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Endocrinology Vol. 141, No. 6 2145-2152
Copyright © 2000 by The Endocrine Society

ARTICLES

Nongenomic Action of Progesterone: Activation of XenopusOocyte Phospholipase C through a Plasma Membrane-Associated Tyrosine Kinase1

Thomas Morrison, Leslie Waggoner, Laura Whitworth-Langley and Bradley J. Stith

Biology Department, University of Colorado, Denver, Colorado 80217

Address all correspondence and requests for reprints to: Dr. Bradley J. Stith, University of Colorado, Biology 171, 1224 Fifth Street, Denver, Colorado 80204-3364. E-mail: bstith{at}carbon.cudenver.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using a plasma membrane-cortex preparation (wherein the nucleus and >90% of the total cell protein are removed), progesterone stimulated tyrosine kinase activity that stimulated phospholipase C. Although it has been known for over 20 yr that progesterone acts at the plasma membrane of Xenopus oocytes to induce oocyte maturation, this is the first report that progesterone stimulates this tyrosine kinase activity that is associated with the oocyte plasma membrane and cortex. A tyrosine kinase inhibitor (tyrphostin B46) inhibited steroid stimulation of tyrosine kinase and phospholipase C (PLC) activities, but did not block lipase C stimulation by G protein activators. A fusion protein that contains tandem N- and C-terminal SH2 domains of PLC{gamma} also blocked progesterone stimulation of PLC (a fusion protein with the SH2 domain from Shc was ineffective). Lowering the Ca2+ concentration in the medium inhibited progesterone, but not guanosine 5'-O-(3-thiotriphosphate), stimulation of PLC, and the effects of progesterone and a G protein agonist were additive. However, neither progesterone nor insulin increased phosphotyrosine on PLC{gamma}. To evaluate another tyrosine kinase path involving phosphatidylinositol 3-kinase, we added wortmannin to our membrane preparation, but wortmannin did not inhibit progesterone’s ability to activate PLC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE PAST 5 yr, increasing attention has been drawn to nongenomic action of steroids in reproductive tissues (1). The Xenopus oocyte is one of the first systems where steroid action at the plasma membrane (independent of transcription changes) has been described (2, 3, 4). Upon addition of progesterone to Xenopus oocytes, we and others have shown that the steroid induces rapid increases in sn-1,2-diacylglycerol (5), the mitogen-activated protein kinase pathway and phosphorylation of ribosomal protein S6 (6, 7), and intracellular pH (6) and a decrease in adenylate cyclase activity and cAMP (2, 8). Progesterone also induces progression of meiotic cell division in Xenopus oocytes (2).

We reported that progesterone stimulates Xenopus oocyte phospholipase C (PLC) to break down phosphatidylinositol 4,5-bisphosphate (PIP2) to increase inositol 1,4,5-trisphosphate (IP3) mass (9). Progesterone (or insulin) increased IP3 from about 29 to 47 fmol/oocyte (or from an estimated concentration of ~60 to ~100 nM). Although detection of a localized intracellular calcium ([Ca2+]i) increase has been controversial (10), an IP3 increase of this magnitude may release [Ca2+]i based on the efficacy of similar IP3 concentrations to induce [Ca2+]i-dependent membrane depolarization of Xenopus oocytes (11) and to release [Ca2+]i from microsomal fractions from Xenopus oocytes (12). The production of IP3 and a localized elevation of [Ca2+]i may be important in the ability of progesterone to induce meiotic cell division in Xenopus oocytes (12, 13, 14, 15). Another steroid, 17ß-estradiol, appears to release intracellular calcium in human oocytes and increases the success rate of subsequent fertilization (16).

In this study we examine the nongenomic mechanism of the stimulation of PLC by progesterone. The ß-isoform of PLC is regulated by G proteins, whereas PLC{gamma} is regulated by tyrosine phosphorylation, phosphatidylinositol 3-kinase (PI 3-kinase), or other signaling paths (17). PLC{delta} regulation is not well described (18, 19).

