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Phospholipid-dependent Ca2+-activated protein kinase (C-kinase) in the pituitary: Further characterization and endogenous redistribution
Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland,
201
47 (1986) 201-208 Ltd.
MCE 01529
Phospholipid-dependent Ca2+- activated protein kinase (C-kinase) in the pituitary: further characterization and endogenous redistribution Jacob Hermon Departments
‘,*, Anat Azrad ‘, Nachum
of ’ Hormone Research and .’ Chemical Immunology, (Received
Key words: protein
kinase C; diacylglycerol;
phorbol
Reiss ’ and Zvi Naor ’
The Weirmann
4 April 1986; accepted
Institute of Scrence, Rehooot 76100 (Israel)
6 June 1986)
ester.
Summary Phospholipid-dependent, Ca*+ -activated protein kinase (C-kinase) was recently shown to be expressed in rat pituitary. The enzyme is activated by Ca2+ and phosphatidylserine (PS). Diacylglycerol (DG), which is liberated during phosphoinositide turnover, and the potent tumor promoter 12-O-tretadecanoyl-phorbol13-acetate (TPA) activate pituitary C-kinase in the presence of PS, even at resting levels of intracellular concentraCa2+ (lo-’ M), and increase the apparent affinity of the enzyme for Ca *+ . While micromolar tion of Ca*+ had no effect on the apparent affinity of the enzyme for PS (K, - 15 pg/ml), elevation of Ca2+ to the millimolar range produced a sharp increase in the apparent affinity for PS (K, - 5 pg/ml). Elevation of PS (up to 500 pgg/ml) could not replace Ca 2+ in supporting maximal enzyme activity even in the presence of DG. Cytosolic pituitary C-kinase (70% of total enzyme activity) is recovered in an inactive state and can be activated without further purification. The particulate enzyme (30%) is recovered in a cofactors-insensitive form but can be activated after detergent-solubilization and anion exchange chromatography. Endogenous redistribution of soluble pituitary C-kinase to the membrane does not convert it here to its proteolytic product which is insensitive to Ca *+ , PS and DG. Pituitary C-kinase characterized most likely plays a key role in signal transduction mechanisms involved in pituitary functions.
Introduction The hypothalamic hormones stimulate pituitary function via the formation of ‘second messengers’ (for review see Gershengorn, 1982; Naor, 1982). While cyclic AMP might be a major mediator of
* In partial fulfillment of the requirements for a Ph.D. degree of the Feinberg Graduate School of the Weizmann Institute of Science. Address correspondence and reprint requests to: Dr. Zvi Naor, Department of Hormone Research, The Weizmann Institute of Science, Rehovot, 76100, Israel. 0303-7207/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
corticotropin releasing factor (CRF) and growth hormone releasing factor (GHRF) action (Brazeau et al., 1982; Giguere et al., 1982; Bilezikjian and Vale, 1983; Schettini et al., 1984), enhanced phosphoinositide turnover was implicated in gonadotropin releasing hormone ‘(GnRH) and thyrotropin releasing hormone (TRH) action (Naor and Catt, 1981; Gershengorn, 1982; Naor, 1982; Snyder and Bleasdale, 1982; Martin, 1983; Rebecchi and Gershengorn, 1983; Naor et al., 1985a). The Ca*+/phospholipid-dependent protein kinase (C-kinase) (Takai et al., 1979) is activated by DG which is released during phosphoinositide turnover Ltd
202
(Nishizuka, 1984). Activation of pituitary C-kinase might therefore represent a key step in signal transduction mechanisms involved in pituitary function. Preliminary studies described the presence of C-kinase in the pituitary (Turgeon et al., 1984) and modulation of its activity by GnRH (Hirota et al., 1985; Naor et al., 1985b) and TRH (Drust and Martin, 1985; Fearon and Tashjian, 1985). Nevertheless, no detailed characterization of the pituitary enzyme has yet been performed. We therefore decided to further characterize the enzyme in the pituitary. Materials and methods Materials Phosphatidylserine (PS, bovine brain), lysinerich calf thymus histone (type III-s), 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 1,2-diolein were from Sigma. [y- 32P]ATP ( > 5000 Ci/mmol) was purchased from Amersham. Methods Enzyme preparation. Anterior pituitaries from adult Wistar-derived female rats from the departmental colony, collected in ice, were homogenized in ice-cold 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 50 mM 2-mercaptoethanol (buffer A). After centrifugation at 4°C for 10 min at 100000 X g (Beckman Airfuge, 30 psi), the supernatant was collected as the source of crude soluble C-kinase. The pellet was washed, solubilized in buffer A containing 0.3% Triton X-100 for 1 h at 4°C and centrifuged as above for 10 min. The second supernatant was used as the crude particulate enzyme preparation. For chromatography, pituitaries were homogenized in buffer A containing 20% sucrose (buffer B). After centrifugation at 650 X g for 10 min, the soluble and particulate preparations were obtained as above and passed through a DEAE-cellulose (DE-52) column (8.5 X 3 cm) equilibrated with buffer B. The column was first washed with 15 ml of buffer B and C-kinase was eluted with a 30 ml linear concentration gradient of NaCl (O-O.4 M) in buffer B. Protein was determined by the Bradford method (1976). Enzyme assay. C-kinase was assayed by measuring the incorporation of 32P from [y- 32P]ATP into calf thymus histone (type III-s), essentially as
described by Takai et al. (1979). The reaction mixture (0.150 ml) contained 5 pmol of Tris-HCI pH 7.5, 1.25 pmol of MgCl,, 50 pg histone, 2.5 nmol of [y-‘3P]ATP (5-10 x lo4 cpm/nmol), 50 pg/ml phosphatidylserine (PS), 3.2 pg/ml diolein (DG), lop5 or lo-’ M of CaCl, for crude and purified enzyme preparation respectively, unless otherwise indicated, and enzyme preparation as indicated. Occasionally, PS and DG concentrations were varied in order to obtain optimal conditions for C-kinase activation by DG. PS and DG were first dissolved in chloroform, dried under nitrogen, and resuspended in 20 mM Tris-HCl pH 7.5 by sonication for 10 min at 4’C. The assay was carried out for 3 min at 30°C and aliquots of 100 ~1 were transferred to 2.5 cm squares of phosphocellulose paper. The papers were washed several times in a large volume of ice-cold TCA (lo%), followed by wash in ethanol and ether. The papers were then dried and the radioactivity was measured by liquid scintillation spectrometry. Results Presence of C-kinase in rat pituitary Table 1 shows C-kinase activity in rat pituitary, cerebral cortex and ovarian preparations. Basal activity ( - Ca’+) was markedly enhanced by Ca’+, in the crude cytosolic, but not in the crude particulate preparations of the tissues examined. In the presence of Ca’+, C-kinase activity was markedly stimulated by addition of PS + DG in all the cytosolic, but not in all the crude particulate preparations examined. In the ovary, Ckinase activity was mostly recovered from the particulate fraction (60%), while in cerebral cortex most of the C-kinase activity is soluble (80%). In both ovary and cerebral cortex we could demonstrate C-kinase activity in crude detergent-solubilized particulate preparations. However, no PSsensitive protein kinase activity could be observed in the crude pituitary particulate fraction before or after detergent solubilization. The presence of Triton X-100 in pituitary particulate extracts is not responsible for the lack of C-kinase activity, since the detergent causes just a slight inhibition when added to soluble C-kinase (Table l), and permits the activation of brain and ovarian particulate C-kinase. The presence of protease and
