman 8000 liquid scintillation counter. Protein concentration was measured by the method of Lowry et al. (1951). RESULTS
Polyphosphoinositide content in full-grown prophasearrested oocytes and in plasma membrane-enriched Protein phosphorylation was examined under similar fractions isolated therefrom experimental conditions except that the pH of the medium The analysis of glycerophospholipid content in was 7.9 and the (?-32p)ATP specific activity was 0.27 mCi/pmol. Membrane samples (about 70 #g protein) cell-free homogenates from full-grown oocytes of were preincubated for I min after 2 rain of sonication. For Bufo arenarum showed that phosphatidylcholine the determination of total protein labelling, incubation was (PC) and phosphatidylethanolamine (PE) comprised stopped with 20% trichloroacetic acid. After successive nearly 82% of the total phospholipids, followed by NaOH extractions and trichloroacetic acid precipitations, phosphatidylinositol (PI), sphingomyelin and phosradioactivity was measured in the protein-enriched pellet by phatidylserine (Table 1). Phosphatidic acid, a key liquid scintillation counting. Samples subjected to sodium dodecyl sulfate 10% acrylamide gel electrophoresis were intermediate in the biosynthetic lipid pathways, repprocessed under the same conditions, but the reaction was resented only 0,15% of the total. The polyphosphostopped by addition of 30#1 of Laemmli solubilization inositides were also minor components of whole buffer (Laemmli, 1970). Differences between duplicate full-grown oocytes (together they comprised 0.1% of experiments were lower than 5%. the total). A subcellular fraction enriched in plasma memLipid extraction, isolation and analysis brane was prepared in order to examine the content Lipid extracts from whole oocytes and plasma membrane- and metabolism of polyphosphoinositides and proenriched fractions were prepared according to the method of tein phosphorylation. Since endoplasmic reticulum is Folch et al. (1957). In order to extract polyphosphoinosiscarce in oocytes and eggs, extensive purification was tides the procedure of Uma and Ramakrishnan (1983) was not considered necessary for the present purposes. employed. PIP and PIP 2 were separated by thin-layer chromatog- The subcellular fractionation was performed as deraphy on 1% potassium oxalate-impregnated silica gel H scribed by Blondeau and Baulieu (1985) with some plates, developed as described by Shaik and Palmer (1977). additional steps that were important in this case to Other phospholipids were isolated by two-dimensional thin- eliminate pigment granules and yolk platelets. The layer chromatography (Rouser et al., 1970). Lipids were presence of diphosphatidylglycerol (cardiolipin), a located by exposure of the plates to iodine vapors. Pure lipid component typically associated with mitochonsubstances as standards were used. The silica was scraped dria, could indicate a slight contamination with these off into vials containing 0.4 ml water and 10 ml 0.4% Omnifluor in toluene-Triton X-100 (800:200, v/v) was organelles. The specific activity of a marker enzyme added (Rodriguez de Turco and Aveldafio de Caldironi, for plasma membranes, 5'-nucleotidase, was en1980). Radioactivity was quantitated by liquid scintillation hanced 12-fold in the plasma membrane-enriched counting, applying the corresponding calculation for 32p fraction with respect to the crude homogenate. The plasma membrane fractions had a characterdecay. Lipid phosphorus was determined according to Rouser et al. (1970). istic phospholipid profile (Table 1). The major components were PC and PE but there was Protein identification .and quantification more phosphatidate, phosphatidylserine and sphinMembrane samples were subjected to polyacrylamide gel gomyelin in this fraction than in the whole oocytes. electrophoresis. Gels were stained with 0.25% Coomassie The content of PIP and PIP2 was also higher (18- and Blue as described by Laemmli (1970). Autoradiography was carried out using 3 M Medical 40-fold respectively) in membranes than in whole X-ray film type R and a Gevaert intensifying screen at oocytes. This, together with the higher activity of the -70°C (Bonner and Laskey, 1974). The 32p incorporated plasma membrane marker enzyme (about 12-fold into individual bands was quantitated by excising them higher 5'-nucleotidase activity) is indicative of the from wet stained gels, treating the polyacrylamide slices enrichment achieved in the subcellular fractionation.
v/v).
Table 1. Phospholipid content in cell-free homogenates and plasma membrane-enriched fractions of amphibian full-grown prophase-arrested oocytes Cell-free homogenate (nmol/mg protein) (%) Phosphatidic acid Phosphatidylserine Phosphatidylinositol Sphingomyelin Phosphatidylcholine Phosphatidylethanolamine Diphosphatidylglycerol Phosphatidylinositol 4-phosphate Phosphatidylinositol 4,5-bisphosphate
0.14 -- 0,002 1.46 -- 0.25 8.58 + 0,70 6.03 ,, 1.06 52.91 ,, 6,67 26.42 ,, 1,24 0.65 + 0,12 0.09 -- 0,01 0.01 + 0.003
0.15 1.52 8.91 6,26 54,91 27,44 0,68 0,09 0,01
Plasma membrane (nmol/mg protein) 1.80 -- 0.50 23.7t + 4.02 41.93 ,, 5.82 45.72 ,, 2.33 194.80 ,, 30.54 115.31 ,, 20.30 24.10 _+ 6.23 1.60 ,, 0.61 0.42 _+ 0.10
(%) 0.40 5.28 9.33 10.17 43.35 25.66 5.36 0.36 0.09
Plasma membrane-enriched fractions were obtained as described in Materials and Methods. Phospholipids were isolated by bidimensional thin-layer chromatography (Rouser et al., 1970) and polyphosphoinositides by monodimensional thin-layer chromatography (Shaik and Palmer, 1977). Phospholipidic phosphorus was measured according to Rouser et aL (1970). Data are expressed as mean -- SD from six independent samples.