One path for PLC{gamma} activation involves a hormone activating a tyrosine kinase that would autophosphorylate tyrosine residues and create a binding site for the two SH2 domains of PLC{gamma}. Once bound to the phosphotyrosines of the tyrosine kinase, PLC{gamma} would be phosphorylated on at least two tyrosines and activated. Thus, a fusion protein containing at least one SH2 domain from PLC{gamma} would competitively inhibit PLC{gamma} activation. An SH2 fusion protein inhibited the [Ca2+]i increase at fertilization in starfish (20).

To study the nongenomic action of progesterone, we took advantage of one of the unique properties of the Xenopus oocyte, the ability to make a plasma membrane-cortex or cortex (PMC) preparation. Similar in appearance to a red blood cell ghost, preparation of the PMC involves removal of the nucleus and 99% of cell protein (2). PMC preparations are on the order of 10 µm thick and include cytoskeletal fibers and vesicles (21, 22, 23). This preparation maintains responsiveness to hormones (i.e. functional hormone receptors) and receptor coupling to downstream agents. For example, addition of progesterone to the PMC preparation inhibits Gs and adenylate cyclase (2). Another advantage of the use of the PMC is that we have removed any influence of progesterone-induced calcium influx across the plasma membrane. Calcium influx can activate PLC, and this would complicate experiments examining progesterone activation of PLC by other cell-signaling pathways. With PMC isolation, there is a lowering of background signals from the cytoplasm, and we can easily add substrate or inhibitors to examine early signaling events located at the plasma membrane.

Another strength of these studies is that we record IP3 mass as a measure of PLC activity. Other methods for recording PLC activity have often involved measurement of downstream events such as the release of calcium (calcium levels are dependent upon events other than PLC activity).

Using this PMC preparation, we report that progesterone stimulated both tyrosine kinase and PLC activities. Tyrphostin B46 (a tyrosine kinase inhibitor) and a glutathione-S-transferase (GST) fusion protein containing the two tandem SH2 domains of PLC{gamma} blocked progesterone stimulation of PLC. Experiments with insulin, a hormone that is known to act through tyrosine kinase activity, produced results similar to those with progesterone. There is evidence that progesterone does not act through a G protein to activate PLC; stimulation of PLC by a G protein agonist and that by progesterone were additive, and buffering calcium concentrations to low levels inhibited hormone stimulation of PLC, but not stimulation by G protein agonists.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The isolated plasma membrane and cortex preparation
To study cell-signaling events at or near the plasma membrane, PMC preparations were made from Xenopus oocytes (Xenopus One, Ann Arbor, MI; or Xenopus Express, Homosassa, FL) in the manner described by Sadler and Maller (2). Briefly, oocytes were hand-isolated and kept in O-R2 (83 mM NaCl, 0.5 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.9). Cells were then transferred to ice-cold buffer (10 mM NaCl and 10 mM HEPES, pH 7.2) and torn open with the point of dissecting tweezers. The plasma membrane and associated cortex were washed to remove 99% of cellular protein (e.g. nucleus and most of the cytoplasm). Each group (typically 15 cortexes in 50–100 µl total volume) was kept at 15 C, and usually there were 4–6 groups/treatment. Thus, each point from 1 experiment represents the response of 60–90 cortexes.

Treatments and chemical sources
Wortmannin (Calbiochem, La Jolla, CA), insulin (porcine, Eli Lily & Co., Indianapolis, IN; stock concentration determined spectrophotometrically), progesterone (Sigma, St. Louis, MO), guanosine 5'-O-(3-thiotriphosphate) (GTP-{gamma}-S) or guanosine 5'-O-(2-thiodiphosphate) (GDP-ß-S) (Calbiochem, La Jolla, CA), antibovine PLC{gamma}1 (Upstate Biotechnology, Inc., Lake Placid, NY), tyrphostin B46 [or AG 555 or N-(3-phenylpropyl)-3,4-dihydroxybenzylidenecyanoacetamide], or inactive tyrphostin A1 [or (4-methoxybenzylidene)-malononitrile; Calbiochem] (24) were used with either the PMC or whole oocyte. The calcium concentration in the PMC solution was reduced by the addition of a mixture of EGTA and CaCl2 (typically referred to as the EGTA group). The ratio of EGTA to CaCl2 and the MaxChelator program was used to calculate the free calcium concentration as approximately 70 nM (25). Solutions of chelator and CaCl2 were made by weighing powder to the nearest 1000th of a gram, and the ionic strength of the control groups were set equal to that in the experimental groups by the addition of NaCl.