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TABLE
1
DISTRIBUTION OF CEREBRAL CORTEX
C-KINASE IN AND OVARIAN
RAT PITUITARY, PREPARATIONS
Crude cytosol and solubilized particulate fraction (25-50 pg protein) were assayed for C-kinase activity. Pituitaries and cortex were obtained from adult female rats, while ovaries were taken from 27-day-old female rats treated with 15 IU of pregnant mare serum gonadotropin on day 25 to induce follicular growth. For DEAE-cellulose (DE-52) chromatography crude supernatant and detergent-solubilized particulate fractions were passed through a DE-52 column. The column was washed with 15 ml of column buffer and C-kinase was eluted with a 30 ml linear gradient of NaCl (O-O.4 M). Samples ( - 4 pg protein) taken from the peak of C-kinase activity were used for the assay. Additions were Ca2+ 10 pM for crude and 1 mM for partial purified enzyme preparation, PS 50 pg/ml and DG 3.2 Pg/ml. The values are the mean + SEM of 2-4 experiments each done in duplicate. Sample
Pituitary Cytosol Particulate Particulate a Cytosol b Cytosol c Particulate ’ Cerebral cortex Cytosol Particulate Ovary Cytosol Particulate Pituitary (DE-52) Cytosol Particulate
Protein kinase C activity (pm01 ” P/mg protein/min) -Ca*+
+Ca*+
- PS, DG
- PS, DG
395 5 110+11 N.D. d 17+ 2 39& 4 117+10
146k12 93* 7 25+ 2 136+ 8 118* 10 78k 8
386 f 31 88*10 27+ 3 287 f 30 357 * 25 80* 7
176*20 462 + 50
398 + 35 458 * 50
739*50 578 + 40
N.D. N.D.
125+15 186+18
271 f 30 303 f 35
300 150
328 156
+ PS, DG
(Table 1 and data not shown). However, when the cytosol and the solubilized particulate fractions were chromatographed on DEAE-cellulose, a similar major peak of C-kinase activity, that was separated from other protein kinases, was eluted at about 0.16 M NaCl (Table 1). DE-52 chromatography separated the Ca’+-dependent kinase from C-kinase in the cytosolic preparation, and unmasked the particulate C-kinase (Table 1). Soluble and particulate pituitary C-kinase constitute 70 and 30% of the total enzyme activity respectively. Kinetic properties Reaction velocity was linear for the first 3 min of incubation at 30°C and this time point was chosen for all experiments (Fig. 1). Reaction velocity was also linear with protein concentration in the range examined and routinely used (- 4 pg) (Fig. 2). Pituitary C-kinase is activated by PS and an unsaturated diacylglycerol (DG) such as 1,2-diolein (Fig. 3). The enzyme is dependent on the simultaneous presence of Ca2+ and PS (Fig. 3A), whereas Ca2+ and DG alone are not sufficient for enzyme activation (Fig. 3B). PS increased enzyme activity in the presence of Ca2+ and DG increased the affinity for PS, and also increased slightly maximal enzyme activity (Fig. 3A). A dose-response curve for DG activation of C-kinase could
1052 772
’ Without Triton X-100 solubilization. b Cytosolic fraction was incubated with Ttiton X-100,0.3% for 1 h at 4OC before the assay of C-kinase. ’ Soluble and particulate enzyme were prepared, solubilized and assayed in the presence of the protease and phosphatase inhibitors: leupeptin 0.01% phenylmethylsulfonyl fluoride (PMSF) 2 mM, and NaF 10 mM. d Not determined.
gp*,,
,
2
4 Time
phosphatase inhibitors such as leupeptin, phenylmethylsulfonyl fluoride (PMSF) and NaF, or the Ca’+-chelator EGTA, during enzyme preparation and assay are not sufficient to demonstrate Ckinase activity in the crude particulate fraction
,
6
/
8
,I IO
(mtn)
Fig. 1. Time-course of C-kinase activation. Crude cytosolic enzyme preparation (20 pg protein) was incubated in the presence (open circles) or absence (closed circles) of 2 mM Ca2+, 24 pg phosphatidylserine (PS), 0.8 pg diolein (DG). C-kinase activity was measured as described in Methods.
204
PS+DG
J
OLb CoC12(log M) Protein (pg) Fig. 2. C-kinase activation as function of protein concentration. Crude soluble enzyme preparation was fractionated on DE-52 column as described in Methods. The respective peak of C-kinase was used for the assay. The assay was carried out for 3 min at 30°C in the presence of Ca2+ alone (1 mM, triangles) or in the presence of Ca2+ (1 mM), PS (50 pg/ml) and DG (3.2 pg/ml) (squares). Each point is the mean of two samples, each done in duplicate, and similar results were observed in two other experiments.
be demonstrated only in the presence of both Ca2+ and PS (Fig. 3B). As shown in Fig. 4, increasing concentrations of Ca2+ alone, or with DG, had only a small effect on enzyme activity; however, in the presence of PS, an increase in
Fig. 3. Effect of phosphatidylserine (PS) and diolein (DG) on C-kinase activity. Activity was determined in crude cytosolic enzyme preparation in (A) the presence of various concentrations of PS and Ca 2+ (10 PM, squares), or Ca (10 PM) and diolein (1.6 pg/ml, triangles), or (B) at various concentrations of diolein and Ca’ + (1 mM, squares), or Ca*+ (1 mM) and PS (12 pg/ml, triangles).
Fig. 4. Effect of diolein (DC) on pituitary C-kinase. Partial purified C-kinase (4 pg/tube) was used for the assay and various concentrations of Ca2+ were added. Ca2+ alone (open circles), with diolein (DC, 3.2 ag/ml, open triangles). with PS (12 pg/ml, filled squares). or with PS+ DG (filled triangles). Each point is the mean of two samples each done in duplicate. and similar results were obtained in two other experiments.
enzyme activity was observed. The presence of DG, in addition to PS, reduced the apparent K, for Ca2+ from 4 I_IM to 1.6 PM with little effect on maximal reaction velocity at saturating Ca2+ concentrations (Fig. 4). Enzyme activity was enhanced even at lo-’ M of Ca2+, only when DG was present in the reaction with PS. The tumor promoters phorbol esters exert pleiotropic effects on a variety of cells and recent evidence suggests that C-kinase is a major initial cellular binding site and transducer of their actions (Niedel et al., 1983; Nishizuka, 1984; Anderson et al., 1985). Since phorbol esters were reported to stimulate pituitary hormone release (Naor and Catt, 1981; Smith and Vale, 1981; Naor and Eli, 1985) it was of great interest to examine whether they activate pituitary C-kinase. Indeed, the phorbol ester, TPA, could replace DG in pituitary C-kinase activation (Fig. 5). In the presence of Ca2+, TPA on its own was a weak stimulant of enzyme activity. However, in the presence of Ca2+ and PS, TPA markedly increased enzyme activity in particular at low Ca2+ concentrations. Note that at lo-’ M of Ca2+, which is near the resting level of intracellular Ca2+ in the pituitary as monitored with Quin-2 fluorescence (- 150 nM, data not shown), C-kinase is activated only when DG or TPA are present (Figs. 4 and 5).