PPI synthesis by Bufo oocytes
587
A
e-
6O0
1200
"O
0. Im
E
A00
800
O
E 200
"6 4oo E 10
i
i
20
30
0.2
0.~
0.G
protein Imgl
time (rain)
Fig. 1. PI kinase and PIP kinase activities in oocyte plasma membrane. (A) time-course of PI kinase (A)-, and PIP kinase (0)- catalyzed reactions. (B) effect of protein concentration on PI kinase (A) and PIP kinase ( 0 ) reactions after l0 min incubation. Plasma membrane-enriched fractions obtained as described in Materials and Methods were incubated with 50/~M 0,-32p)ATP (spec. act. 0.67 mCi/pmol), I0 mM MgCI2, 62 mM NaC1 and 7.5 mM HEPES, pH 7.9, in a final volume of 250/~1 at 30°C. Membranes were preincubated in this medium for 5 min at 30°C after 2 rnin of sonication. Incubation was stopped by addition of 2.25 ml ice-cold distilled water followed by 2.50 ml ice-cold 20% trichloroacetic acid. Lipids were extracted from the pellets with acidified solvents (Uma and Ramakrishnan, 1983). Phosphoinositides were isolated by thin-layer chromatography (Shaik and Palmer, 1977). The values of incorporation represent the average of two separate samples.
Synthesis of acidic glycerophospholipids in plasma membrane -enriched fractions
Concentration-dependent effects of A TP on polyphosphoinositide and phosphatidate synthesis
Incubation of oocyte plasma membrane-enriched fractions with (?-32P)ATP in the presence of Mg 2+ promoted the labelling of PIP, PA and PIP2, in that order (32p incorporation could not be detected when the magnesium concentration was reduced to about one-tenth). The order of distribution of radioactivity appears to be logical, since the concentration of the substrate for PI kinase should not be rate-limiting, given that PI was present in sufficient amounts in the membrane preparation. This may not have been the case for PIP kinase since the concentration of PIP was low, and the PIP kinase depends on the first kinase for the provision of substrate. As far as the labelling of PA is concerned, since no exogenous substrate was added to the incubation medium, diacylglycerol kinase must necessarily have phosphorylated the endogenous diacylglycerol. The time-dependence of the labelling by (?32P)ATP of PIP and of PIP2 in plasma membranes is shown in Fig. 1A. Both were labelled as early as 2 min after the addition of the precursor. The rate of formation of PIP increased during the first 10 min and subsequently reached a maximum of about 130 fmol min- 1 m g - l protein, slowly declining thereafter. The amount of PIP 2 labelled was lower than that of PIP, reaching a maximum of about 20 fmol min-~mg -~ protein after 5rain. The labelling of polyphosphoinositides was also dependent on the amount of membrane (Fig. 1B). The formation of PIP was higher than that of PIP 2 in the range between 100 and 600/~g membrane protein. The time-course of phosphatidate labelling after incubation of oocyte plasma membrane with (~32p)ATP is depicted in Fig. 2. The incorporation increased linearly up to about 10 rain of incubation, reaching a maximum of about 45 fmol min-~mg -] protein. Protein concentration also affected the incorporation of 32p into PA (Fig. 2, inset).
Since the enzymatic conversion of PI to PIP and the subsequent phosphorylation to produce PIP2 require ATP as substrate, the effect of the concentration of this nucleotide on PIP and PIP2 labelling was analyzed (Fig. 3). Increasing concentrations of ATP in the range between 25 and 300 p M stimulated the activities of phosphatidylinositol kinase and PIP kinase 9- and 6-fold respectively. Diacylglycero] phosphorylation was stimulated 11-fold in the same ATP concentration range.
Protein phosphorylation of oocyte plasma membraneenriched fractions Plasma membrane fractions obtained from fullgrown prophase-arrested oocytes were incubated with (~J2P)ATP to analyze total protein labelling. The time-course of such phosphorylation is depicted
[] ,
D
¢- (00
'~, O.. 300
'U 200
.& 200
• J 100
E
L: O
E
100 0.2
O.&
0.6
protei n I mgl
w-
i 10
i
i 20
,
i
i 30
t i m e (mini
Fig. 2. Biosynthesis of phosphatidic acid in oocyte plasma membrane-enriched fractions. Experimental conditions as in Fig. 1. The effect of protein concentration is depicted in the inset. Values represent the average of two separate samples.
588
TELMA S. ALONSO
25 E '~ 20 E ,?
15 x
:g ~
10
,45
100
200
300
ATPluM)
Fig. 3. Effect of ATP concentration on PI kinase (A), PIP kinase ( 0 ) and diacylglyceride kinase (D) activities. Membranes were incubated for 10rain as described in Fig. I. Values represent the average of two separate samples. in Fig. 4. A net 32p incorporation (as early as 2 min after the addition of the labelled nucleotide) was detected, a plateau being reached after 10min incubation. Polyacrylamide (10%) gel electrophoresis of the membrane samples revealed at least 40 protein bands (Fig. 5A). The major ones corresponded to groups of proteins with an Mr of about 48, 70 and 97 kDa. The time-course of 32p incorporation into individual polypeptide chains was analyzed by autoradiography and by radioactive counting of excised individual bands. Autoradiograms exposed for 96 hr enabled the detection of minor phosphorylated bands. A conspicuous band of Mr 87 kDa and a strong band migrating at the front of the gel were most significantly labelled by 32p (Fig. 5B); four additional bands (Mr 29, 45, 61 and 76 kDa) were weakly labelled. The degree of phosphorylation of some bands increased during the incubation period (mainly in the Mr 61 and 76 kDa groups). Quantitative data could be furnished by radioactivity measurements of the individual bands excised from the gel (data not shown). The radioactivity was mainly associated with proteins migrating at the front of the gel and a group of Mr 80-95 kDa, and labelling increased as a function of incubation time. A similar change could be observed in the 40-45, 60-70 and 70-80 kDa groups of proteins.
DISCUSSION
et al.
