Molecular and Cellular Endocn’nology, 0 1992 Elsevier Scientific Publishers
MOLCEL
125
86 (1992) 125-132 Ireland, Ltd. 030?-7207/92/$05.00
02784
Protein kinase C stimulatory activity in the pseudopregnant
rat ovary
KM. Eyster, M.S. Waller and M.J. Johnson Department
of Physiology and
Pharmacology, Uni~~er.s@ofSouth Dakota. Vermillion, SD, USA
(Received
Key words: Protein
kinase
C; Ovary;
Stimulation;
13 March
1992; accepted
3 April 1992)
Modulator
Summary Ovarian cytosol from pseudopregnant rats was heated to SO-90°C for 2 min and precipitated proteins removed by centrifugation. The supernatant of the heated ovarian cytosol contained no protein kinase C activity but when added to a control preparation containing protein kinase C, enzyme activity was increased to 200% of control. The stimulatory activity was stable to heating for 10 min, was retained on a centrifugal filtration device with a 100,000 M, cut-off, did not affect CAMP-dependent protein kinase, was not extractable in petroleum ether or chloroform/methanol (2: 11, and enhanced the phosphorylation of protein kinase C-specific peptide substrates. The stimulatory factor was calcium-dependent and could substitute for phosphatidylserine and diacylglycerol in the protein kinase C assay. This stimulatory factor may provide a mechanism whereby the response of protein kinase C to hormonal activation could be regulated by the cell.
Introduction Protein kinase C, the calcium- and lipid-dependent protein kinase, is an important intracellular messenger in diverse cellular activities from mediating the mechanism of hormone action to cell division (Macara, 1985; Nishizuka, 1986; Olashaw and Pledger, 19881, and its regulation in various tissues is equally diverse and complicated. The multiple isoforms of protein kinase C include calcium-dependent and calcium-independent forms (One et al., 1988) and exhibit differential sensitivity to activation by phospholipids and diacylglycerol (One et al., 1988). In addition, there is
Correspondence to: Kathleen M. Eyster, Department of Physiology and Pharmacology, University of South Dakota School of Medicine, Vermillion, SD 57069, USA. Tel. (605) 677-5159: Fax (605) 677-5124.
differential distribution of the various isoforms across tissue types (Knopf et al., 1986; Ono et al., 1988). Regulation of protein kinase C is further complicated by the presence of endogenous inhibitors (Eyster, 1990; Pearson et al., 1990) and stimulators (Hsu et al., 1991) of the enzyme in some tissues. Although these many regulatory mechanisms have been documented, their physiological roles have yet to be well-defined. The regulation of ovarian function involves the complex interaction of multiple endocrine and paracrine factors, many of which utilize second messenger pathways such as the protein kinase C pathway to convey their messages to intracellular actions. The pseudopregnant rat model has often been used for studies of the mechanism of hormone action in the ovary. Prostaglandin F,, (Leung et al., 1986) and gonadotropin releasing hormone (Leung et al., 1983) stimulate phospho-
126
lipase C-induced phosphatidylinositol turnover, the transduction pathway which activates protein kinase C, in the pseudopregnant rat ovary, and this pathway is hormonally activated in other ovarian cell types as well (Davis et al., 1986; Ranta et al., 1986). We have used the pseudopregnant rat model for studies of protein kinase C activity (Eyster, 1990, 1991). In the course of our work we found that heat treatment of pseudopregnant rat ovarian cytosol resulted in protein kinase C-stimulatory activity. The current studies were undertaken to examine this protein kinase C-stimulatory factor. Methods
and materials
Pseudopregnancy was induced in immature female rats by injection of pregnant mares’ serum gonadotropin and chorionic gonadotropin (Eyster, 1990). The ovaries were removed on day 7 of pseudopregnancy and stored at -70°C. Cytosol was prepared as described (Eyster and Clark, 1989). Briefly, the ovaries were homogenized with a Polytron homogenizer in a 20 mM Tris-HCI buffer, pH 7.4, with 2 mM EDTA, 5 mM EGTA, 0.25 M sucrose, and 50 mM 2-mercaptoethanol at a dilution of 1 : 5 (w/v). The tissues were homogenized for 30 s at full speed, allowed to cool for 1 min, then homogenized for a second 30 s period. The homogenate was centrifuged for 600,000 X g min and the resulting supernatant was saved as the cytosolic preparation. Protein kinase C activity was measured by the transfer of “P from [Y-~~P]ATP to histone III-S as described (Eyster and Clark, 1989). The assay mixture contained 20 mM Tris-HCI, pH 7.4, 5 mM magnesium acetate, 10 PM ATP (with lo6 cpm [Y-‘~P]ATP), 0.2 mg/ml histone III-S, 20 mM 2-mercaptoethanol, 100 mM sucrose, 0.8 mM EDTA and 2.0 mM EGTA. Enzyme activity was measured in the absence and presence of 2.9 mM calcium (approximately 0.1 mM free calcium), 0.8 Fg/ml 1,2-dioleoyl rat-glycerol, and 20 pg/ml phosphatidylserine (standard lipid concentration). Incubations were initiated with the addition of the tissue sample and were carried out for 3 min at 30°C. The reaction was stopped by addition of 1 ml ice-cold 25% trichloroacetic acid. Precipitated proteins were collected by filtration on ni-