Measurement of tyrosine kinase activity
Tyrosine kinase activity in the PMC was measured in the presence of a modified peptide sequence from pp60-Src. This tyrosine kinase substrate, RR-SRC (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), was added to the group of 15 PMCs to a concentration of 0.5 mM in the presence of [32P]ATP (500 µM; 50 µCi) and 5 mM MgCl2 for an optimal time (15 min). RR-SRC (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) does not have serine or threonine, binds P81 filter paper, is poorly dephosphorylated (26), and has been shown to be an appropriate substrate for many different tyrosine kinases (27, 28). Reactions were stopped by transferring an aliquot to P81 paper and placing the papers into 300 ml 75 mM phosphoric acid. After three washes, the papers were placed into scintillation vials, and radiation was quantified by Cherenkov counting. Addition of equivalent amounts of ethanol (carrier for progesterone) did not alter tyrosine kinase activity (data not shown).

PLC activity was measured by recording IP3 mass
IP3 mass in the PMC preparation was measured 15 min (an optimal time) after hormone stimulation. Trichloroacetic acid (25%; 200 µl) was added, the PMC groups were homogenized, and a binding assay was used to quantify IP3 mass with an IP3 receptor binding assay (New England Nuclear, Boston, MA) (9).

Microinjection of fusion proteins
One GST fusion protein contained the N- and C-terminal SH2 domains of PLC{gamma}, and another contained the SH2 domain from Shc (both made and purified from Escherichia coli) (29). Using a PV380 pneumatic Picopump (World Precision Instruments, Sarasota, FL), individual oocytes were pressure-injected with 50 nl fusion protein solutions.

Immunoprecipitation of PLC{gamma}
We used immunoprecipitation procedures similar to those proven to purify Xenopus PLC{gamma} (30, 31, 32). Oocytes were lysed on ice in 1 ml 20 mM HEPES, 1% (wt/vol) Triton X-100, 50 mM NaF, 1 mM phenylmethylsufonylfluoride, 80 mM ß-glycerophosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM Na3V04, and 20 mM EGTA (pH 7.2). After centrifugation (10 min at 14,000x g), the supernatant was precleared with 50 µl 50% protein A-Sepharose (Sigma; 1 h, 4 C) and centrifuged again. The precleared supernatant was incubated with 10 µl antibovine PLC{gamma}1 (Upstate Biotechnology, Inc.) for 14 h (4 C). After the addition of 40 µl 50% protein A-Sepharose suspension and incubation (1 h, 4 C), the antigen-antibody complex was pelleted (14,000 x g, 5 min). The immunoprecipitate was washed with a low salt solution [20 mM Tris (pH 7.4), 0.1% (vol/vol) Triton X-100, 5 mM EDTA, and 100 mM NaCl], a high salt solution [20 mM Tris (pH 7.4), 0.1% (vol/vol) Triton X-100, 5 mM EDTA, and 1 M NaCl], and 20 mM HEPES (1 h each). At this point, some samples were run on an 8.5% polyacrylamide gel. Similar to published results from three different laboratories, protein staining or autoradiography detected one major band at 140,000 kDa (data not shown).