and PIP2, an effect which was abolished upon diminishing the Mg 2÷ concentration 10-fold. This observation strongly suggests that MgE+-dependent kinases are operative in the oocyte. The 32p labelling of either PIP or PIP 2 in rat erythrocyte membrane could be privileged depending on the magnesium concentration in the incubation medium (Marche et al., 1982). In rat brain Mg 2+ is an essential cofactor for PIP kinase with optimal stimulation between 10 and 30 mM (Van Dongen et al., 1984). Because of the relationship between the activation of calcium-mobilizing receptors and inositol lipid breakdown (Abdel-Latif, 1989) it has been assumed that the synthesis of polyphosphoinositides occurs in the plasma membrane. In rat liver it has been shown that the enzyme that phosphorylates PI to PIP is present in Golgi membranes and lysosomes as well as in the plasma membrane (Cockcroft et al., 1985). Recently, PIP kinase activity in bovine brain was found in both the cytosolic and membrane fractions (Moritz et aL, 1990). The present study demonstrates that the oocyte plasma membrane possesses the enzymes for phosphorylation of PI and PIP although the possibility that enzymes are also located in other subcellular sites cannot be discarded. In addition, we found a significant activity of diacylglycerol kinase which initiates the resynthesis of phosphatidylinositol lipids and plays a major role in the control of the intracellular concentration of the second messenger, diacylglycerol. Although a translocation from soluble to membrane-bound kinase has been observed in rat brain (Besterman et al., 1986) and Amoeba (Jimenez et al., 1988), there is strong evidence in support of the membrane-bound nature of diacylglycerol kinase in Swiss 3T3 cells (MacDonald et al., 1988). In agreement with data reported for other biological systems (Stubbs et aL, 1988), the loss of radioactivity from phosphoinositides after an initially active labelling phase indicates that these lipids are subject to an active turnover (Fig. 1A). It is therefore likely that in addition to kinases, phosphomonoesterases and phosphodiesterases are also present in these membranes. Such a possibility implies that the amount of phosphorylated lipids formed may, in fact, be underestimated. In rat liver plasma membrane fractions an actual decline with time in the amount of
E
~o O.
E 20 "6 E I
The major conclusion of the present results is that full-grown Bufo arenarum oocytes blocked in the meiotic prophase possess the capacity to biosynthesize polyphosphoinositides and to phosphorylate membrane proteins. Incubation of plasma membrane-enriched fractions with 0'-32p)ATP in a medium containing 10 mM MgCI 2 promotes the labelling of PIP, phosphatidate
i
z5
time (rnin)
Fig. 4. 32p incorporation in total proteins of oocyte plasma membrane-enriched fractions. Membranes were incubated with 50pM (~,-32p)ATP (spec. act. 0.27mCi/#mol) in a 7.5 mM HEPES, pH 7.9, containing 10mM MgC12 and 62mM NaC1. Reaction was stopped with 20% trichloroacetic acid. Data are the average of two separate samples.
PPI synthesis by Bufo oocytes
A
589
B
-205K-
i~~i!i!~ii~i~
-116- 97-
- 67-
-45! ii! ¸ ?
i i!!~i~! ~i~
ili~ ~ii~ i~!i
C
2
5
I0
15
Time (min) Fig. 5. Coomassie Blue and autoradiographic patterns of (T-32p)ATP labelled plasma membrane from full-grown prophase-arrested oocytes. (A) Coomassie Blue-stained electropherogram, (B) autoradiogram of membranes incubated for different times with labelled precursor, (C) membrane control. The following molecular weight markers were run in parallel: myosin (205,000), fl-galactosidase (I 16,000), phosphorylase b (97,400), bovine serum albumin (66,500) and ovoalbumin (45,000).
the two labelled species has been reported (Cockcroft et al., 1985). This was concomitant with the nearly total disappearance of ATP from the reaction mixture after 10min (short incubation times were recommended in order to minimize the effect of ATPase activity). PIP content was shown to be higher than that of PIP2 in whole oocytes, in close agreement with data reported for Xenopus laevis oocytes arrested at first meiotic prophase (Le Peuch et al., 1985). The levels of both inositol lipids in our plasma membrane preparations were higher than in whole cells with a relative enrichment in PIP 2. We report a net phosphorylation of plasma membrane proteins registered as early as 2 min after incubation with (~-32p)ATP. Similarly, a burst of protein phosphorylation has been reported to occur shortly before germinal vesicle breakdown in starfish oocytes (Meijer et al., 1986; Sano, 1985), this being taken as clear evidence that an elevated level of phosphorylated proteins is required in order for the maturation-promoting factor to become active, and for maturation to occur (Varnold and Smith, 1990). The different time-course of the protein labelling
profile in our plasma preparations suggests that the proteins were probably phosphorylated by other kinases or that an autophosphorylation process occurred. Phosphorylating enzymes appear to be dependent on Mg 2+ and to require a low calcium concentration, since the Ca 2÷ content measured by atomic absorption spectroscopy in similar membrane preparations from Xenopus oocytes has been reported to be as low as 5 0 # M (Blondeau and Baulieu, 1985). The occurrence of protein kinase C and AMPc-dependent types of phosphorylation can be discarded, due to the experimental conditions in which lipid and protein phosphorylation were assayed. Differences in protein phosphorylation between this and previous work in the amphibian oocytes (Blondeau and Baulieu, 1985; Kostellow et al., 1987) can be ascribed to differences either in the amphibian oocyte membrane sources or to the experimental procedures followed. It remains to be elucidated whether the reported protein phosphorylation is related to the observed phosphorylation of inositol lipids and whether such a relationship continues to exist when oocyte maturation is induced by progesterone.
590
TELMA S. ALONSO et al.
Acknowledgements--We are grateful to Dr Norma Giusto and Dr Marta Aveldafio for their critical reading of the manuscript and helpful discussions REFERENCES
Abdel-Latif A. A. (1989) Calcium-mobilizing receptors, polyphosphoinositides, generation of second messengers and contraction in the mammalian iris smooth muscle: historical perspectives and current status. Life Sci. 45, 757-786. Abdel-Latif A. A. (1986) Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmac. Rev. 38, 227-272. Besterman J. M., Pollenz R. S., Booker E. L. and Cuatrecasas P. (1986) Diacylglycerol-induced translocation of diacylglycerol kinase: use of affinity-purified enzyme in a reconstitution system. Proc. natn. Acad. Sci. U.S.A. 83, 9378-9382. Blondeau J. P. and Baulieu E. E. (1985) Progesterone-inhibited phosphorylation of a unique M r 48,000 protein in the plasma membrane of Xenopus laevis oocytes. J. biol. Chem. 260, 361~3625. Bonner W. M. and Laskey R. A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-87. Cockcroft S., Taylor J. A. and Judah J. D. (1985) Subcellular localisation of inositol lipid kinases in rat liver. Biochim. biophys. Acta 845, 163-170. Dascal N., Yekuel R. and Oron Y. (1984) Acetylcholine promotes progesterone-induced maturation of Xenopus laevis oocytes. J. exp. Zool. 230, 131-135. Folch J., Lees M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. Jimenez B., Van Lookeren Campagne M. M., Prestana A. and Fernandez-Renart M. (1988) Regulation of diacylglycerol kinase in the transition from quiescence to proliferation in Dictyostelium discoideum. Biochem. biophys. Res. Commun. 150, 118-125. Kostellow A. B., Chien E. J. and Morrill G. A. (1987) Calcium-dependent phosphorylation of the amphibian oocyte plasma membrane: an early event in initiating the meiotic divisions. Biochem. biophys. Res. Commun. 147, 863-869. Lacal J. C., De La Pena P., Moscat J., Garcia-Barreno P., Anderson P. S. and Aaronson S. A. (1987) Rapid stimulation of diacylglycerol production in Xenopus oocytes by microinjection of H-ras p 21. Science 238, 533-536. Laemmli U. K. (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. Le Peuch C. J., Picard A. and Doree M. (1985) Parthenogenetic activation decreases the polyphosphoinositide content of frog eggs. FEBS Lett. 187, 61-64. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275.