trocellulose membranes. Enzyme activity was linear with respect to time and tissue concentrations as previously reported (Clark et al., 1983). Protein kinase activity in the absence of calcium and lipid was subtracted from protein kinase activity in the presence of calcium and lipid to yield specific protein kinase C activity. For the heat treatment, ovarian cytosol was heated to X0-90°C for 2 min and precipitated proteins removed by centrifugation for 5 min at 16,000 X g. The supernatant was mixed with a control preparation of protein kinase C and preincubated for 5 min at 30°C before beginning the phosphorylation assay. The control preparation consisted of rat brain cytosol which had been partially purified on DEAE in a mini-batchwise test tube technique (Eyster, 1990) analogous to conventional column purification with DEAE. The phosphorylation of protein kinase Cspecific peptide substrates (Yasuda et al., 1990) was examined by substituting the various substrates for the histone III-S. The peptides tested were a synthetic peptide derived from myelin basic protein consisting of amino acids 4-14 with the N-terminal acetylated (AC-MBP+,,,), a peptide derived from the pseudosubstrate region of the protein kinase C molecule in which the 25 position has been replaced with a serine so that the peptide can serve as a substrate for protein kinase C ([Ser”]PKC(,,~,,,), and a peptide derived from glycogen synthase ([Ala”,‘~‘,Lys”,‘21were obtained from GS (,_,2). Peptide substrates Life Technologies (Gaithersburg, MD, USA). The final concentration of peptide substrates in the assay was 51 PM. In assays in which the peptide substrates were substituted for histone III-S, the samples were spotted on 4 X 4 cm squares of P-81 paper (Whatman) after the incubation. The papers were placed in 0.1 M H,PO, and washed 3 times for 5 min each in a bath of 0.1 M H,PO,, then counted in scintillation fluid (Yasuda et al., 1990). For assays involving cyclic AMP-dependent protein kinase, the enzyme was partially purified from rat ovarian cytosol using a similar DEAE test tube technique to that used for protein kinase C, except the CAMP-dependent protein kinase was eluted from the DEAE with 0.5 M NaCI. Cyclic AMP-dependent protein kinase ac-
tivity was measured as described (Davis and Clark, 1983). For all enzyme assays, data are expressed as picomoles of 32P transferred per min (pmol/min). Except where mentioned, chemicals and materials were obtained as previously described (Eyster, 1990). Results When pseudopregnant rat ovarian cytosol was heated to 80-90°C for 2 min all protein kinase C activity was destroyed (0 pmol/min). When the heat-treated ovarian cytosol was mixed with the control preparation of protein kinase C from rat brain, enzyme activity was substantially increased (control 11.7 pmol/min vs. control + heated ovarian cytosol 22.4 pmol/min). The stimulatory factor was relatively heat stable; there was no change in stimulation in ovarian cytosol heated to 8090°C for 2, 5, or 10 min or boiled for 10 min (Fig. 1). Protein kinase C activity was directly correlated with the volume of heat-treated ovarian cytosol added (Fig. 2). When heated cytosol was separated on a centrifugal filtration device with a 100,000 M, cut-off (CentriCon 100; Amicon Division of WR Grace and Co., Beverly, MA, USA), the stimulatory activity was located in the reten-
O/A 0
10
Volume
20
30
Heated
40
50
Cytosol
60 (pi)
Fig. 1. Protein kinase C activity was measured in the control preparation (CTL, open bar), and after mixing the control preparation with ovarian cytosol that had been heated for 2, 5, or 10 min, or boiled for 10 min (B-10). The values expressed are the mean&SE of four separate measurements. The asterisk denotes enzyme activities significantly different from control.
0 CTL Lerlqth
of
10’
2
5
Yeat
Treatment
B-IO (min)
Fig. 2. Protein kinase C activity was measured in the absence (0) and presence of increasing volumes (~1) of ovarian cytosol heated to SO-90°C for 2 min. The standard volume of heated ovarian cytosol used was 28 ~1. The values shown are the mean f SE of three separate measurements.
tate (Control (Ctl) 14.4 pmol/min, Ctl + retentate 36.7 pmol/min, Ctl + filtrate 14.1 pmol/min). The stimulatory activity was not extractable in petroleum ether (Ctl 10.4 pmol/min, Ctl + heated ovarian cytosol 20.2 pmol/min; Ctl + extract 9.4 pmol/min, Ctl + aqueous phase 25.3 pmol/min), chloroform/methanol (2 : 1; Ctl 10.4 pmol/min; Ctl + extract 13.3 pmol/min, Ctl + aqueous phase 18.0 pmol/min), or acidified chloroform/ methanol (2: 1, pH 1.0; Ctl 10.4 pmol/min; Ctl + extract 12.4 pmol/min, Ctl + aqueous phase 17.4 pmol/min). When heated ovarian cytosol was mixed with partially purified CAMP-dependent protein kinase, enzyme activity fell to 5.8 pmol/min from a control of 7.9 pmol/min. The heated ovarian cytosol enhanced the phosphorylation of protein kinase C-specific substrates and (Ala’,“‘, AC-MBP+,,,, [Ser2’lPKC(,,_,,,, by 166%, 201% and 166%, reLYS”,‘~IGS+,,, spectively (Fig. 3). The calcium, lipids (phosphatidylserine and 1,2-dioleoyl rat-glycerol), and histone substrate were, each in turn, removed from the reaction mixture and replaced with the heated ovarian cytosol. Removal of the histone substrate from the reaction mixture reduced measurable protein kinase C activity to 8% of control enzyme activity. Substitution of heated ovarian cytosol for the histone resulted in an increase of protein kinase C activity to 16% of control. Removal of lipids
.;
25
\ F
20
a ;
15
> 2 0 y CL
10 5 0 Histone
MBP
PKC
GS
Fig. 3. Protein kinase C activity was measured in the absence (crosshatched bars) and presence (solid bars) of heated ovarian cytosol with different substrates for enzyme phosphorylation. The substrates were histone 111-S (the standard substrate), a peptide derived from myelin basic protein, Ac(MBP), a modified peptide derived from the pseuMBP+,,, dosubstrate region of protein kinase C, [Ser’“]PKC~,,,_,,, (PKC). and a modified peptide derived from glycogen synthase, [Ala’~~“‘,Lys”~‘~]GS,,~,~, (GS). The values are expressed as the mean* SE of three separate measurements. Enzyme activities in the absence vs. the presence of heated ovarian cytosol that are significantly different are designated with an asterisk.