Phosphoamino acid analysis of PLC{gamma}
To record any hormone-induced change in the phosphorylation state of PLC{gamma}, groups of 50–150 oocytes were incubated in 0.5 mCi 32P04 (6–15 h), washed twice with fresh O-R2, and incubated with either 1 µM insulin or 10 µM progesterone for various times (typically 15 min). After PLC{gamma} immunoprecipitation, the peptide bonds in the immunoprecipitate were hydrolyzed in 50 µl 5.7 N constant boiling HCl (Sigma) for 1 h (110 C). After evaporation of HCl with nitrogen, samples were resolubilized in 5 µl of pH 1.9 buffer (50 ml 88% formic acid, 156 ml acetic acid, and 1794 ml deionized water) and 5 µl 0.1 µg/ml phosphoamino acid standards (Sigma). Samples were spotted on thin layer cellulose plates (DC-Plastikfolien Cellulose, EM Science, Gibbstown, NJ) and electrophoresed in two dimensions using the Hunter thin layer electrophoresis system (HTLE-7000, CBS Scientific Co., Del Mar, CA). The samples were electrophoresed in the first dimension for 35 min at 1500 V in the pH 1.9 buffer and, after air-drying, in a second dimension for 20 min at 1200 V in the pH 3.5 buffer (100 ml acetic acid, 10 ml pyridine, and 1890 ml deionized water). After electrophoresis, plates were sprayed with 0.25% ninhydrin (Sigma) and baked (65 C, 10–15 min) to detect phosphoamino acid standards. To quantify radiation visualized by autoradiography (Biomax MS, Eastman Kodak Co., Rochester, NY), film was digitalized (ScanJet with light source in the lid, Hewlett-Packard Co., Palo Alto, CA), and spot density was determined with SigmaGel (Jandel Scientific, San Rafael, CA). In some experiments the phosphoserine spot on the TLC plate was cut out, and radiation was quantified by liquid scintillation counting. As a measure of successful labeling, phosphoserine spots from control groups from three experiments had 1935 ± 134 cpm (n = 3).

Statistical analyses
Asterisks in the figures denote significance at P < 0.05, analyzed with a two-sided pooled t test, and the SEM is reported in both figures and text. The letter n represents the number of determinations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tyrphostin inhibition of tyrosine kinase and PLC activities in the PMC
To activate PLC{gamma}, it would be predicted that progesterone would have to stimulate a tyrosine kinase. Using the PMC preparation, we were able to show that progesterone stimulated a membrane-associated tyrosine kinase activity and that this activity was inhibitable by tyrphostin B46 (Fig. 1AGo). Similar results were obtained with a hormone, insulin, that is known to act through a tyrosine kinase. Tyrphostin B46 is a tyrosine kinase inhibitor; it was chosen for these studies because there is an inactive derivative tyrphostin A1, and in other studies (not shown) we found that genistein was a weaker inhibitor. The inactive tyrphostin derivative A1 was without effect on tyrosine kinase activity.



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  		Figure 1. Progesterone or insulin stimulates tyrosine kinase and PLC activities. A, Tyrosine kinase activity in the PMC was measured 15 min after the addition of 1 µM insulin (n = 14) or 5 µM progesterone (n = 11). The INS group received insulin and inactive tyrphostin A1, and the PROG group received progesterone and tyrphostin A1. In the TYR + INS group, tyrphostin B46, a tyrosine kinase inhibitor, was added 10 min before insulin to a final concentration of 200 µM (n = 3). In the TYR + PROG group, progesterone was added after tyrphostin B46 (n = 3). In three other experiments, it was found that inactive tyrphostin A1 did not alter basal tyrosine kinase activity. B, PLC activity in the PMC was recorded by measuring IP3 mass 15 min after hormone addition. The CON group received no treatment. Progesterone (PROG) or insulin (INSULIN) was added 5 min after groups received either inactive tyrphostin A1 (TA1) or tyrphostin A25 or B46 (TYR). There were 6–16 determinations/treatment (with 15 PMCs used in each determination).

 
Similar to what we reported with whole cells (9), we found that progesterone or insulin addition to the PMC stimulated PLC activity (as measured by IP3 mass; Fig. 1BGo). PLC activation by either progesterone or insulin was inhibited by tyrphostin B46 (tyrphostin A1 had no effect on hormone-stimulated IP3 levels; Fig. 1BGo).