MacDonald M. L., Mack K. F., Williams B. W., King W. C. and Glomset J. A. (1988) A membrane-bound diacylglycerol kinase that selectively phosphorylates arachidonoyl--diacylglycerol. J. biol. Chem. 263, 1584-1592. Marche P., Koutouzov S. and Meyer P. (1982) Metabolism of phosphoinositides in rat erythrocyte membrane. A reappraisal of the effect of magnesium on the 32p incorporation into polyphosphoinositides. Biochim. biophys. Acta 710, 332-340. Meijer L., Pondaven P., Tung H. Y. L., Cohen P. and Wallace R. W. (1986) Protein phosphorylation and oocyte maturation. II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase. Expl Cell Res. 163, 489 499. Michell R. H. and Hawthorne J. N. (1965) The site of diphosphoinositide synthesis in rat liver. Biochem. biophys. Res. Commun. 21, 333-338. Moritz A., De Graan P. N. E., Ekhart P. F., Gispen W. H. and Wirtz K. W. A. (1990) Purification of a phosphatidylinositol 4-phosphate kinase from bovine brain membranes. J. Neurochem. 54, 351-354. Oron Y., Dascal N., Nadler E. and Lupu M. (1985) Inositol 1,4,5-trisphosphate mimics muscarinic response in Xenopus oocytes. Nature, Lond. 313, 141-143. Rodriguez de Turco E. B. and Aveldafio de Caldironi M. I. (1980) Counting efficiency in the radioassay of 3H and 14C labeled lipids adsorbed on silica gel by liquid scintillation. Analyt. Biochem. 104, 62-69. Rouser G., Fleischer S. and Yamamoto A. (1970) A twodimensional thin-layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5, 494 496. Sano K. (1985) Calcium and cyclic AMP-independent labile protein kinase appearing during starfish oocyte maturation: its extraction and partial characterization. Devl. Growth Diff. 27, 263-275. Shaik N. A. and Palmer F. B. S. (1977) Phosphoinositide kinases in chick brain and sciatic nerve, a developmental study. J. Neurochem. 28, 395-402. Stubbs E. B. Jr, Keheller J. A. and Sun G. Y. (1988) Phosphatidylinositol kinase, phosphatidylinositol-4phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions. Biochem. biophys. Acta 958, 247-254. Uma S. and Ramakrishnan C. V. (1983) Studies on polyphosphoinositides in developing rat brain. J. Neurochem. 40, 914-916. Valdez Toledo C. L. and Pisano A. (1980) Studies of oogenesis in Bufo arenarum. Reproduccion 4, 15-33. Van Dongen C. J., Zwiers H. and Gispen W. H. (1984) Purification and partial characterization of the phosphatidylinositol 4-phosphate kinase from rat brain. Biochem. J. 223, 197-203. Varnold R. L. and Smith L. D. (1990) Protein kinase C and progesterone-induced maturation in Xenopus oocytes. Development 109, 597-604.
0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
POLYPHOSPHOINOSITIDE SYNTHESIS AND PROTEIN PHOSPHORYLATION IN THE PLASMA MEMBRANE FROM FULL-GROWN BUFO A R E N A R U M OOCYTES TELMA S. ALONSO*, IDA C. BONINI DE ROMANELLI,ANA M. ROCCAMODE FERNANDEZ and FRANCISCOJ. BARRANTES Instituto de Investigaciones Bioquimicas, Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET) and Universidad Nacional del Sur, C. C. 857, 8000 Bahia Blanca, Argentina (Tel.: 09 131342; Fax: 054 91 27876)
(Received 4 November 1991) Abstract--1. Polyphosphoinositide content and phosphorylation of lipids and proteins were analyzed in oocytes of the toad Bufo arenarum Hensel. 2. Plasma membrane-enriched fractions obtained from full-grown, prophase-arrested oocytes incorporated 32p into both phospholipids and proteins after incubation with [?-32p]ATP in an Mg2+-containing medium. Phosphatidylinositol 4-phosphate (PIP), phosphatidate (PA) and phosphatidylinositol-4,5-bisphosphate (PIPa) were the only labelled lipids. The 32p incorporation depended on incubation time, the amount of protein, and the ATP concentration. 3. Autoradiography of polyacrylamide gel electropherograms and scintillation counting showed that the radioactivity was mainly associated with a group of membrane proteins having an M r of 87,000. 4. This paper provides evidence for the capacity of prophase-arrested oocytes from Bufo arenarum to synthesize polyphosphoinositides and to phosphorylate distinct membrane proteins.
INTRODUCTION Although polyphosphoinositides have been found to be only minor constituents of the animal tissues examined to date, increased attention is being given to their metabolism and function in a variety of membranes (Abdel-Latif, 1989). There is general agreement now that activation of some receptors leads to the phosphodiesteratic breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2), through the action of phospholipase C, into myo-inositol 1,4,5triphosphate and 1,2-diacylglycerol, which may serve as second messengers in a number of calciumdependent functions (Abdel-Latif, 1986). In amphibian oocytes it has been suggested that polyphosphoinositides play a possible role in the maturation process. Thus, the oocyte maturation induced by progesterone is promoted by cholinergic muscarinic stimulation (Dascal et al., 1984) which in Xenopus laevis has been shown to induce the hydrolysis of PIP 2 (Oron et al., 1985). Ras-injected oocytes have shown changes in inositol lipid metabolites in response to induction of maturation (Lacal et al., 1987). D a t a on inositol lipid content and metabolism in full-grown oocytes capable of undergoing maturation are scarce. This paper presents evidence for the presence of an active biosynthesis of polyphosphoinositides in a plasma membrane-enriched fraction from full-grown, prophase-arrested Bufo arenarum
oocytes. Phosphorylation of proteins is also demonstrated.