(phosphatidylserine and 1,2-dioleoyl rat-glycerol) from the reaction mixture resulted in reduction of protein kinase C activity to 6% of control. When heated ovarian cytosol was substituted for lipids, enzyme activity increased to 110% of control. Protein kinase C activity was then measured in the presence of decreasing concentrations of lipids, in the absence and the presence of a constant volume of the heated ovarian cytosol and at 100% (100 PM) of the standard calcium concentration (Fig. 4). In the absence of the heated ovarian cytosol, protein kinase C activity decreased as the concentration of lipids decreased from the standard concentration of lipid (20 pg/ml phosphatidylserine and 0.8 pg/ml 1,2-dioleoyl rat-glycerol) to zero lipid. In the presence of heated ovarian cytosol, protein kinase C activity remained constant as the concentration of lipids decreased. Removal of all calcium from the reaction mixture did not result in loss of protein kinase C activity (105% of control). However, there was no effect of the heated ovarian cytosol on protein
kinase C activity in the absence of calcium (108% of control). Protein kinase C activity was measured in the presence of decreasing concentrations of calcium, in the absence and presence of a constant volume of heated ovarian cytosol and at 100% of the standard lipid concentration (Fig. 5). As the concentration of calcium decreased from 100 PM to 0 PM, the activity of protein kinase C decreased at 50 PM to 25 PM, then returned to control levels at 12.5 PM and 0 PM calcium. In the presence of the heated ovarian cytosol, increases in protein kinase C activity above control occurred at calcium concentrations between 65 PM and 100 PM, but not at calcium concentrations of 50 PM or less (Fig. 5). The requirement for 65 PM or greater calcium to effect stimulatory activity of the heated ovarian cytosol per-’ sisted at all concentrations of lipid tested (Fig. 6, A-F). In the absence of the stimulators of pro-
I
Fig. 4. Protein kinase C activity was measured in the absence (closed circles) and presence (open bars) of heated ovarian cytosol, both in the presence of increasing concentrations of lipids (phosphatidylserine and IJdioleoyl rat-glycerol). The concentration of lipids increased from 0 to 100% of the standard concentration used in the protein kinase C assay (see Methods and materials). Assays were performed in 100 PM calcium. Values are expressed as the mean k SE of five separate measurements. Protein kinase C activities in the presence vs. the absence of heated ovarian cytosol that are significantly different are designated by an asterisk (p < 0.05, paired Student’s I-test).
I29
24
20
16
12
8
4
0 12.5
25
Fig. 5. Protein kinase C activity was measured in the absence (closed circles) and presence (open bars) of heated ovarian cytosol, both in the presence of increasing concentrations of calcium from 0 to 100 FM (the standard concentration of calcium in the assay). Values are the mean+SE of three separate determinations. Protein kinase C activities in the absence vs. the presence of heated ovarian cytosol which are significantly different are designated by an asterisk (p < 0.05, paired Student’s r-test).
tein kinase C, calcium and lipids, there was no effect of heated ovarian cytosol on protein phosphorylation (Fig. 6A ). Discussion Heat treatment of pseudopregnant rat ovarian cytosol resulted in the manifestation of a factor with protein kinase C-stimulatory activity. There was no stimulation of CAMP-dependent protein kinase activity, suggesting that the stimulation did not occur at a site common to all kinases such as the ATP-binding site. The enhancement of phosphorylation of protein kinase C-specific peptides by the stimulatory factor, and the absence of stimulation of basal phosphorylation (phosphorylation in the absence of calcium and lipids) corroborate the specificity of the stimulatory factor for protein kinase C. Retention of the factor on a 100,000 M, cut-off filter suggested that it had a M, greater than 100,000 or that it formed an
aggregate. The factor was also heat stable. Although the standard heat treatment was 2 min at 80-9072, boiling for 10 min did not produce a significant decrease in stimulatory activity. Nonpolar molecules are extracted from aqueous solution by organic solvents; however, the stimulatory activity was located in the aqueous phase after extraction by petroleum ether, chloroform/methanol, and acidified chloroform/ methanol. These data suggest that the stimulatory factor is a polar, heat-stable molecule (or aggregate) with a M, greater than 100,000. It was clearly not a supersubstrate for protein kinase C, as it did not substitute for histone in the assay. Not only did the factor maintain protein kinase C activity in the absence of lipids (phosphatidylserine and diacylglycerol), enzyme activity was greater than that stimulated by saturating concentrations of lipids. Whether the activation by the stimulator-y factor occurred through binding at one of the lipid-binding sites on protein kinase C, or at another site, remains undetermined. However, the factor clearly does not synergize with the lipids, as protein kinase C activity remained constant in the presence of the stimulatory factor regardless of the concentration of phosphatidylserine and 1,2-dioleoyl rat-glycerol in the reaction mixture. Although the lipids known to stimulate protein kinase C have a relatively low molecular weight, lipids form micelles which may have a relatively high apparent M,. Micellar formation, in addition to aggregation, could explain the apparent high M, of the stimulatory factor. However, lipids and micelles would have been extracted in the solvents used (i.e., chloroform/methanol) but the stimulatory factor was not extracted; rather, the stimulatory activity remained in the water-soluble phase of the extracted samples. Therefore, although the stimulatory factor substituted for lipids in the stimulation of protein kinase C, the extraction data do not indicate that it has a lipid nature. The rat brain contains both calcium-dependent and calcium-independent protein kinases C as described in the literature (One et al., 1988) and as indicated by the biphasic nature of protein kinase C activity in the absence of the stimulatory factor in Fig. 4. The activity of the stimulatory factor was clearly calcium-dependent. This can be
I30
interpreted as a direct calcium dependence of the stimulator-y factor, or an insensitivity of the calcium-independent protein kinases C to the stimulatory factor. Since the loss of stimulation of protein kinase C by the factor and the increase in protein kinase C activity that signifies the calcium-independent protein kinases C occur at dif-
ferent concentrations of calcium (65 PM vs. O12.5 FM), it is likely that the first interpretation is correct. If so, then the activation constant of the stimulatory factor for calcium is 65 PM in the in vitro assay. If the stimulatory factor serves a physiological role in regulation of protein kinase C. then one would expect that the stimulatory
3.