A protein with the SH2 domains from PLC{gamma} blocks progesterone activation of PLC
We then postulated that if progesterone acted through a tyrosine kinase, the steroid may activate the {gamma}-isoform of PLC. One accepted method of PLC{gamma} activation involves binding of its SH2 regions to phosphorylated tyrosines of an upstream activator. Of the three isoforms of PLC, only PLC{gamma} contains SH2 regions (19). Thus, addition of a protein containing the SH2 domains (but not the catalytic domain) of PLC{gamma} should block activation of PLC{gamma}, but not that of other PLC isozymes.

Addition of a protein containing the two SH2 domains from PLC{gamma} inhibited progesterone action in PMC and whole cells (Fig. 2Go, A and B). As a control, addition of a fusion protein with the single SH2 domain from Shc to the PMC was unable to block progesterone activation of PLC (Fig. 2CGo).



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  		Figure 2. A GST fusion protein containing the N- and C-terminal SH2 domains of PLC{gamma} inhibits progesterone stimulation of PLC. A, Addition of a GST fusion protein containing the two tandem SH2 domains of PLC{gamma} to PMCs inhibits progesterone’s ability to increase IP3 mass. The PLC SH2 fusion protein (final concentration, 21 µg/ml) and, after a preincubation period of 15 min, progesterone (10 µM) were added to PMCs. Fifteen minutes after hormone addition, PLC activity was recorded by measuring IP3 mass (n = 10; each determination had 15 PMCs). CON, Control group; PROG, group that received progesterone; PROG + PLC SH2, group that was incubated with the SH2-containing fusion protein and progesterone. B, Microinjection of the PLC SH2 domain fusion protein into whole Xenopus oocytes inhibits progesterone’s ability to raise IP3 mass. Fifty microliters of the stock solution of the PLC SH2 fusion protein (520 µg/ml) were microinjected to a final intracellular concentration of 54 µg/ml in whole oocytes (free volume of 480 nl) (58 ). After a preincubation period of 15 min, progesterone (5 µM) was added to the whole cells. Fifteen minutes after hormone addition, the cells (each treatment had 3–4 groups, with 15 cells/group) were homogenized and analyzed for IP3 mass (9 ). The IP3 produced in the PROG + PLC SH2 group was less than that produced in the group that received progesterone alone (P < 0.056). C, A GST fusion protein containing the SH2 domain from Shc did not prevent progesterone from elevating PLC activity. In a manner similar to the procedure described in B, a different fusion protein, one containing the single SH2 domain from Shc, was added to the PMC preparation (final concentration, 20 µg/ml) 15 min before progesterone (10 µM) addition. After another 15-min period, PLC activity was measured (n = 6–9; 15 PMCs/determination). CON, Control group; PROG, group treated with progesterone; PROG + Shc SH2, groups in which the Shc SH2 fusion protein was added before progesterone.

 
G protein and progesterone activation of PLC is additive
As PLCß can be activated by G proteins, we examined whether progesterone acts through a G protein to activate PLCß activity. First, we demonstrated the presence of PLCß in the PMC, as the G protein agonist GTP-{gamma}-S increased IP3 production (Fig. 3AGo). Significantly, the tyrosine kinase inhibitor tyrphostin B46 was unable to inhibit GTP-{gamma}-S stimulation of PLC (Table 1Go).



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  		Figure 3. Addition of G protein agonist GTP-{gamma}-S. A, GTP-{gamma}-S, a G protein agonist, was added to groups of 15 PMCs (or cortex), and after 15 min, PLC activity was followed by recording IP3 mass per PMC (n = 6–20). A PLC activatable by a G protein agonist (presumably PLCß) was present in the PMC (i.e. PLCß was not washed away when the PMCs were prepared). B, Addition of a G protein agonist, GDP-ß-S, and progesterone together produced an IP3 increase that was the sum of IP3 increases obtained when the agonist and progesterone were added individually. CON, The group that did not receive any treatment. With PLC activity in the PMC or cortex, GDP-ß-S (GDPBS) or progesterone (PROG) addition produced responses that were less than when both were added together (BOTH is the group that received both GDP-ß-S and progesterone; n = 7 for each). As asterisks denote a comparison between the control group and the experimental group, we also compared experimental groups. The IP3 value for the BOTH group was significantly higher than that for either the GDPBS or PROG group (P < 0.05).