205
I
I 1
I
-1
5
I
I
-6
-5 CoCIz
-4
(IogM)
Fig. 5. Effect of the tumor promoter phorbol ester, TPA, on pituitary C-kinase activity. Partial purified enzyme (4 pg/tube) was used for the assay. Various concentrations of Ca2+ were added with TPA (20 ng/ml, circles), or with PS (12 pg/ml, squares) or both (triangles). Each point is the mean of two samples, each done in duplicate, and similar results were obtained at least in two other experiments.
We then investigated whether elevated PS levels (> 100 pg/ml) could replace Ca2+ in activating pituitary C-kinase. Elevation of PS (100-500
Oo+--o_I I /[PSI Fig. 6. Kinetic analysis of C-kinase activation by PS at various Ca2+ concentrations. Partial purified enzyme (4 pg/tube) was used for the assay in the absence of DC. The data was analyzed as a reciprocal plot of l/V - V. against l/PS, where V = activity at a given PS concentration and V, = activity for PS = 0. The apparent K, values derived from the curves are 16.6, 14.2, 6.25 and 4.5 pg/ml of PS at Ca’+ concentrations of 10e6, 10m5, 10m4 and 1O-3 M, respectively.
pg/ml) in the presence of DG and the absence of Ca2+, was not sufficient for maximal enzyme activation. Furthermore, high concentrations of PS (> 100 pg/ml) were inhibitory to C-kinase activation at low Ca2+ concentrations (lop7 to 10V5 M) in the presence or absence of DG (data not shown). We also investigated whether elevated Ca2+ levels could replace DG in increasing the affinity of the enzyme for PS (Fig. 6). At the micromolar range, elevation of Ca” did not change the apparent affinity for PS (K, = 14.2 and 16.6 pg/ml at 10d5 and low6 M of Ca*+ respectively). Howrange ever, raising Ca *+ levels to the millimolar increased the apparent affinity to PS with no effect on maximal velocity (K, = 4.5 and 6.25 pg/ml at 10-j and 10m4 M of Ca*+ respectively). Discussion Neurotransmitters, peptide hormones and growth factors activate cellular functions via the formation of ‘second messengers’. The major cyclic nucleotides (CAMP and cGMP) and Ca2+ activate a respective protein kinase which is responsible for phosphorylation of specific substrate proteins (Greengard, 1978). The newly discovered C-kinase (for review see Nishizuka, 1984) is activated by DG in the presence of Ca2+ and PS. Since DG is liberated during phospoinositide turnover, activation of C-kinase is a potential key step in the action of hypophysiotropic hormones which stimulate the inositol phospholipid cycle. Pituitary C-kinase is mostly soluble (70%) and partly particulate (30%), similar to the distribution of C-kinase in the cerebral cortex (Table 1 and Kuo et al., 1984). The soluble enzyme is recovered in an inactive state and can be activated by Ca2+, PS and DG even in the crude form (Table 1). Unlike the soluble enzyme, the crude pituitary particulate enzyme was not responsive to the addition of Ca’+, PS and DG. We ruled out the possibility that the presence of Triton X-100 in the crude preparation was responsible for lack of measurable activity by demonstrating that the detergent was not harmful to the pituitary soluble enzyme or to the particulate ovarian and brain enzymes (Table 1). Also, the presence of protease and phosphatase inhibitors, or chelating agents,
206
did not reveal C-kinase activity in crude pituitary particulate fractions. However, solubilization of the particulate fraction followed by anion exchange chromatography enabled us to demonstrate the particulate enzyme activity. It is possible therefore that the pituitary particulate fraction contains inhibitory activity to C-kinase which was removed during detergent solubilization and fractionation. Alternatively. it is possible that the particulate enzyme is already bound to PS and DG and is recovered in the active form. Chromatography will then remove the PS and DG and enable detection of its activity by the addition of the cofactors. The third possibility is that endogenous C-kinase was converted to ‘M-kinase’ (a proteolytic product of C-kinase which is fully active in the absence of Ca*+, PS and DG (Kishimoto et al., 1983; Naor et al., 1985b)), upon redistribution to the membrane (Tapley and Murray, 1984; Melloni et al., 1985). It was recently reported that TPA activates C-kinase by translocating the enzyme from the cytosol to the particulate fraction in parietal yolk sac cells (Kraft and Anderson, 1983), human erythrocytes (Palfrey and Waseem, 1985) and rabbit platelets (Uratsuji et al., 1985). Moreover, TPA-induced translocation of erythrocyte and platelets C-kinase to the membrane resulted in an activated form of the enzyme which was not dependent on exogenous Ca*+ and PS (Palfrey and Waseem, 1985; Uratsuji et al., 1985). Tapley and Murray (1984) demonstrated that TPA translocates human platelet C-kinase to the membrane and converts it to ‘M-kinase’. Similarly, Ca’+-induced binding of C-kinase to human neutrophil membranes results in its conversion to ‘M-kinase’ (Melloni et al., 1985). Thus, endogenous C-kinase can be translocated to the membrane upon hormonal stimulation (Drust and Martin, 1985; Fearon and Tashjian, 1985; Hirota et al., 1985; Naor et al., 1985b), or elevation in [Ca*+], (Melloni et al., 1985; Wolf et al., 1985) and might be destined to irreversible activation by limited proteolysis and formation of the active ‘M-kinase’ (Tapley and Murray, 1984; Melloni et al., 1985). Our results on the intracellular distribution of pituitary C-kinase suggest that the particulate form escaped limited proteolysis, since after detergent-solubilization and chromatography, the enzyme was further activated by Ca’+, PS