12.5
0 F
Fig. 6. Protein kinase C activity was measured A-F.
was performed
measurements.
Significant
at a different differences
50
25 yM
Cati
Co
75
100 *
+7
-I-
in the absence (closed circles) and presence (open bars) of heated ovarian cytosol.
both in the presence of increasing concentrations parts
80
pM
of lipids from 0 to 100% of the standard
concentration
of calcium.
as designated.
Values
assay lipid concentration. are the mean+SE
Each a~rve,
of three separate
in enzyme activities in the absence vs. the presence of heated ovarian cytosol are designated by an asterisk ( p < 0.05. Student’s
r-test).
131
factor would also be regulated. The calcium dependence of the stimulator-y factor presents a physiological mechanism for regulation of the stimulatory factor. The physiological role of the stimulatory factor may be to enhance the ability of protein kinase C to respond to subsequent stimuli after an initial stimulus in specific, regulated conditions. Such a response has been documented in adrenal glomerulosa cells. In these cells there is a timedependent potentiation of protein kinase C activation after previous stimulation of the enzyme (Bollag et al., 1991); perhaps the stimulatory factor is involved in similar ‘cell memory’ responses in the rat ovary. Alternatively, the physiological importance of this factor may not be that it increases protein kinase C activity to greater levels than phosphatidylserine, diacylglycerol, and calcium, but that it is a water-soluble factor which can maintain protein kinase C activity in the absence of membrane lipids. Although it has become the accepted dogma that activation of protein kinase C by diacylglycerol results in translocation of the enzyme from the cytosol to the plasma membrane (Olashaw and Pledger, 19881, we know that protein kinase C phosphorylates cytoplasmic and cytoskeletal proteins (Nishizuka, 1986; Graff et al., 1989) as well as DNA transcription factors in the nucleus (Hoeffler and Habener, 1990). Perhaps the role of the ovarian protein kinase C-stimulatory factor is to allow protein kinase C to be active in the cytosol or in the nuclei of cells under specific conditions. The manifestation of stimulatory activity only after heat treatment in the rat ovary may be due to association of the factor with a heat-sensitive regulatory protein, or to association of the factor with nonspecific proteins during homogenization of the tissue. There have been reports of other endogenous molecules which stimulate protein kinase C activity. Endogenous lipids such as polyphosphoinositides (O’Brian et al., 1987; Chauhan et al., 19X9>, unsaturated fatty acids, i.e., oleic and arachidonic acids (McPhail et al., 1984; Murakami and Routtenberg, 198.5; El Touny et al., 1990; Murakami et al., 19901, and others (Molleyres and Rando, 1988; Walker and Sando, 1988; Roman0 and Hawiger, 1990) have been reported to stimulate protein
kinase C. These factors are, in general, both lipid soluble and heat labile. In addition, two novel ether aminophosphoglycerides (modulators 1 and 2) which modulate the glucocorticoid-receptor complex are also reported to stimulate protein kinase C (Hsu et al., 1991). In contrast to the protein kinase C-stimulatory factor in heated ovarian cytosol, modulators 1 and 2 require the presence of all protein kinase C cofactors (calcium and lipids) for their stimulation of protein kinase C activity. Thus comparisons between the protein kinase C-stimulator-y factor from the rat ovary and previously reported stimulators indicate that the ovarian factor is different from those previously reported. Our laboratory has reported the presence of an endogenous inhibitor of protein kinase C in the pseudopregnant rat ovary (Eyster, 1990, 1991). The presence of endogenous inhibitors of protein kinase C in other tissues has also been reported (Schwantke and LePeuch, 1984; McDonald and Walsh, 1985; Huang and Oshana, 1986; Hucho et al., 1987; Pearson et al., 1990). The presence of both an endogenous stimulator and an endogenous inhibitor of protein kinase C in the pseudopregnant rat ovary suggests that these cells may be able to modulate the response of protein kinase C to extracellular activation in either a negative or a positive manner.
Acknowledgments This work was supported in part by NIH HD26640 and USDSM Parson’s Fund. The authors extend their appreciation to Dr. A.A. Hagen and Dr. M.R. Clark for their encouragement and suggestions about the manuscript.