 

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  		Table 1. Phospholipase C activation by GTP-{gamma}-S is independent of lowered calcium and tyrphostin B46 levels

 
GDP-ß-S is a G protein antagonist in some cells, but an agonist in other cell types (33). We examined its action in Xenopus PMCs. Similar to its role in sea urchin and rabbit eggs (34, 35), GDP-ß-S proved to be a G protein agonist, as it stimulated IP3 production in the Xenopus PMC preparation (Fig. 3BGo) and induced membrane potential depolarization after microinjection into Xenopus eggs (membrane depolarization is an indirect measure of increased [Ca2+]i in Xenopus oocytes) (B. J. Stith, unpublished data). Furthermore, IP3 production in the presence of both the G protein agonist GDP-ß-S and progesterone was additive (i.e. equivalent to the sum of the IP3 produced when each agonist was added separately; Fig. 3BGo). This additivity suggested that progesterone does not act through G proteins.

Lowering [Ca2+]i levels inhibits hormone activation of PLC, but not PLC activation by G protein agonist
As another method of examining whether progesterone acts through a G protein path, we searched for conditions that would not affect one path (e.g. the G protein path), but would inhibit another (e.g. the tyrosine kinase path). We found that lowering the free calcium concentration inhibited hormonal stimulation of IP3 production in the PMC (Fig. 4Go), but this did not inhibit PLC activation by a G protein agonist (Table 1Go). Although lowered calcium could inhibit multiple steps, this result suggests that progesterone does not act through activation of a G protein and PLCß.



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  		Figure 4. Buffering the free calcium concentration to low levels inhibits hormonal stimulation of PLC activity. IP3 mass, a measure of PLC activity in the PMC (or cortex), was measured 15 min after hormone addition (n = 3 each treatment; 15 PMCs/treatment). Addition of hormone is denoted by I for insulin or P for progesterone. Hormones were added to PMCs when calcium was not lowered (denoted by a - to the right of EGTA/CA) and when calcium was lowered by EGTA addition (+) to about 100 nM.

 
Progesterone increases phosphoserine in Xenopus PLC{gamma}
One accepted method of PLC{gamma}1 activation involves the binding of SH2 domains of PLC{gamma}1 to phosphorylated tyrosines of a tyrosine kinase followed by PLC{gamma}1 phosphorylation on tyrosines 771, 783, and 1254 (regulation of PLC{gamma}2 is not fully described, although it may be similar) (18). Based on this model, insulin or progesterone should increase phosphorylation of tyrosines in PLC{gamma}. However, than five Western blots with two different antiphosphotyrosine antibodies produced negative results (e.g. neither progesterone nor insulin increased phosphotyrosine in PLC{gamma}). Due to differing efficacies of antiphosphotyrosine antibodies and a concern about the sensitivity of the Western blot, we measured phosphoamino acids in PLC{gamma} directly. After prelabeling cells with 32P04, immunoprecipitation and hydrolysis of PLC{gamma}, and two-dimensional TLC electrophoresis of phosphoamino acids, we were unable to show any progesterone-induced increase in phosphotyrosine on PLC{gamma} (Fig. 5Go). Similar negative results were obtained with insulin, with different time points after the addition of either hormone (30 sec and 1, 5, 15, or 30 min) and longer incubation times in higher concentrations (2 mCi/ml) of radioisotope or after microinjection of concentrated [32P]ATP. In addition, no phosphotyrosine on PLC{gamma} was found after hormone treatment of PMCs prelabeled with [32P]ATP. However, both progesterone and insulin were able to increase the amount of phosphoserine in PLC{gamma} (Fig. 6BGo). Increased phosphoserine is believed to be associated with the inhibition of PLC{gamma} (41, 42).