and DG, which is characteristic of C-kinase but not of ‘M-kinase’ (Kishimoto et al., 1983; Naor et al., 1985b). It is possible that under normal physiological conditions the [Ca*+], rise does not reach the concentration needed for limited proteolysis of C-kinase. Since the brain and ovarian particulate preparations revealed C-kinase activity in the crude form, it seems most likely that the pituitary particulate fraction contains inhibitory activity to Ckinase which was removed during detergent-solubilization and chromatography. In the pituitary, the potent tumor promoter TPA activates C-kinase (Fig. 5) and stimulates hormone release (Naor and Catt, 1981; Smith and Vale, 1981; Naor and Eli, 1985) suggesting that translocation of C-kinase might occur following hormone-receptor activation. Indeed, the early phosphoinositide turnover stimulated by TRH and GnRH (Snyder and Bleasdale, 1982; Martin, 1983; Rebecchi and Gershengorn, 1983; Naor et al., 1985a), is associated with rapid release of DG (Martin, 1983; Rebecchi and Gershengorn, 1983) and recruitment of C-kinase to the membrane (Drust and Martin, 1985; Fearon and Tashjian, 1985; Hirota et al., 1985; Naor et al., 1985b). The presence of DG increases the affinity of the enzyme for Ca*+ and PS, thus activation can occur in the face of resting levels of cellular Ca2+ (lo-’ M). Since DG is rapidly metabolized to phosphatidic acid via the PI cycle, or acted upon by diglyceride lipase, it was interesting to investigate whether the hormone-induced rise in cytoplasmic Ca2+ (Gershengorn and Thaw, 1983; Albert and Tashjian, 1984) may have implication on the enzyme affinity to PS. If elevated Ca2’ will increase the affinity to PS, as DG does, enzymatic activity could proceed even in the absence of DG. Our findings suggest that changes in the concentration of Ca2’ at near physiological range (0.1-10 PM) have no effect on the affinity to PS (K,, - 15 pg/ml). However, elevation of Ca’+ to the millimolar range (0.1-l mM) caused an increase in the affinity to PS (K,, - 5 pg/ml). We cannot rule out the possibility that high concentrations of Ca*+ will be generated at specific areas in the plasma membrane, and hence the observation that mM concentrations of Ca2+ affect the affinity of the enzyme to PS might have some implications. A similar line of arguments exists regarding the irre-
versible proteolytic activation of C-kinase by Ca*+-dependent neutral protease (Kishimoto et al., 1983). It was previously shown that Ca*+ induces aggregation of sonicated PS vesicles (Portis et al., 1979). Fusion of the vesicles occurs at a threshold of Ca*+ concentration of about 1 mM corresponding to binding of one Ca2+ per two PS molecules. It is possible that the increase in the affinity of C-kinase to PS by mM concentrations of Ca*+, observed in this study, results from the ability of Ca*+ to fuse PS vesicles. We therefore suggest that while DG is the rate-limiting step in the early activation of C-kinase, Ca*+ may become the rate-limiting step once DG is metabolized. Our results also suggest that the requirement for Ca*+ in pituitary C-kinase activation is absolute since elevation in PS concentrations even to supramaximal levels (500 pg/ml) could not support maximal C-kinase activity in the absence of Ca2+ and the presence of DG. This is interesting since recent reports have demonstrated that in some secretory cells a role for Ca2+ in exocytosis can be obviated (Di Virgilio et al., 1984; Barrowman et al., 1986). It is therefore possible that under certain conditions where exocytosis is fully activated independent of changes in [Ca*+],, the active species involved was ‘M-kinase’ which is not dependent on Ca *+ for its activation. Pituitary C-kinase characterized here might have a key regulatory role in signal transduction mechanisms in the pituitary, in particular in mechanisms involved in GnRH and TRH action which are known to activate phosphoinositide turnover, during which DG is generated. Acknowledgements We thank Mrs. T. Hannoch for excellent technical assistance, and Mrs. M. Kopelowitz for typing the manuscript. Supported by NIH grant HD16279 and by the United States Israel Binational Science Foundation. References Albert, P.R. and Tashjian, Jr., A.H. (1984) J. Biol. Chem. 259, 5827-5832. Anderson, W.B., Estival, A., Tapiovaara, H. and Gopalakishna, R. (1985) In: Advances in Cyclic Nucleotide and
Protein Phosphorylation Research, Vol. 19, Raven Press, New York, pp. 287-304. Barrowman, M.M., Cockcroft, S. and Gomperts, B.D. (1986) Nature 319, 5044507. Bilezikjian, L.M. and Vale, W.W. (1983) Endocrinology 113, 657-662. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Brazeau, P., Ling, N., Esch, F., Bohlen, P., Mougin, C. and Guillemin, R. (1982) Biochem. Biophys. Res. Commun. 109, 588-594. Di Virgilio, F., Lew, D.P. and Pozzan, T. (1984) Nature 310, 691-693. Drust, D.A. and Martin, T.F.J. (1985) Biochem. Biophys. Res. Commun. 128, 531-537. Fearon, C.W. and Tashjian, Jr., A.H. (1985) J. Biol. Chem. 260, 8366-8371. Gershengorn, M.C. (1982) Mol. Cell. Biochem. 45, 163-179. Gershengorn, M.C. and Thaw, C. (1983) Endocrinology 113. 1522-1524. Giguere, V., Labrie, F., Cote, J., Coy, D.H., Sueiras-Diaz, J. and Schally, A.V. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3466-3469. Greengard, P. (1978) Science 199, 146-152. Hirota, K., Hirota, T., Aguilera, G. and Catt, K.J. (1985) J. Biol. Chem. 260, 3243-3246. Kishimoto, A., Kajikawa, N., Shiota, M. and Nishizuka, Y. (1983) J. Biol. Chem. 25, 1156-1164. Kraft, A.S. and Anderson, W.B. (1983) Nature 301, 621-623. Kuo, J.F., Schatzman, R.C., Turner, R.C. and Mazzei, J. (1984) Mol. Cell. Endocrinol. 35, 65-73. Martin, T.F.J. (1983) J. Biol. Chem. 258, 14816-14822. Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F. and Horecker, B.L. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6435-6439. Naor, 2. (1982) In: Multihormonal Regulation in Neuroendocrine Cells, Vol. 110, Eds.: A. Tixier-Vidal and P. Richard (Inserm, Paris) pp. 395-418. Naor, Z. and Catt, K.J. (1981) J. Biol. Chem. 256, 2226-2229. Naor. Z. and Ely, Y. (1985) Biochem. Biophys. Res. Commun. 130, 848-853. Naor, Z., Molcho, J., Zakut, H. and Yavin, E. (1985a) Biothem. J. 231, 19-23. Naor, Z., Zer, J., Zakut, H. and Hermon, J. (1985b) Proc. Natl. Acad. Sci. U.S.A. 83, 8203-8207. Niedel, J.E., Kuhn, L.J. and Vandenbark, G.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 36-40. Nishizuka, Y. (1984) Nature 308, 693-698. Palfrey. H.C. and Waseem, A. (1985) J. BioI. Chem. 260, 16021-16029. Portis, A., Newton, C., Pangborn, W. and Papahadjopoulos, D. (1979) Biochemistry 18, 780-790. Rebecchi, M.J. and Gershengorn, M.C. (1983) B&hem. J. 216, 287-294. Schettini, G., Cronin. M.J., Hewlett, E.L., Thorner, M.O. and Macleod, R.M. (1984) Endocrinology 115, 1308-1314. Smith, M.A. and Vale, W.W. (1981) Endocrinology 108, 752-759. Snyder, G.D. and Bleasdale, J.E. (1982) Mol. Cell. Endocrinol. 28, 55-63.