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MOLCEL
125
86 (1992) 125-132 Ireland, Ltd. 030?-7207/92/$05.00
02784
Protein kinase C stimulatory activity in the pseudopregnant
rat ovary
KM. Eyster, M.S. Waller and M.J. Johnson Department
of Physiology and
Pharmacology, Uni~~er.s@ofSouth Dakota. Vermillion, SD, USA
(Received
Key words: Protein
kinase
C; Ovary;
Stimulation;
13 March
1992; accepted
3 April 1992)
Modulator
Summary Ovarian cytosol from pseudopregnant rats was heated to SO-90°C for 2 min and precipitated proteins removed by centrifugation. The supernatant of the heated ovarian cytosol contained no protein kinase C activity but when added to a control preparation containing protein kinase C, enzyme activity was increased to 200% of control. The stimulatory activity was stable to heating for 10 min, was retained on a centrifugal filtration device with a 100,000 M, cut-off, did not affect CAMP-dependent protein kinase, was not extractable in petroleum ether or chloroform/methanol (2: 11, and enhanced the phosphorylation of protein kinase C-specific peptide substrates. The stimulatory factor was calcium-dependent and could substitute for phosphatidylserine and diacylglycerol in the protein kinase C assay. This stimulatory factor may provide a mechanism whereby the response of protein kinase C to hormonal activation could be regulated by the cell.
Introduction Protein kinase C, the calcium- and lipid-dependent protein kinase, is an important intracellular messenger in diverse cellular activities from mediating the mechanism of hormone action to cell division (Macara, 1985; Nishizuka, 1986; Olashaw and Pledger, 19881, and its regulation in various tissues is equally diverse and complicated. The multiple isoforms of protein kinase C include calcium-dependent and calcium-independent forms (One et al., 1988) and exhibit differential sensitivity to activation by phospholipids and diacylglycerol (One et al., 1988). In addition, there is
Correspondence to: Kathleen M. Eyster, Department of Physiology and Pharmacology, University of South Dakota School of Medicine, Vermillion, SD 57069, USA. Tel. (605) 677-5159: Fax (605) 677-5124.
differential distribution of the various isoforms across tissue types (Knopf et al., 1986; Ono et al., 1988). Regulation of protein kinase C is further complicated by the presence of endogenous inhibitors (Eyster, 1990; Pearson et al., 1990) and stimulators (Hsu et al., 1991) of the enzyme in some tissues. Although these many regulatory mechanisms have been documented, their physiological roles have yet to be well-defined. The regulation of ovarian function involves the complex interaction of multiple endocrine and paracrine factors, many of which utilize second messenger pathways such as the protein kinase C pathway to convey their messages to intracellular actions. The pseudopregnant rat model has often been used for studies of the mechanism of hormone action in the ovary. Prostaglandin F,, (Leung et al., 1986) and gonadotropin releasing hormone (Leung et al., 1983) stimulate phospho-
126
lipase C-induced phosphatidylinositol turnover, the transduction pathway which activates protein kinase C, in the pseudopregnant rat ovary, and this pathway is hormonally activated in other ovarian cell types as well (Davis et al., 1986; Ranta et al., 1986). We have used the pseudopregnant rat model for studies of protein kinase C activity (Eyster, 1990, 1991). In the course of our work we found that heat treatment of pseudopregnant rat ovarian cytosol resulted in protein kinase C-stimulatory activity. The current studies were undertaken to examine this protein kinase C-stimulatory factor. Methods
and materials
Pseudopregnancy was induced in immature female rats by injection of pregnant mares’ serum gonadotropin and chorionic gonadotropin (Eyster, 1990). The ovaries were removed on day 7 of pseudopregnancy and stored at -70°C. Cytosol was prepared as described (Eyster and Clark, 1989). Briefly, the ovaries were homogenized with a Polytron homogenizer in a 20 mM Tris-HCI buffer, pH 7.4, with 2 mM EDTA, 5 mM EGTA, 0.25 M sucrose, and 50 mM 2-mercaptoethanol at a dilution of 1 : 5 (w/v). The tissues were homogenized for 30 s at full speed, allowed to cool for 1 min, then homogenized for a second 30 s period. The homogenate was centrifuged for 600,000 X g min and the resulting supernatant was saved as the cytosolic preparation. Protein kinase C activity was measured by the transfer of “P from [Y-~~P]ATP to histone III-S as described (Eyster and Clark, 1989). The assay mixture contained 20 mM Tris-HCI, pH 7.4, 5 mM magnesium acetate, 10 PM ATP (with lo6 cpm [Y-‘~P]ATP), 0.2 mg/ml histone III-S, 20 mM 2-mercaptoethanol, 100 mM sucrose, 0.8 mM EDTA and 2.0 mM EGTA. Enzyme activity was measured in the absence and presence of 2.9 mM calcium (approximately 0.1 mM free calcium), 0.8 Fg/ml 1,2-dioleoyl rat-glycerol, and 20 pg/ml phosphatidylserine (standard lipid concentration). Incubations were initiated with the addition of the tissue sample and were carried out for 3 min at 30°C. The reaction was stopped by addition of 1 ml ice-cold 25% trichloroacetic acid. Precipitated proteins were collected by filtration on ni-