distinct
membrane
MATERIALS AND METHODS
Preparation of full-grown oocytes The ovarian tissue was removed from adult Bufo arenarum Hensel female toads, placed in a Ca2+-free Barth's medium, rinsed, and cut into small pieces. Full-grown oocytes (1.7-1.8 mm in diameter) in stage V were selected under a stereo-microscope following the criteria of Valdez Toledo and Pisano (1980). They were treated with 0.25% (w/v) collagenase (Sigma type I) in a Ca2+-free Barth's medium with agitation for 1.5 hr at 23°C. After this treatment, oocytes free from follicular cells were rinsed several times with Barth's medium, now containing Ca 2+ (1 mM). Isolation of the plasma membrane -enriched fractions The procedure described by Blondeau and Baulieu (1985) was followed with some additional steps. Oocytes and eggs were rinsed three times and homogenized in 10 mM HEPES buffer, pH 7.9, containing 83 mM NaCI and 1 mM MgCI2. Homogenates were centrifuged at 1000g for 10min. The supernatants were centrifuged at 10,000g for 20 min. The pellets containing the plasma membrane were rehomogenized in the same buffer and centrifuged at 1000g for 10min. The supernatants were centrifuged at 10,000g for another 20 rain. The resulting pellets were resuspended in the phosphorylation buffer and used immediately. The activity of 5'-nucleotidase was used as an enzyme marker for membrane enrichment, following the procedure of Michell and Hawthorne (1965). Phosphorylation of plasma membrane-enriched fractions The standard medium used for lipid phosphorylation was 7.5mM HEPES, pH 6.8 10mM MgCI2 62mM NaC1. Membrane samples (about 0.3 mg of protein) were preincubated in this medium for 5 min at 30°C after 2rain of
*To whom all correspondence should be addressed. Abbreviations--PA, phosphatidic acid; PI, phosphatidylinositol; PC, phosphatidylcholine; PE phosphatidylethanolamine; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5 bisphosphate. 585
586
TELMAS. ALONSOet al.
sonication. The phosphorylation assay was initiated by the addition of 50 ,aM (7-32P)ATP (spec. act. 0,67 mCi//~mol). Incubation was carried out in a final volume of 250 #1 and was ended by addition of 2.25 ml ice-cold distilled water followed by 2.50 ml ice-cold 20% trichloroacetic acid. The precipitated material was centrifuged in a benchtop centrifuge. The supernatant was removed and the pellet extracted with chloroform-methanol-concentrated HCI (400 : 200 : 3,
with H202 at 70°C overnight, and counting their radioactivity in a Beckman 8000 liquid scintillation counter. Protein concentration was measured by the method of Lowry et al. (1951). RESULTS
Polyphosphoinositide content in full-grown prophasearrested oocytes and in plasma membrane-enriched Protein phosphorylation was examined under similar fractions isolated therefrom experimental conditions except that the pH of the medium The analysis of glycerophospholipid content in was 7.9 and the (?-32p)ATP specific activity was 0.27 mCi/pmol. Membrane samples (about 70 #g protein) cell-free homogenates from full-grown oocytes of were preincubated for I min after 2 rain of sonication. For Bufo arenarum showed that phosphatidylcholine the determination of total protein labelling, incubation was (PC) and phosphatidylethanolamine (PE) comprised stopped with 20% trichloroacetic acid. After successive nearly 82% of the total phospholipids, followed by NaOH extractions and trichloroacetic acid precipitations, phosphatidylinositol (PI), sphingomyelin and phosradioactivity was measured in the protein-enriched pellet by phatidylserine (Table 1). Phosphatidic acid, a key liquid scintillation counting. Samples subjected to sodium dodecyl sulfate 10% acrylamide gel electrophoresis were intermediate in the biosynthetic lipid pathways, repprocessed under the same conditions, but the reaction was resented only 0,15% of the total. The polyphosphostopped by addition of 30#1 of Laemmli solubilization inositides were also minor components of whole buffer (Laemmli, 1970). Differences between duplicate full-grown oocytes (together they comprised 0.1% of experiments were lower than 5%. the total). A subcellular fraction enriched in plasma memLipid extraction, isolation and analysis brane was prepared in order to examine the content Lipid extracts from whole oocytes and plasma membrane- and metabolism of polyphosphoinositides and proenriched fractions were prepared according to the method of tein phosphorylation. Since endoplasmic reticulum is Folch et al. (1957). In order to extract polyphosphoinosiscarce in oocytes and eggs, extensive purification was tides the procedure of Uma and Ramakrishnan (1983) was not considered necessary for the present purposes. employed. PIP and PIP 2 were separated by thin-layer chromatog- The subcellular fractionation was performed as deraphy on 1% potassium oxalate-impregnated silica gel H scribed by Blondeau and Baulieu (1985) with some plates, developed as described by Shaik and Palmer (1977). additional steps that were important in this case to Other phospholipids were isolated by two-dimensional thin- eliminate pigment granules and yolk platelets. The layer chromatography (Rouser et al., 1970). Lipids were presence of diphosphatidylglycerol (cardiolipin), a located by exposure of the plates to iodine vapors. Pure lipid component typically associated with mitochonsubstances as standards were used. The silica was scraped dria, could indicate a slight contamination with these off into vials containing 0.4 ml water and 10 ml 0.4% Omnifluor in toluene-Triton X-100 (800:200, v/v) was organelles. The specific activity of a marker enzyme added (Rodriguez de Turco and Aveldafio de Caldironi, for plasma membranes, 5'-nucleotidase, was en1980). Radioactivity was quantitated by liquid scintillation hanced 12-fold in the plasma membrane-enriched counting, applying the corresponding calculation for 32p fraction with respect to the crude homogenate. The plasma membrane fractions had a characterdecay. Lipid phosphorus was determined according to Rouser et al. (1970). istic phospholipid profile (Table 1). The major components were PC and PE but there was Protein identification .and quantification more phosphatidate, phosphatidylserine and sphinMembrane samples were subjected to polyacrylamide gel gomyelin in this fraction than in the whole oocytes. electrophoresis. Gels were stained with 0.25% Coomassie The content of PIP and PIP2 was also higher (18- and Blue as described by Laemmli (1970). Autoradiography was carried out using 3 M Medical 40-fold respectively) in membranes than in whole X-ray film type R and a Gevaert intensifying screen at oocytes. This, together with the higher activity of the -70°C (Bonner and Laskey, 1974). The 32p incorporated plasma membrane marker enzyme (about 12-fold into individual bands was quantitated by excising them higher 5'-nucleotidase activity) is indicative of the from wet stained gels, treating the polyacrylamide slices enrichment achieved in the subcellular fractionation.
v/v).