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  		Figure 5. Addition of progesterone or insulin to whole oocytes did not increase tyrosine phosphorylation of immunoprecipitated PLC{gamma}. A, PLC{gamma} was analyzed for phosphoamino acids in a control group of oocytes (CON) and a group treated with progesterone (PROG). Neither progesterone nor insulin (data not shown) induced an increase in phosphotyrosine on PLC{gamma}. For the CON and PROG groups, the location of standards are circled with a dotted line: phosphoserine (P-SER) is top left, phosphothreonine (P-THR) is to the right, and phosphotyrosine (P-TYR) is to the lower right. Groups of 50 oocytes were incubated in 0.5 mCi/ml 32P04 for 6 h, and after washing, progesterone (10 µM) was added for 15 min, then the cells were homogenized. PLC{gamma} was immunoprecipitated, and the peptide bonds were hydrolyzed. Phosphoamino acids were separated by two-dimensional thin layer electrophoresis. B, Fifteen minutes after addition of progesterone (10 µM) or insulin (2 µM), there was an increase in the amount of phosphoserine in PLC{gamma}. Data were taken from two-dimensional TLCs, such as that shown in part A. After autoradiography and digitalizing the phosphoamino acid spots (using an HP ScanJet with a light source in the scanner lid), the densities of the phosphoamino acid spots on the x-ray film were quantified by use of SigmaGel (Jandel Scientific). To minimize differences between experiments with different cells, we divided all values by the control density value (thus, control is 1.0). Incorporation of label into control values was high (1935 ± 134 cpm). Phosphoserine spot density was compared with a two-sided one-sample t test, comparing the experimental values to a control value of 1.0 (n = 8 for each treatment).

 


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  		Figure 6. Wortmannin, a PI 3 kinase inhibitor, did not inhibit the ability of progesterone to stimulate PLC activity in PMC or whole cells. A, Wortmannin (100 nM) was added to the PMCs, and after a preincubation period of 15 min, progesterone (10 µM) was added. Fifteen minutes after hormone addition, the samples were analyzed for IP3 mass (for each treatment, there were 4 groups of 15 PMCs). CON, Control group; PROG, progesterone-treated group; PROG + WORT., group pretreated with wortmannin and then progesterone. B, Wortmannin (100 nM) was added to whole Xenopus oocytes for 30 min (this treatment was able to inhibit insulin induction of meiotic cell division and has been shown to inhibit Xenopus PI 3 kinase) (39 ). Progesterone (10 µM) was then added, and after 15 min, cells were homogenized and analyzed for IP3 mass.

 
Progesterone does not act through PI 3-kinase to activate PLC
Although we have evidence that a tyrosine kinase is involved in PLC activation by progesterone, PLC{gamma} can also be activated through a tyrosine kinase path involving PI 3- kinase (17, 36, 37, 38). Activation of PI 3-kinase would increase phosphatidylinositol 3,4,5-trisphosphate in the plasma membrane, and the SH2 or PH domain of PLC would bind the phospholipid and translocate PLC to a location near its substrate PIP2. This would increase IP3 production. However, wortmannin, at a concentration that inhibits Xenopus PI3 kinase and blocks insulin-induced meiotic cell division (39) (our unpublished data), did not inhibit progesterone- induced activation of PLC{gamma} in the PMC or whole cells (Fig. 6Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Progesterone and insulin act through a tyrosine kinase to activate PLC
Although it is known whether progesterone acts at a plasma membrane location (3, 4) [the 110-kDa protein thought to be the plasma membrane progesterone receptor has been found to be a contaminating ribosomal protein (40)], this is the first demonstration that progesterone stimulates and acts through a tyrosine kinase located in or near the Xenopus oocyte membrane. However, it is not the first report that progesterone acts through a tyrosine kinase to activate PLC; progesterone addition to human sperm activates a tyrosine kinase to stimulate PLC (41, 42). The most common nongenomic action of steroids is an increase in [Ca2+]i (1), so our results showing that progesterone increases IP3 in whole cells (9) and in the PMC (this report) is not unexpected. In addition, our suggestion that progesterone does not act through a G protein to activate oocyte PLC is similar to the conclusion that the increase in [Ca2+] in sperm by progesterone does not involve G proteins (43, 44).