trocellulose membranes. Enzyme activity was linear with respect to time and tissue concentrations as previously reported (Clark et al., 1983). Protein kinase activity in the absence of calcium and lipid was subtracted from protein kinase activity in the presence of calcium and lipid to yield specific protein kinase C activity. For the heat treatment, ovarian cytosol was heated to X0-90°C for 2 min and precipitated proteins removed by centrifugation for 5 min at 16,000 X g. The supernatant was mixed with a control preparation of protein kinase C and preincubated for 5 min at 30°C before beginning the phosphorylation assay. The control preparation consisted of rat brain cytosol which had been partially purified on DEAE in a mini-batchwise test tube technique (Eyster, 1990) analogous to conventional column purification with DEAE. The phosphorylation of protein kinase Cspecific peptide substrates (Yasuda et al., 1990) was examined by substituting the various substrates for the histone III-S. The peptides tested were a synthetic peptide derived from myelin basic protein consisting of amino acids 4-14 with the N-terminal acetylated (AC-MBP+,,,), a peptide derived from the pseudosubstrate region of the protein kinase C molecule in which the 25 position has been replaced with a serine so that the peptide can serve as a substrate for protein kinase C ([Ser”]PKC(,,~,,,), and a peptide derived from glycogen synthase ([Ala”,‘~‘,Lys”,‘21were obtained from GS (,_,2). Peptide substrates Life Technologies (Gaithersburg, MD, USA). The final concentration of peptide substrates in the assay was 51 PM. In assays in which the peptide substrates were substituted for histone III-S, the samples were spotted on 4 X 4 cm squares of P-81 paper (Whatman) after the incubation. The papers were placed in 0.1 M H,PO, and washed 3 times for 5 min each in a bath of 0.1 M H,PO,, then counted in scintillation fluid (Yasuda et al., 1990). For assays involving cyclic AMP-dependent protein kinase, the enzyme was partially purified from rat ovarian cytosol using a similar DEAE test tube technique to that used for protein kinase C, except the CAMP-dependent protein kinase was eluted from the DEAE with 0.5 M NaCI. Cyclic AMP-dependent protein kinase ac-
tivity was measured as described (Davis and Clark, 1983). For all enzyme assays, data are expressed as picomoles of 32P transferred per min (pmol/min). Except where mentioned, chemicals and materials were obtained as previously described (Eyster, 1990). Results When pseudopregnant rat ovarian cytosol was heated to 80-90°C for 2 min all protein kinase C activity was destroyed (0 pmol/min). When the heat-treated ovarian cytosol was mixed with the control preparation of protein kinase C from rat brain, enzyme activity was substantially increased (control 11.7 pmol/min vs. control + heated ovarian cytosol 22.4 pmol/min). The stimulatory factor was relatively heat stable; there was no change in stimulation in ovarian cytosol heated to 8090°C for 2, 5, or 10 min or boiled for 10 min (Fig. 1). Protein kinase C activity was directly correlated with the volume of heat-treated ovarian cytosol added (Fig. 2). When heated cytosol was separated on a centrifugal filtration device with a 100,000 M, cut-off (CentriCon 100; Amicon Division of WR Grace and Co., Beverly, MA, USA), the stimulatory activity was located in the reten-
O/A 0
10
Volume
20
30
Heated
40
50
Cytosol
60 (pi)
Fig. 1. Protein kinase C activity was measured in the control preparation (CTL, open bar), and after mixing the control preparation with ovarian cytosol that had been heated for 2, 5, or 10 min, or boiled for 10 min (B-10). The values expressed are the mean&SE of four separate measurements. The asterisk denotes enzyme activities significantly different from control.
0 CTL Lerlqth
of
10’
2
5
Yeat
Treatment
B-IO (min)
Fig. 2. Protein kinase C activity was measured in the absence (0) and presence of increasing volumes (~1) of ovarian cytosol heated to SO-90°C for 2 min. The standard volume of heated ovarian cytosol used was 28 ~1. The values shown are the mean f SE of three separate measurements.
tate (Control (Ctl) 14.4 pmol/min, Ctl + retentate 36.7 pmol/min, Ctl + filtrate 14.1 pmol/min). The stimulatory activity was not extractable in petroleum ether (Ctl 10.4 pmol/min, Ctl + heated ovarian cytosol 20.2 pmol/min; Ctl + extract 9.4 pmol/min, Ctl + aqueous phase 25.3 pmol/min), chloroform/methanol (2 : 1; Ctl 10.4 pmol/min; Ctl + extract 13.3 pmol/min, Ctl + aqueous phase 18.0 pmol/min), or acidified chloroform/ methanol (2: 1, pH 1.0; Ctl 10.4 pmol/min; Ctl + extract 12.4 pmol/min, Ctl + aqueous phase 17.4 pmol/min). When heated ovarian cytosol was mixed with partially purified CAMP-dependent protein kinase, enzyme activity fell to 5.8 pmol/min from a control of 7.9 pmol/min. The heated ovarian cytosol enhanced the phosphorylation of protein kinase C-specific substrates and (Ala’,“‘, AC-MBP+,,,, [Ser2’lPKC(,,_,,,, by 166%, 201% and 166%, reLYS”,‘~IGS+,,, spectively (Fig. 3). The calcium, lipids (phosphatidylserine and 1,2-dioleoyl rat-glycerol), and histone substrate were, each in turn, removed from the reaction mixture and replaced with the heated ovarian cytosol. Removal of the histone substrate from the reaction mixture reduced measurable protein kinase C activity to 8% of control enzyme activity. Substitution of heated ovarian cytosol for the histone resulted in an increase of protein kinase C activity to 16% of control. Removal of lipids
.;
25
\ F
20
a ;
15
> 2 0 y CL
10 5 0 Histone
MBP
PKC
GS
Fig. 3. Protein kinase C activity was measured in the absence (crosshatched bars) and presence (solid bars) of heated ovarian cytosol with different substrates for enzyme phosphorylation. The substrates were histone 111-S (the standard substrate), a peptide derived from myelin basic protein, Ac(MBP), a modified peptide derived from the pseuMBP+,,, dosubstrate region of protein kinase C, [Ser’“]PKC~,,,_,,, (PKC). and a modified peptide derived from glycogen synthase, [Ala’~~“‘,Lys”~‘~]GS,,~,~, (GS). The values are expressed as the mean* SE of three separate measurements. Enzyme activities in the absence vs. the presence of heated ovarian cytosol that are significantly different are designated with an asterisk.