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Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara. Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. Tapley, P.M. and Murray, A.W. (1984) B&hem. Biophys. Res. Commun. 122, 158-164. Turgeon, J.L., Ashcroft, S.J.H.. Waring, D.W., Milewski, M.A. and Walsh, D.A. (1984) Mol. Cell. Endocrinol. 34, 107-112.
Uratsuji, Y., Nakanishi, H., Takeyama, Y., Kishimoto, A. and Nishizuka, Y. (1985) Biochem. Biophys. Res. Commun. 130, 654-661. Wolf, M.. Levine, III, H., May, Jr., W.S., Cuatrecasas, P. and Sahyoun. N. (1985) Nature 317, 546-549.
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47 (1986) 201-208 Ltd.
MCE 01529
Phospholipid-dependent Ca2+- activated protein kinase (C-kinase) in the pituitary: further characterization and endogenous redistribution Jacob Hermon Departments
‘,*, Anat Azrad ‘, Nachum
of ’ Hormone Research and .’ Chemical Immunology, (Received
Key words: protein
kinase C; diacylglycerol;
phorbol
Reiss ’ and Zvi Naor ’
The Weirmann
4 April 1986; accepted
Institute of Scrence, Rehooot 76100 (Israel)
6 June 1986)
ester.
Summary Phospholipid-dependent, Ca*+ -activated protein kinase (C-kinase) was recently shown to be expressed in rat pituitary. The enzyme is activated by Ca2+ and phosphatidylserine (PS). Diacylglycerol (DG), which is liberated during phosphoinositide turnover, and the potent tumor promoter 12-O-tretadecanoyl-phorbol13-acetate (TPA) activate pituitary C-kinase in the presence of PS, even at resting levels of intracellular concentraCa2+ (lo-’ M), and increase the apparent affinity of the enzyme for Ca *+ . While micromolar tion of Ca*+ had no effect on the apparent affinity of the enzyme for PS (K, - 15 pg/ml), elevation of Ca2+ to the millimolar range produced a sharp increase in the apparent affinity for PS (K, - 5 pg/ml). Elevation of PS (up to 500 pgg/ml) could not replace Ca 2+ in supporting maximal enzyme activity even in the presence of DG. Cytosolic pituitary C-kinase (70% of total enzyme activity) is recovered in an inactive state and can be activated without further purification. The particulate enzyme (30%) is recovered in a cofactors-insensitive form but can be activated after detergent-solubilization and anion exchange chromatography. Endogenous redistribution of soluble pituitary C-kinase to the membrane does not convert it here to its proteolytic product which is insensitive to Ca *+ , PS and DG. Pituitary C-kinase characterized most likely plays a key role in signal transduction mechanisms involved in pituitary functions.
Introduction The hypothalamic hormones stimulate pituitary function via the formation of ‘second messengers’ (for review see Gershengorn, 1982; Naor, 1982). While cyclic AMP might be a major mediator of
* In partial fulfillment of the requirements for a Ph.D. degree of the Feinberg Graduate School of the Weizmann Institute of Science. Address correspondence and reprint requests to: Dr. Zvi Naor, Department of Hormone Research, The Weizmann Institute of Science, Rehovot, 76100, Israel. 0303-7207/86/$03.50
0 1986 Elsevier Scientific
Publishers
Ireland,
corticotropin releasing factor (CRF) and growth hormone releasing factor (GHRF) action (Brazeau et al., 1982; Giguere et al., 1982; Bilezikjian and Vale, 1983; Schettini et al., 1984), enhanced phosphoinositide turnover was implicated in gonadotropin releasing hormone ‘(GnRH) and thyrotropin releasing hormone (TRH) action (Naor and Catt, 1981; Gershengorn, 1982; Naor, 1982; Snyder and Bleasdale, 1982; Martin, 1983; Rebecchi and Gershengorn, 1983; Naor et al., 1985a). The Ca*+/phospholipid-dependent protein kinase (C-kinase) (Takai et al., 1979) is activated by DG which is released during phosphoinositide turnover Ltd
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(Nishizuka, 1984). Activation of pituitary C-kinase might therefore represent a key step in signal transduction mechanisms involved in pituitary function. Preliminary studies described the presence of C-kinase in the pituitary (Turgeon et al., 1984) and modulation of its activity by GnRH (Hirota et al., 1985; Naor et al., 1985b) and TRH (Drust and Martin, 1985; Fearon and Tashjian, 1985). Nevertheless, no detailed characterization of the pituitary enzyme has yet been performed. We therefore decided to further characterize the enzyme in the pituitary. Materials and methods Materials Phosphatidylserine (PS, bovine brain), lysinerich calf thymus histone (type III-s), 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 1,2-diolein were from Sigma. [y- 32P]ATP ( > 5000 Ci/mmol) was purchased from Amersham. Methods Enzyme preparation. Anterior pituitaries from adult Wistar-derived female rats from the departmental colony, collected in ice, were homogenized in ice-cold 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 50 mM 2-mercaptoethanol (buffer A). After centrifugation at 4°C for 10 min at 100000 X g (Beckman Airfuge, 30 psi), the supernatant was collected as the source of crude soluble C-kinase. The pellet was washed, solubilized in buffer A containing 0.3% Triton X-100 for 1 h at 4°C and centrifuged as above for 10 min. The second supernatant was used as the crude particulate enzyme preparation. For chromatography, pituitaries were homogenized in buffer A containing 20% sucrose (buffer B). After centrifugation at 650 X g for 10 min, the soluble and particulate preparations were obtained as above and passed through a DEAE-cellulose (DE-52) column (8.5 X 3 cm) equilibrated with buffer B. The column was first washed with 15 ml of buffer B and C-kinase was eluted with a 30 ml linear concentration gradient of NaCl (O-O.4 M) in buffer B. Protein was determined by the Bradford method (1976). Enzyme assay. C-kinase was assayed by measuring the incorporation of 32P from [y- 32P]ATP into calf thymus histone (type III-s), essentially as