The identity of the progesterone-stimulated tyrosine kinase in the Xenopus oocyte may be similar to that of the receptor tyrosine kinase Hu9 found in human sperm (see discussion in Ref. 45) or involve a soluble tyrosine kinase such as pp60-src. Progesterone does not act through the insulin or insulin-like growth factor I receptor (both tyrosine kinases), because an inhibitory antibody that binds to these receptors does not block progesterone induction of meiosis (46).

The ability of a tyrosine kinase to activate Xenopus PLC has been found in prior studies using the oocyte as an expression system for exogenous tyrosine kinase receptors (47). Vanadate (a tyrosine phosphatase inhibitor) also increased PLC activity in the PMC (Stith B. J., manuscript in preparation) and is able to induce meiotic cell division (46).

The ability of progesterone to activate a tyrosine kinase may play a role in induction of maturation of the Xenopus oocyte. Microinjection of a tyrosine phosphatase (48) or antibodies to phosphotyrosine (49) into Xenopus oocytes or the addition of high levels of tyrphostin B47 (50) inhibited progesterone-induced maturation.

Progesterone regulation of PLC
There are many advantages to the use of the PMC preparation in the study of PLC regulation. There has been criticism of many in vitro studies of PLC due to the use of artificial water-soluble substrates and "which [artificial] substrate is used and how the homogeneous solution of a substrate is prepared can significantly affect the outcome of studies on effector molecules" (53). In most studies, the "use of detergents may entangle studies on the effects of potential regulators" and "agonist-sensitive activation of PLC has been difficult to demonstrate ... " in the presence of detergents (51). In support of later statement, Xenopus PLC activity has been measured in a membrane-rich fraction after homogenization, differential centrifugation, and addition of exogenous substrate ([3H]PIP2 in a sonicated mixture of phosphatidylserine and phosphatidylethanolamine) (52). This PLC activity required above normal levels of calcium (100 µM) and was insensitive to progesterone, insulin, and GTP-{gamma}-S (52). In contrast to these in vitro results, our work examined PLC in a relatively intact preparation and used natural substrate and no detergents, and we were successful in showing stimulation of PLC activity by hormones and GTP-{gamma}-S.

As we are unable to demonstrate any hormone-induced increase in phosphotyrosine on PLC{gamma}, we suggest the following model for progesterone activation of PLC in Xenopus. The hormone would activate a tyrosine kinase that phosphorylates an unknown protein. The phosphotyrosine domain of this protein would then bind to the SH2 domain(s) of PLC{gamma} and activate the lipase. The unknown protein would not be a tyrosine kinase, because no phosphotyrosine is found on PLC{gamma}. Instead of activation by tyrosine phosphorylation, PLC{gamma} may be activated by translocation from sites near the plasma membrane to membranes with the substrate PIP2. Instead of a translocation, PLC{gamma} activation may be through a conformational change induced by binding of its two tandem SH2 domains (no other PLC isozymes have SH2 domains) (19). Similar to activation of PTP2 by agents binding to its two SH2 domains and due to enhanced binding specificity with two (not one) tandem SH2 domains (53, 54), tyrosine phosphorylation of PLC{gamma} would not have to take place. Many other laboratories have suggested that activation of PLC{gamma} can occur in the absence of tyrosine phosphorylation (17, 55, 56, 57).

In summary, progesterone stimulates a tyrosine kinase activity that is located at or near the plasma membrane. The tyrosine kinase is responsible for stimulation of PLC activity. This model is based in part on the inhibition of progesterone stimulation of PLC by the tyrosine kinase inhibitor tyrphostin B46 and an SH2-containing fusion protein.


   Acknowledgments
 
Thanks for help from Keith Woronoff, Jennifer Hall, Ronald Espinoza, Michelle Rich, Lisa Swise, and Leslie Edwards. A special thanks to N. Webster and Ayse Kayali (University of California, San Diego) for the fusion proteins and valuable discussions.


   Footnotes
 
1 This work was supported by NSF Grants MCB-9220108 and IBN-9722938, and two Undergraduate Research Opportunities Program awards from the University of Colorado-Denver. Back

Received December 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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