(phosphatidylserine and 1,2-dioleoyl rat-glycerol) from the reaction mixture resulted in reduction of protein kinase C activity to 6% of control. When heated ovarian cytosol was substituted for lipids, enzyme activity increased to 110% of control. Protein kinase C activity was then measured in the presence of decreasing concentrations of lipids, in the absence and the presence of a constant volume of the heated ovarian cytosol and at 100% (100 PM) of the standard calcium concentration (Fig. 4). In the absence of the heated ovarian cytosol, protein kinase C activity decreased as the concentration of lipids decreased from the standard concentration of lipid (20 pg/ml phosphatidylserine and 0.8 pg/ml 1,2-dioleoyl rat-glycerol) to zero lipid. In the presence of heated ovarian cytosol, protein kinase C activity remained constant as the concentration of lipids decreased. Removal of all calcium from the reaction mixture did not result in loss of protein kinase C activity (105% of control). However, there was no effect of the heated ovarian cytosol on protein
kinase C activity in the absence of calcium (108% of control). Protein kinase C activity was measured in the presence of decreasing concentrations of calcium, in the absence and presence of a constant volume of heated ovarian cytosol and at 100% of the standard lipid concentration (Fig. 5). As the concentration of calcium decreased from 100 PM to 0 PM, the activity of protein kinase C decreased at 50 PM to 25 PM, then returned to control levels at 12.5 PM and 0 PM calcium. In the presence of the heated ovarian cytosol, increases in protein kinase C activity above control occurred at calcium concentrations between 65 PM and 100 PM, but not at calcium concentrations of 50 PM or less (Fig. 5). The requirement for 65 PM or greater calcium to effect stimulatory activity of the heated ovarian cytosol per-’ sisted at all concentrations of lipid tested (Fig. 6, A-F). In the absence of the stimulators of pro-
I
Fig. 4. Protein kinase C activity was measured in the absence (closed circles) and presence (open bars) of heated ovarian cytosol, both in the presence of increasing concentrations of lipids (phosphatidylserine and IJdioleoyl rat-glycerol). The concentration of lipids increased from 0 to 100% of the standard concentration used in the protein kinase C assay (see Methods and materials). Assays were performed in 100 PM calcium. Values are expressed as the mean k SE of five separate measurements. Protein kinase C activities in the presence vs. the absence of heated ovarian cytosol that are significantly different are designated by an asterisk (p < 0.05, paired Student’s I-test).
I29
24
20
16
12
8
4
0 12.5
25
Fig. 5. Protein kinase C activity was measured in the absence (closed circles) and presence (open bars) of heated ovarian cytosol, both in the presence of increasing concentrations of calcium from 0 to 100 FM (the standard concentration of calcium in the assay). Values are the mean+SE of three separate determinations. Protein kinase C activities in the absence vs. the presence of heated ovarian cytosol which are significantly different are designated by an asterisk (p < 0.05, paired Student’s r-test).
tein kinase C, calcium and lipids, there was no effect of heated ovarian cytosol on protein phosphorylation (Fig. 6A ). Discussion Heat treatment of pseudopregnant rat ovarian cytosol resulted in the manifestation of a factor with protein kinase C-stimulatory activity. There was no stimulation of CAMP-dependent protein kinase activity, suggesting that the stimulation did not occur at a site common to all kinases such as the ATP-binding site. The enhancement of phosphorylation of protein kinase C-specific peptides by the stimulatory factor, and the absence of stimulation of basal phosphorylation (phosphorylation in the absence of calcium and lipids) corroborate the specificity of the stimulatory factor for protein kinase C. Retention of the factor on a 100,000 M, cut-off filter suggested that it had a M, greater than 100,000 or that it formed an
aggregate. The factor was also heat stable. Although the standard heat treatment was 2 min at 80-9072, boiling for 10 min did not produce a significant decrease in stimulatory activity. Nonpolar molecules are extracted from aqueous solution by organic solvents; however, the stimulatory activity was located in the aqueous phase after extraction by petroleum ether, chloroform/methanol, and acidified chloroform/ methanol. These data suggest that the stimulatory factor is a polar, heat-stable molecule (or aggregate) with a M, greater than 100,000. It was clearly not a supersubstrate for protein kinase C, as it did not substitute for histone in the assay. Not only did the factor maintain protein kinase C activity in the absence of lipids (phosphatidylserine and diacylglycerol), enzyme activity was greater than that stimulated by saturating concentrations of lipids. Whether the activation by the stimulator-y factor occurred through binding at one of the lipid-binding sites on protein kinase C, or at another site, remains undetermined. However, the factor clearly does not synergize with the lipids, as protein kinase C activity remained constant in the presence of the stimulatory factor regardless of the concentration of phosphatidylserine and 1,2-dioleoyl rat-glycerol in the reaction mixture. Although the lipids known to stimulate protein kinase C have a relatively low molecular weight, lipids form micelles which may have a relatively high apparent M,. Micellar formation, in addition to aggregation, could explain the apparent high M, of the stimulatory factor. However, lipids and micelles would have been extracted in the solvents used (i.e., chloroform/methanol) but the stimulatory factor was not extracted; rather, the stimulatory activity remained in the water-soluble phase of the extracted samples. Therefore, although the stimulatory factor substituted for lipids in the stimulation of protein kinase C, the extraction data do not indicate that it has a lipid nature. The rat brain contains both calcium-dependent and calcium-independent protein kinases C as described in the literature (One et al., 1988) and as indicated by the biphasic nature of protein kinase C activity in the absence of the stimulatory factor in Fig. 4. The activity of the stimulatory factor was clearly calcium-dependent. This can be
I30
interpreted as a direct calcium dependence of the stimulator-y factor, or an insensitivity of the calcium-independent protein kinases C to the stimulatory factor. Since the loss of stimulation of protein kinase C by the factor and the increase in protein kinase C activity that signifies the calcium-independent protein kinases C occur at dif-
ferent concentrations of calcium (65 PM vs. O12.5 FM), it is likely that the first interpretation is correct. If so, then the activation constant of the stimulatory factor for calcium is 65 PM in the in vitro assay. If the stimulatory factor serves a physiological role in regulation of protein kinase C. then one would expect that the stimulatory
3.