described by Takai et al. (1979). The reaction mixture (0.150 ml) contained 5 pmol of Tris-HCI pH 7.5, 1.25 pmol of MgCl,, 50 pg histone, 2.5 nmol of [y-‘3P]ATP (5-10 x lo4 cpm/nmol), 50 pg/ml phosphatidylserine (PS), 3.2 pg/ml diolein (DG), lop5 or lo-’ M of CaCl, for crude and purified enzyme preparation respectively, unless otherwise indicated, and enzyme preparation as indicated. Occasionally, PS and DG concentrations were varied in order to obtain optimal conditions for C-kinase activation by DG. PS and DG were first dissolved in chloroform, dried under nitrogen, and resuspended in 20 mM Tris-HCl pH 7.5 by sonication for 10 min at 4’C. The assay was carried out for 3 min at 30°C and aliquots of 100 ~1 were transferred to 2.5 cm squares of phosphocellulose paper. The papers were washed several times in a large volume of ice-cold TCA (lo%), followed by wash in ethanol and ether. The papers were then dried and the radioactivity was measured by liquid scintillation spectrometry. Results Presence of C-kinase in rat pituitary Table 1 shows C-kinase activity in rat pituitary, cerebral cortex and ovarian preparations. Basal activity ( - Ca’+) was markedly enhanced by Ca’+, in the crude cytosolic, but not in the crude particulate preparations of the tissues examined. In the presence of Ca’+, C-kinase activity was markedly stimulated by addition of PS + DG in all the cytosolic, but not in all the crude particulate preparations examined. In the ovary, Ckinase activity was mostly recovered from the particulate fraction (60%), while in cerebral cortex most of the C-kinase activity is soluble (80%). In both ovary and cerebral cortex we could demonstrate C-kinase activity in crude detergent-solubilized particulate preparations. However, no PSsensitive protein kinase activity could be observed in the crude pituitary particulate fraction before or after detergent solubilization. The presence of Triton X-100 in pituitary particulate extracts is not responsible for the lack of C-kinase activity, since the detergent causes just a slight inhibition when added to soluble C-kinase (Table l), and permits the activation of brain and ovarian particulate C-kinase. The presence of protease and
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TABLE
1
DISTRIBUTION OF CEREBRAL CORTEX
C-KINASE IN AND OVARIAN
RAT PITUITARY, PREPARATIONS
Crude cytosol and solubilized particulate fraction (25-50 pg protein) were assayed for C-kinase activity. Pituitaries and cortex were obtained from adult female rats, while ovaries were taken from 27-day-old female rats treated with 15 IU of pregnant mare serum gonadotropin on day 25 to induce follicular growth. For DEAE-cellulose (DE-52) chromatography crude supernatant and detergent-solubilized particulate fractions were passed through a DE-52 column. The column was washed with 15 ml of column buffer and C-kinase was eluted with a 30 ml linear gradient of NaCl (O-O.4 M). Samples ( - 4 pg protein) taken from the peak of C-kinase activity were used for the assay. Additions were Ca2+ 10 pM for crude and 1 mM for partial purified enzyme preparation, PS 50 pg/ml and DG 3.2 Pg/ml. The values are the mean + SEM of 2-4 experiments each done in duplicate. Sample
Pituitary Cytosol Particulate Particulate a Cytosol b Cytosol c Particulate ’ Cerebral cortex Cytosol Particulate Ovary Cytosol Particulate Pituitary (DE-52) Cytosol Particulate
Protein kinase C activity (pm01 ” P/mg protein/min) -Ca*+
+Ca*+
- PS, DG
- PS, DG
395 5 110+11 N.D. d 17+ 2 39& 4 117+10
146k12 93* 7 25+ 2 136+ 8 118* 10 78k 8
386 f 31 88*10 27+ 3 287 f 30 357 * 25 80* 7
176*20 462 + 50
398 + 35 458 * 50
739*50 578 + 40
N.D. N.D.
125+15 186+18
271 f 30 303 f 35
300 150
328 156
+ PS, DG
(Table 1 and data not shown). However, when the cytosol and the solubilized particulate fractions were chromatographed on DEAE-cellulose, a similar major peak of C-kinase activity, that was separated from other protein kinases, was eluted at about 0.16 M NaCl (Table 1). DE-52 chromatography separated the Ca’+-dependent kinase from C-kinase in the cytosolic preparation, and unmasked the particulate C-kinase (Table 1). Soluble and particulate pituitary C-kinase constitute 70 and 30% of the total enzyme activity respectively. Kinetic properties Reaction velocity was linear for the first 3 min of incubation at 30°C and this time point was chosen for all experiments (Fig. 1). Reaction velocity was also linear with protein concentration in the range examined and routinely used (- 4 pg) (Fig. 2). Pituitary C-kinase is activated by PS and an unsaturated diacylglycerol (DG) such as 1,2-diolein (Fig. 3). The enzyme is dependent on the simultaneous presence of Ca2+ and PS (Fig. 3A), whereas Ca2+ and DG alone are not sufficient for enzyme activation (Fig. 3B). PS increased enzyme activity in the presence of Ca2+ and DG increased the affinity for PS, and also increased slightly maximal enzyme activity (Fig. 3A). A dose-response curve for DG activation of C-kinase could
1052 772
’ Without Triton X-100 solubilization. b Cytosolic fraction was incubated with Ttiton X-100,0.3% for 1 h at 4OC before the assay of C-kinase. ’ Soluble and particulate enzyme were prepared, solubilized and assayed in the presence of the protease and phosphatase inhibitors: leupeptin 0.01% phenylmethylsulfonyl fluoride (PMSF) 2 mM, and NaF 10 mM. d Not determined.
gp*,,
,
2
4 Time
phosphatase inhibitors such as leupeptin, phenylmethylsulfonyl fluoride (PMSF) and NaF, or the Ca’+-chelator EGTA, during enzyme preparation and assay are not sufficient to demonstrate Ckinase activity in the crude particulate fraction
,
6
/
8
,I IO
(mtn)
Fig. 1. Time-course of C-kinase activation. Crude cytosolic enzyme preparation (20 pg protein) was incubated in the presence (open circles) or absence (closed circles) of 2 mM Ca2+, 24 pg phosphatidylserine (PS), 0.8 pg diolein (DG). C-kinase activity was measured as described in Methods.
204
PS+DG
J
OLb CoC12(log M) Protein (pg) Fig. 2. C-kinase activation as function of protein concentration. Crude soluble enzyme preparation was fractionated on DE-52 column as described in Methods. The respective peak of C-kinase was used for the assay. The assay was carried out for 3 min at 30°C in the presence of Ca2+ alone (1 mM, triangles) or in the presence of Ca2+ (1 mM), PS (50 pg/ml) and DG (3.2 pg/ml) (squares). Each point is the mean of two samples, each done in duplicate, and similar results were observed in two other experiments.
be demonstrated only in the presence of both Ca2+ and PS (Fig. 3B). As shown in Fig. 4, increasing concentrations of Ca2+ alone, or with DG, had only a small effect on enzyme activity; however, in the presence of PS, an increase in
Fig. 3. Effect of phosphatidylserine (PS) and diolein (DG) on C-kinase activity. Activity was determined in crude cytosolic enzyme preparation in (A) the presence of various concentrations of PS and Ca 2+ (10 PM, squares), or Ca (10 PM) and diolein (1.6 pg/ml, triangles), or (B) at various concentrations of diolein and Ca’ + (1 mM, squares), or Ca*+ (1 mM) and PS (12 pg/ml, triangles).