Table 1. Phospholipid content in cell-free homogenates and plasma membrane-enriched fractions of amphibian full-grown prophase-arrested oocytes Cell-free homogenate (nmol/mg protein) (%) Phosphatidic acid Phosphatidylserine Phosphatidylinositol Sphingomyelin Phosphatidylcholine Phosphatidylethanolamine Diphosphatidylglycerol Phosphatidylinositol 4-phosphate Phosphatidylinositol 4,5-bisphosphate
0.14 -- 0,002 1.46 -- 0.25 8.58 + 0,70 6.03 ,, 1.06 52.91 ,, 6,67 26.42 ,, 1,24 0.65 + 0,12 0.09 -- 0,01 0.01 + 0.003
0.15 1.52 8.91 6,26 54,91 27,44 0,68 0,09 0,01
Plasma membrane (nmol/mg protein) 1.80 -- 0.50 23.7t + 4.02 41.93 ,, 5.82 45.72 ,, 2.33 194.80 ,, 30.54 115.31 ,, 20.30 24.10 _+ 6.23 1.60 ,, 0.61 0.42 _+ 0.10
(%) 0.40 5.28 9.33 10.17 43.35 25.66 5.36 0.36 0.09
Plasma membrane-enriched fractions were obtained as described in Materials and Methods. Phospholipids were isolated by bidimensional thin-layer chromatography (Rouser et al., 1970) and polyphosphoinositides by monodimensional thin-layer chromatography (Shaik and Palmer, 1977). Phospholipidic phosphorus was measured according to Rouser et aL (1970). Data are expressed as mean -- SD from six independent samples.
PPI synthesis by Bufo oocytes
587
A
e-
6O0
1200
"O
0. Im
E
A00
800
O
E 200
"6 4oo E 10
i
i
20
30
0.2
0.~
0.G
protein Imgl
time (rain)
Fig. 1. PI kinase and PIP kinase activities in oocyte plasma membrane. (A) time-course of PI kinase (A)-, and PIP kinase (0)- catalyzed reactions. (B) effect of protein concentration on PI kinase (A) and PIP kinase ( 0 ) reactions after l0 min incubation. Plasma membrane-enriched fractions obtained as described in Materials and Methods were incubated with 50/~M 0,-32p)ATP (spec. act. 0.67 mCi/pmol), I0 mM MgCI2, 62 mM NaC1 and 7.5 mM HEPES, pH 7.9, in a final volume of 250/~1 at 30°C. Membranes were preincubated in this medium for 5 min at 30°C after 2 rnin of sonication. Incubation was stopped by addition of 2.25 ml ice-cold distilled water followed by 2.50 ml ice-cold 20% trichloroacetic acid. Lipids were extracted from the pellets with acidified solvents (Uma and Ramakrishnan, 1983). Phosphoinositides were isolated by thin-layer chromatography (Shaik and Palmer, 1977). The values of incorporation represent the average of two separate samples.
Synthesis of acidic glycerophospholipids in plasma membrane -enriched fractions
Concentration-dependent effects of A TP on polyphosphoinositide and phosphatidate synthesis
Incubation of oocyte plasma membrane-enriched fractions with (?-32P)ATP in the presence of Mg 2+ promoted the labelling of PIP, PA and PIP2, in that order (32p incorporation could not be detected when the magnesium concentration was reduced to about one-tenth). The order of distribution of radioactivity appears to be logical, since the concentration of the substrate for PI kinase should not be rate-limiting, given that PI was present in sufficient amounts in the membrane preparation. This may not have been the case for PIP kinase since the concentration of PIP was low, and the PIP kinase depends on the first kinase for the provision of substrate. As far as the labelling of PA is concerned, since no exogenous substrate was added to the incubation medium, diacylglycerol kinase must necessarily have phosphorylated the endogenous diacylglycerol. The time-dependence of the labelling by (?32P)ATP of PIP and of PIP2 in plasma membranes is shown in Fig. 1A. Both were labelled as early as 2 min after the addition of the precursor. The rate of formation of PIP increased during the first 10 min and subsequently reached a maximum of about 130 fmol min- 1 m g - l protein, slowly declining thereafter. The amount of PIP 2 labelled was lower than that of PIP, reaching a maximum of about 20 fmol min-~mg -~ protein after 5rain. The labelling of polyphosphoinositides was also dependent on the amount of membrane (Fig. 1B). The formation of PIP was higher than that of PIP 2 in the range between 100 and 600/~g membrane protein. The time-course of phosphatidate labelling after incubation of oocyte plasma membrane with (~32p)ATP is depicted in Fig. 2. The incorporation increased linearly up to about 10 rain of incubation, reaching a maximum of about 45 fmol min-~mg -] protein. Protein concentration also affected the incorporation of 32p into PA (Fig. 2, inset).
Since the enzymatic conversion of PI to PIP and the subsequent phosphorylation to produce PIP2 require ATP as substrate, the effect of the concentration of this nucleotide on PIP and PIP2 labelling was analyzed (Fig. 3). Increasing concentrations of ATP in the range between 25 and 300 p M stimulated the activities of phosphatidylinositol kinase and PIP kinase 9- and 6-fold respectively. Diacylglycero] phosphorylation was stimulated 11-fold in the same ATP concentration range.
Protein phosphorylation of oocyte plasma membraneenriched fractions Plasma membrane fractions obtained from fullgrown prophase-arrested oocytes were incubated with (~J2P)ATP to analyze total protein labelling. The time-course of such phosphorylation is depicted
[] ,
D
¢- (00
'~, O.. 300
'U 200
.& 200
• J 100
E
L: O
E
100 0.2
O.&
0.6
protei n I mgl
w-
i 10
i
i 20
,
i
i 30
t i m e (mini
Fig. 2. Biosynthesis of phosphatidic acid in oocyte plasma membrane-enriched fractions. Experimental conditions as in Fig. 1. The effect of protein concentration is depicted in the inset. Values represent the average of two separate samples.