12.5
0 F
Fig. 6. Protein kinase C activity was measured A-F.
was performed
measurements.
Significant
at a different differences
50
25 yM
Cati
Co
75
100 *
+7
-I-
in the absence (closed circles) and presence (open bars) of heated ovarian cytosol.
both in the presence of increasing concentrations parts
80
pM
of lipids from 0 to 100% of the standard
concentration
of calcium.
as designated.
Values
assay lipid concentration. are the mean+SE
Each a~rve,
of three separate
in enzyme activities in the absence vs. the presence of heated ovarian cytosol are designated by an asterisk ( p < 0.05. Student’s
r-test).
131
factor would also be regulated. The calcium dependence of the stimulator-y factor presents a physiological mechanism for regulation of the stimulatory factor. The physiological role of the stimulatory factor may be to enhance the ability of protein kinase C to respond to subsequent stimuli after an initial stimulus in specific, regulated conditions. Such a response has been documented in adrenal glomerulosa cells. In these cells there is a timedependent potentiation of protein kinase C activation after previous stimulation of the enzyme (Bollag et al., 1991); perhaps the stimulatory factor is involved in similar ‘cell memory’ responses in the rat ovary. Alternatively, the physiological importance of this factor may not be that it increases protein kinase C activity to greater levels than phosphatidylserine, diacylglycerol, and calcium, but that it is a water-soluble factor which can maintain protein kinase C activity in the absence of membrane lipids. Although it has become the accepted dogma that activation of protein kinase C by diacylglycerol results in translocation of the enzyme from the cytosol to the plasma membrane (Olashaw and Pledger, 19881, we know that protein kinase C phosphorylates cytoplasmic and cytoskeletal proteins (Nishizuka, 1986; Graff et al., 1989) as well as DNA transcription factors in the nucleus (Hoeffler and Habener, 1990). Perhaps the role of the ovarian protein kinase C-stimulatory factor is to allow protein kinase C to be active in the cytosol or in the nuclei of cells under specific conditions. The manifestation of stimulatory activity only after heat treatment in the rat ovary may be due to association of the factor with a heat-sensitive regulatory protein, or to association of the factor with nonspecific proteins during homogenization of the tissue. There have been reports of other endogenous molecules which stimulate protein kinase C activity. Endogenous lipids such as polyphosphoinositides (O’Brian et al., 1987; Chauhan et al., 19X9>, unsaturated fatty acids, i.e., oleic and arachidonic acids (McPhail et al., 1984; Murakami and Routtenberg, 198.5; El Touny et al., 1990; Murakami et al., 19901, and others (Molleyres and Rando, 1988; Walker and Sando, 1988; Roman0 and Hawiger, 1990) have been reported to stimulate protein
kinase C. These factors are, in general, both lipid soluble and heat labile. In addition, two novel ether aminophosphoglycerides (modulators 1 and 2) which modulate the glucocorticoid-receptor complex are also reported to stimulate protein kinase C (Hsu et al., 1991). In contrast to the protein kinase C-stimulatory factor in heated ovarian cytosol, modulators 1 and 2 require the presence of all protein kinase C cofactors (calcium and lipids) for their stimulation of protein kinase C activity. Thus comparisons between the protein kinase C-stimulator-y factor from the rat ovary and previously reported stimulators indicate that the ovarian factor is different from those previously reported. Our laboratory has reported the presence of an endogenous inhibitor of protein kinase C in the pseudopregnant rat ovary (Eyster, 1990, 1991). The presence of endogenous inhibitors of protein kinase C in other tissues has also been reported (Schwantke and LePeuch, 1984; McDonald and Walsh, 1985; Huang and Oshana, 1986; Hucho et al., 1987; Pearson et al., 1990). The presence of both an endogenous stimulator and an endogenous inhibitor of protein kinase C in the pseudopregnant rat ovary suggests that these cells may be able to modulate the response of protein kinase C to extracellular activation in either a negative or a positive manner.
Acknowledgments This work was supported in part by NIH HD26640 and USDSM Parson’s Fund. The authors extend their appreciation to Dr. A.A. Hagen and Dr. M.R. Clark for their encouragement and suggestions about the manuscript.
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