Fig. 4. Effect of diolein (DC) on pituitary C-kinase. Partial purified C-kinase (4 pg/tube) was used for the assay and various concentrations of Ca2+ were added. Ca2+ alone (open circles), with diolein (DC, 3.2 ag/ml, open triangles). with PS (12 pg/ml, filled squares). or with PS+ DG (filled triangles). Each point is the mean of two samples each done in duplicate. and similar results were obtained in two other experiments.
enzyme activity was observed. The presence of DG, in addition to PS, reduced the apparent K, for Ca2+ from 4 I_IM to 1.6 PM with little effect on maximal reaction velocity at saturating Ca2+ concentrations (Fig. 4). Enzyme activity was enhanced even at lo-’ M of Ca2+, only when DG was present in the reaction with PS. The tumor promoters phorbol esters exert pleiotropic effects on a variety of cells and recent evidence suggests that C-kinase is a major initial cellular binding site and transducer of their actions (Niedel et al., 1983; Nishizuka, 1984; Anderson et al., 1985). Since phorbol esters were reported to stimulate pituitary hormone release (Naor and Catt, 1981; Smith and Vale, 1981; Naor and Eli, 1985) it was of great interest to examine whether they activate pituitary C-kinase. Indeed, the phorbol ester, TPA, could replace DG in pituitary C-kinase activation (Fig. 5). In the presence of Ca2+, TPA on its own was a weak stimulant of enzyme activity. However, in the presence of Ca2+ and PS, TPA markedly increased enzyme activity in particular at low Ca2+ concentrations. Note that at lo-’ M of Ca2+, which is near the resting level of intracellular Ca2+ in the pituitary as monitored with Quin-2 fluorescence (- 150 nM, data not shown), C-kinase is activated only when DG or TPA are present (Figs. 4 and 5).
205
I
I 1
I
-1
5
I
I
-6
-5 CoCIz
-4
(IogM)
Fig. 5. Effect of the tumor promoter phorbol ester, TPA, on pituitary C-kinase activity. Partial purified enzyme (4 pg/tube) was used for the assay. Various concentrations of Ca2+ were added with TPA (20 ng/ml, circles), or with PS (12 pg/ml, squares) or both (triangles). Each point is the mean of two samples, each done in duplicate, and similar results were obtained at least in two other experiments.
We then investigated whether elevated PS levels (> 100 pg/ml) could replace Ca2+ in activating pituitary C-kinase. Elevation of PS (100-500
Oo+--o_I I /[PSI Fig. 6. Kinetic analysis of C-kinase activation by PS at various Ca2+ concentrations. Partial purified enzyme (4 pg/tube) was used for the assay in the absence of DC. The data was analyzed as a reciprocal plot of l/V - V. against l/PS, where V = activity at a given PS concentration and V, = activity for PS = 0. The apparent K, values derived from the curves are 16.6, 14.2, 6.25 and 4.5 pg/ml of PS at Ca’+ concentrations of 10e6, 10m5, 10m4 and 1O-3 M, respectively.
pg/ml) in the presence of DG and the absence of Ca2+, was not sufficient for maximal enzyme activation. Furthermore, high concentrations of PS (> 100 pg/ml) were inhibitory to C-kinase activation at low Ca2+ concentrations (lop7 to 10V5 M) in the presence or absence of DG (data not shown). We also investigated whether elevated Ca2+ levels could replace DG in increasing the affinity of the enzyme for PS (Fig. 6). At the micromolar range, elevation of Ca” did not change the apparent affinity for PS (K, = 14.2 and 16.6 pg/ml at 10d5 and low6 M of Ca*+ respectively). Howrange ever, raising Ca *+ levels to the millimolar increased the apparent affinity to PS with no effect on maximal velocity (K, = 4.5 and 6.25 pg/ml at 10-j and 10m4 M of Ca*+ respectively). Discussion Neurotransmitters, peptide hormones and growth factors activate cellular functions via the formation of ‘second messengers’. The major cyclic nucleotides (CAMP and cGMP) and Ca2+ activate a respective protein kinase which is responsible for phosphorylation of specific substrate proteins (Greengard, 1978). The newly discovered C-kinase (for review see Nishizuka, 1984) is activated by DG in the presence of Ca2+ and PS. Since DG is liberated during phospoinositide turnover, activation of C-kinase is a potential key step in the action of hypophysiotropic hormones which stimulate the inositol phospholipid cycle. Pituitary C-kinase is mostly soluble (70%) and partly particulate (30%), similar to the distribution of C-kinase in the cerebral cortex (Table 1 and Kuo et al., 1984). The soluble enzyme is recovered in an inactive state and can be activated by Ca2+, PS and DG even in the crude form (Table 1). Unlike the soluble enzyme, the crude pituitary particulate enzyme was not responsive to the addition of Ca’+, PS and DG. We ruled out the possibility that the presence of Triton X-100 in the crude preparation was responsible for lack of measurable activity by demonstrating that the detergent was not harmful to the pituitary soluble enzyme or to the particulate ovarian and brain enzymes (Table 1). Also, the presence of protease and phosphatase inhibitors, or chelating agents,
206
did not reveal C-kinase activity in crude pituitary particulate fractions. However, solubilization of the particulate fraction followed by anion exchange chromatography enabled us to demonstrate the particulate enzyme activity. It is possible therefore that the pituitary particulate fraction contains inhibitory activity to C-kinase which was removed during detergent solubilization and fractionation. Alternatively. it is possible that the particulate enzyme is already bound to PS and DG and is recovered in the active form. Chromatography will then remove the PS and DG and enable detection of its activity by the addition of the cofactors. The third possibility is that endogenous C-kinase was converted to ‘M-kinase’ (a proteolytic product of C-kinase which is fully active in the absence of Ca*+, PS and DG (Kishimoto et al., 1983; Naor et al., 1985b)), upon redistribution to the membrane (Tapley and Murray, 1984; Melloni et al., 1985). It was recently reported that TPA activates C-kinase by translocating the enzyme from the cytosol to the particulate fraction in parietal yolk sac cells (Kraft and Anderson, 1983), human erythrocytes (Palfrey and Waseem, 1985) and rabbit platelets (Uratsuji et al., 1985). Moreover, TPA-induced translocation of erythrocyte and platelets C-kinase to the membrane resulted in an activated form of the enzyme which was not dependent on exogenous Ca*+ and PS (Palfrey and Waseem, 1985; Uratsuji et al., 1985). Tapley and Murray (1984) demonstrated that TPA translocates human platelet C-kinase to the membrane and converts it to ‘M-kinase’. Similarly, Ca’+-induced binding of C-kinase to human neutrophil membranes results in its conversion to ‘M-kinase’ (Melloni et al., 1985). Thus, endogenous C-kinase can be translocated to the membrane upon hormonal stimulation (Drust and Martin, 1985; Fearon and Tashjian, 1985; Hirota et al., 1985; Naor et al., 1985b), or elevation in [Ca*+], (Melloni et al., 1985; Wolf et al., 1985) and might be destined to irreversible activation by limited proteolysis and formation of the active ‘M-kinase’ (Tapley and Murray, 1984; Melloni et al., 1985). Our results on the intracellular distribution of pituitary C-kinase suggest that the particulate form escaped limited proteolysis, since after detergent-solubilization and chromatography, the enzyme was further activated by Ca’+, PS