588
TELMA S. ALONSO
25 E '~ 20 E ,?
15 x
:g ~
10
,45
100
200
300
ATPluM)
Fig. 3. Effect of ATP concentration on PI kinase (A), PIP kinase ( 0 ) and diacylglyceride kinase (D) activities. Membranes were incubated for 10rain as described in Fig. I. Values represent the average of two separate samples. in Fig. 4. A net 32p incorporation (as early as 2 min after the addition of the labelled nucleotide) was detected, a plateau being reached after 10min incubation. Polyacrylamide (10%) gel electrophoresis of the membrane samples revealed at least 40 protein bands (Fig. 5A). The major ones corresponded to groups of proteins with an Mr of about 48, 70 and 97 kDa. The time-course of 32p incorporation into individual polypeptide chains was analyzed by autoradiography and by radioactive counting of excised individual bands. Autoradiograms exposed for 96 hr enabled the detection of minor phosphorylated bands. A conspicuous band of Mr 87 kDa and a strong band migrating at the front of the gel were most significantly labelled by 32p (Fig. 5B); four additional bands (Mr 29, 45, 61 and 76 kDa) were weakly labelled. The degree of phosphorylation of some bands increased during the incubation period (mainly in the Mr 61 and 76 kDa groups). Quantitative data could be furnished by radioactivity measurements of the individual bands excised from the gel (data not shown). The radioactivity was mainly associated with proteins migrating at the front of the gel and a group of Mr 80-95 kDa, and labelling increased as a function of incubation time. A similar change could be observed in the 40-45, 60-70 and 70-80 kDa groups of proteins.
DISCUSSION
et al.
and PIP2, an effect which was abolished upon diminishing the Mg 2÷ concentration 10-fold. This observation strongly suggests that MgE+-dependent kinases are operative in the oocyte. The 32p labelling of either PIP or PIP 2 in rat erythrocyte membrane could be privileged depending on the magnesium concentration in the incubation medium (Marche et al., 1982). In rat brain Mg 2+ is an essential cofactor for PIP kinase with optimal stimulation between 10 and 30 mM (Van Dongen et al., 1984). Because of the relationship between the activation of calcium-mobilizing receptors and inositol lipid breakdown (Abdel-Latif, 1989) it has been assumed that the synthesis of polyphosphoinositides occurs in the plasma membrane. In rat liver it has been shown that the enzyme that phosphorylates PI to PIP is present in Golgi membranes and lysosomes as well as in the plasma membrane (Cockcroft et al., 1985). Recently, PIP kinase activity in bovine brain was found in both the cytosolic and membrane fractions (Moritz et aL, 1990). The present study demonstrates that the oocyte plasma membrane possesses the enzymes for phosphorylation of PI and PIP although the possibility that enzymes are also located in other subcellular sites cannot be discarded. In addition, we found a significant activity of diacylglycerol kinase which initiates the resynthesis of phosphatidylinositol lipids and plays a major role in the control of the intracellular concentration of the second messenger, diacylglycerol. Although a translocation from soluble to membrane-bound kinase has been observed in rat brain (Besterman et al., 1986) and Amoeba (Jimenez et al., 1988), there is strong evidence in support of the membrane-bound nature of diacylglycerol kinase in Swiss 3T3 cells (MacDonald et al., 1988). In agreement with data reported for other biological systems (Stubbs et aL, 1988), the loss of radioactivity from phosphoinositides after an initially active labelling phase indicates that these lipids are subject to an active turnover (Fig. 1A). It is therefore likely that in addition to kinases, phosphomonoesterases and phosphodiesterases are also present in these membranes. Such a possibility implies that the amount of phosphorylated lipids formed may, in fact, be underestimated. In rat liver plasma membrane fractions an actual decline with time in the amount of
E
~o O.
E 20 "6 E I
The major conclusion of the present results is that full-grown Bufo arenarum oocytes blocked in the meiotic prophase possess the capacity to biosynthesize polyphosphoinositides and to phosphorylate membrane proteins. Incubation of plasma membrane-enriched fractions with 0'-32p)ATP in a medium containing 10 mM MgCI 2 promotes the labelling of PIP, phosphatidate
i
z5
time (rnin)
Fig. 4. 32p incorporation in total proteins of oocyte plasma membrane-enriched fractions. Membranes were incubated with 50pM (~,-32p)ATP (spec. act. 0.27mCi/#mol) in a 7.5 mM HEPES, pH 7.9, containing 10mM MgC12 and 62mM NaC1. Reaction was stopped with 20% trichloroacetic acid. Data are the average of two separate samples.
PPI synthesis by Bufo oocytes
A
589
B
-205K-
i~~i!i!~ii~i~
-116- 97-
- 67-
-45! ii! ¸ ?
i i!!~i~! ~i~
ili~ ~ii~ i~!i
C
2
5
I0
15
Time (min) Fig. 5. Coomassie Blue and autoradiographic patterns of (T-32p)ATP labelled plasma membrane from full-grown prophase-arrested oocytes. (A) Coomassie Blue-stained electropherogram, (B) autoradiogram of membranes incubated for different times with labelled precursor, (C) membrane control. The following molecular weight markers were run in parallel: myosin (205,000), fl-galactosidase (I 16,000), phosphorylase b (97,400), bovine serum albumin (66,500) and ovoalbumin (45,000).
the two labelled species has been reported (Cockcroft et al., 1985). This was concomitant with the nearly total disappearance of ATP from the reaction mixture after 10min (short incubation times were recommended in order to minimize the effect of ATPase activity). PIP content was shown to be higher than that of PIP2 in whole oocytes, in close agreement with data reported for Xenopus laevis oocytes arrested at first meiotic prophase (Le Peuch et al., 1985). The levels of both inositol lipids in our plasma membrane preparations were higher than in whole cells with a relative enrichment in PIP 2. We report a net phosphorylation of plasma membrane proteins registered as early as 2 min after incubation with (~-32p)ATP. Similarly, a burst of protein phosphorylation has been reported to occur shortly before germinal vesicle breakdown in starfish oocytes (Meijer et al., 1986; Sano, 1985), this being taken as clear evidence that an elevated level of phosphorylated proteins is required in order for the maturation-promoting factor to become active, and for maturation to occur (Varnold and Smith, 1990). The different time-course of the protein labelling
profile in our plasma preparations suggests that the proteins were probably phosphorylated by other kinases or that an autophosphorylation process occurred. Phosphorylating enzymes appear to be dependent on Mg 2+ and to require a low calcium concentration, since the Ca 2÷ content measured by atomic absorption spectroscopy in similar membrane preparations from Xenopus oocytes has been reported to be as low as 5 0 # M (Blondeau and Baulieu, 1985). The occurrence of protein kinase C and AMPc-dependent types of phosphorylation can be discarded, due to the experimental conditions in which lipid and protein phosphorylation were assayed. Differences in protein phosphorylation between this and previous work in the amphibian oocytes (Blondeau and Baulieu, 1985; Kostellow et al., 1987) can be ascribed to differences either in the amphibian oocyte membrane sources or to the experimental procedures followed. It remains to be elucidated whether the reported protein phosphorylation is related to the observed phosphorylation of inositol lipids and whether such a relationship continues to exist when oocyte maturation is induced by progesterone.
590
TELMA S. ALONSO et al.
Acknowledgements--We are grateful to Dr Norma Giusto and Dr Marta Aveldafio for their critical reading of the manuscript and helpful discussions REFERENCES
Abdel-Latif A. A. (1989) Calcium-mobilizing receptors, polyphosphoinositides, generation of second messengers and contraction in the mammalian iris smooth muscle: historical perspectives and current status. Life Sci. 45, 757-786. Abdel-Latif A. A. (1986) Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmac. Rev. 38, 227-272. Besterman J. M., Pollenz R. S., Booker E. L. and Cuatrecasas P. (1986) Diacylglycerol-induced translocation of diacylglycerol kinase: use of affinity-purified enzyme in a reconstitution system. Proc. natn. Acad. Sci. U.S.A. 83, 9378-9382. Blondeau J. P. and Baulieu E. E. (1985) Progesterone-inhibited phosphorylation of a unique M r 48,000 protein in the plasma membrane of Xenopus laevis oocytes. J. biol. Chem. 260, 361~3625. Bonner W. M. and Laskey R. A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-87. Cockcroft S., Taylor J. A. and Judah J. D. (1985) Subcellular localisation of inositol lipid kinases in rat liver. Biochim. biophys. Acta 845, 163-170. Dascal N., Yekuel R. and Oron Y. (1984) Acetylcholine promotes progesterone-induced maturation of Xenopus laevis oocytes. J. exp. Zool. 230, 131-135. Folch J., Lees M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. Jimenez B., Van Lookeren Campagne M. M., Prestana A. and Fernandez-Renart M. (1988) Regulation of diacylglycerol kinase in the transition from quiescence to proliferation in Dictyostelium discoideum. Biochem. biophys. Res. Commun. 150, 118-125. Kostellow A. B., Chien E. J. and Morrill G. A. (1987) Calcium-dependent phosphorylation of the amphibian oocyte plasma membrane: an early event in initiating the meiotic divisions. Biochem. biophys. Res. Commun. 147, 863-869. Lacal J. C., De La Pena P., Moscat J., Garcia-Barreno P., Anderson P. S. and Aaronson S. A. (1987) Rapid stimulation of diacylglycerol production in Xenopus oocytes by microinjection of H-ras p 21. Science 238, 533-536. Laemmli U. K. (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. Le Peuch C. J., Picard A. and Doree M. (1985) Parthenogenetic activation decreases the polyphosphoinositide content of frog eggs. FEBS Lett. 187, 61-64. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275.
MacDonald M. L., Mack K. F., Williams B. W., King W. C. and Glomset J. A. (1988) A membrane-bound diacylglycerol kinase that selectively phosphorylates arachidonoyl--diacylglycerol. J. biol. Chem. 263, 1584-1592. Marche P., Koutouzov S. and Meyer P. (1982) Metabolism of phosphoinositides in rat erythrocyte membrane. A reappraisal of the effect of magnesium on the 32p incorporation into polyphosphoinositides. Biochim. biophys. Acta 710, 332-340. Meijer L., Pondaven P., Tung H. Y. L., Cohen P. and Wallace R. W. (1986) Protein phosphorylation and oocyte maturation. II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase. Expl Cell Res. 163, 489 499. Michell R. H. and Hawthorne J. N. (1965) The site of diphosphoinositide synthesis in rat liver. Biochem. biophys. Res. Commun. 21, 333-338. Moritz A., De Graan P. N. E., Ekhart P. F., Gispen W. H. and Wirtz K. W. A. (1990) Purification of a phosphatidylinositol 4-phosphate kinase from bovine brain membranes. J. Neurochem. 54, 351-354. Oron Y., Dascal N., Nadler E. and Lupu M. (1985) Inositol 1,4,5-trisphosphate mimics muscarinic response in Xenopus oocytes. Nature, Lond. 313, 141-143. Rodriguez de Turco E. B. and Aveldafio de Caldironi M. I. (1980) Counting efficiency in the radioassay of 3H and 14C labeled lipids adsorbed on silica gel by liquid scintillation. Analyt. Biochem. 104, 62-69. Rouser G., Fleischer S. and Yamamoto A. (1970) A twodimensional thin-layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5, 494 496. Sano K. (1985) Calcium and cyclic AMP-independent labile protein kinase appearing during starfish oocyte maturation: its extraction and partial characterization. Devl. Growth Diff. 27, 263-275. Shaik N. A. and Palmer F. B. S. (1977) Phosphoinositide kinases in chick brain and sciatic nerve, a developmental study. J. Neurochem. 28, 395-402. Stubbs E. B. Jr, Keheller J. A. and Sun G. Y. (1988) Phosphatidylinositol kinase, phosphatidylinositol-4phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions. Biochem. biophys. Acta 958, 247-254. Uma S. and Ramakrishnan C. V. (1983) Studies on polyphosphoinositides in developing rat brain. J. Neurochem. 40, 914-916. Valdez Toledo C. L. and Pisano A. (1980) Studies of oogenesis in Bufo arenarum. Reproduccion 4, 15-33. Van Dongen C. J., Zwiers H. and Gispen W. H. (1984) Purification and partial characterization of the phosphatidylinositol 4-phosphate kinase from rat brain. Biochem. J. 223, 197-203. Varnold R. L. and Smith L. D. (1990) Protein kinase C and progesterone-induced maturation in Xenopus oocytes. Development 109, 597-604.