and DG, which is characteristic of C-kinase but not of ‘M-kinase’ (Kishimoto et al., 1983; Naor et al., 1985b). It is possible that under normal physiological conditions the [Ca*+], rise does not reach the concentration needed for limited proteolysis of C-kinase. Since the brain and ovarian particulate preparations revealed C-kinase activity in the crude form, it seems most likely that the pituitary particulate fraction contains inhibitory activity to Ckinase which was removed during detergent-solubilization and chromatography. In the pituitary, the potent tumor promoter TPA activates C-kinase (Fig. 5) and stimulates hormone release (Naor and Catt, 1981; Smith and Vale, 1981; Naor and Eli, 1985) suggesting that translocation of C-kinase might occur following hormone-receptor activation. Indeed, the early phosphoinositide turnover stimulated by TRH and GnRH (Snyder and Bleasdale, 1982; Martin, 1983; Rebecchi and Gershengorn, 1983; Naor et al., 1985a), is associated with rapid release of DG (Martin, 1983; Rebecchi and Gershengorn, 1983) and recruitment of C-kinase to the membrane (Drust and Martin, 1985; Fearon and Tashjian, 1985; Hirota et al., 1985; Naor et al., 1985b). The presence of DG increases the affinity of the enzyme for Ca*+ and PS, thus activation can occur in the face of resting levels of cellular Ca2+ (lo-’ M). Since DG is rapidly metabolized to phosphatidic acid via the PI cycle, or acted upon by diglyceride lipase, it was interesting to investigate whether the hormone-induced rise in cytoplasmic Ca2+ (Gershengorn and Thaw, 1983; Albert and Tashjian, 1984) may have implication on the enzyme affinity to PS. If elevated Ca2’ will increase the affinity to PS, as DG does, enzymatic activity could proceed even in the absence of DG. Our findings suggest that changes in the concentration of Ca2’ at near physiological range (0.1-10 PM) have no effect on the affinity to PS (K,, - 15 pg/ml). However, elevation of Ca’+ to the millimolar range (0.1-l mM) caused an increase in the affinity to PS (K,, - 5 pg/ml). We cannot rule out the possibility that high concentrations of Ca*+ will be generated at specific areas in the plasma membrane, and hence the observation that mM concentrations of Ca2+ affect the affinity of the enzyme to PS might have some implications. A similar line of arguments exists regarding the irre-
versible proteolytic activation of C-kinase by Ca*+-dependent neutral protease (Kishimoto et al., 1983). It was previously shown that Ca*+ induces aggregation of sonicated PS vesicles (Portis et al., 1979). Fusion of the vesicles occurs at a threshold of Ca*+ concentration of about 1 mM corresponding to binding of one Ca2+ per two PS molecules. It is possible that the increase in the affinity of C-kinase to PS by mM concentrations of Ca*+, observed in this study, results from the ability of Ca*+ to fuse PS vesicles. We therefore suggest that while DG is the rate-limiting step in the early activation of C-kinase, Ca*+ may become the rate-limiting step once DG is metabolized. Our results also suggest that the requirement for Ca*+ in pituitary C-kinase activation is absolute since elevation in PS concentrations even to supramaximal levels (500 pg/ml) could not support maximal C-kinase activity in the absence of Ca2+ and the presence of DG. This is interesting since recent reports have demonstrated that in some secretory cells a role for Ca2+ in exocytosis can be obviated (Di Virgilio et al., 1984; Barrowman et al., 1986). It is therefore possible that under certain conditions where exocytosis is fully activated independent of changes in [Ca*+],, the active species involved was ‘M-kinase’ which is not dependent on Ca *+ for its activation. Pituitary C-kinase characterized here might have a key regulatory role in signal transduction mechanisms in the pituitary, in particular in mechanisms involved in GnRH and TRH action which are known to activate phosphoinositide turnover, during which DG is generated. Acknowledgements We thank Mrs. T. Hannoch for excellent technical assistance, and Mrs. M. Kopelowitz for typing the manuscript. Supported by NIH grant HD16279 and by the United States Israel Binational Science Foundation. References Albert, P.R. and Tashjian, Jr., A.H. (1984) J. Biol. Chem. 259, 5827-5832. Anderson, W.B., Estival, A., Tapiovaara, H. and Gopalakishna, R. (1985) In: Advances in Cyclic Nucleotide and
Protein Phosphorylation Research, Vol. 19, Raven Press, New York, pp. 287-304. Barrowman, M.M., Cockcroft, S. and Gomperts, B.D. (1986) Nature 319, 5044507. Bilezikjian, L.M. and Vale, W.W. (1983) Endocrinology 113, 657-662. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Brazeau, P., Ling, N., Esch, F., Bohlen, P., Mougin, C. and Guillemin, R. (1982) Biochem. Biophys. Res. Commun. 109, 588-594. Di Virgilio, F., Lew, D.P. and Pozzan, T. (1984) Nature 310, 691-693. Drust, D.A. and Martin, T.F.J. (1985) Biochem. Biophys. Res. Commun. 128, 531-537. Fearon, C.W. and Tashjian, Jr., A.H. (1985) J. Biol. Chem. 260, 8366-8371. Gershengorn, M.C. (1982) Mol. Cell. Biochem. 45, 163-179. Gershengorn, M.C. and Thaw, C. (1983) Endocrinology 113. 1522-1524. Giguere, V., Labrie, F., Cote, J., Coy, D.H., Sueiras-Diaz, J. and Schally, A.V. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3466-3469. Greengard, P. (1978) Science 199, 146-152. Hirota, K., Hirota, T., Aguilera, G. and Catt, K.J. (1985) J. Biol. Chem. 260, 3243-3246. Kishimoto, A., Kajikawa, N., Shiota, M. and Nishizuka, Y. (1983) J. Biol. Chem. 25, 1156-1164. Kraft, A.S. and Anderson, W.B. (1983) Nature 301, 621-623. Kuo, J.F., Schatzman, R.C., Turner, R.C. and Mazzei, J. (1984) Mol. Cell. Endocrinol. 35, 65-73. Martin, T.F.J. (1983) J. Biol. Chem. 258, 14816-14822. Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F. and Horecker, B.L. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6435-6439. Naor, 2. (1982) In: Multihormonal Regulation in Neuroendocrine Cells, Vol. 110, Eds.: A. Tixier-Vidal and P. Richard (Inserm, Paris) pp. 395-418. Naor, Z. and Catt, K.J. (1981) J. Biol. Chem. 256, 2226-2229. Naor. Z. and Ely, Y. (1985) Biochem. Biophys. Res. Commun. 130, 848-853. Naor, Z., Molcho, J., Zakut, H. and Yavin, E. (1985a) Biothem. J. 231, 19-23. Naor, Z., Zer, J., Zakut, H. and Hermon, J. (1985b) Proc. Natl. Acad. Sci. U.S.A. 83, 8203-8207. Niedel, J.E., Kuhn, L.J. and Vandenbark, G.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 36-40. Nishizuka, Y. (1984) Nature 308, 693-698. Palfrey. H.C. and Waseem, A. (1985) J. BioI. Chem. 260, 16021-16029. Portis, A., Newton, C., Pangborn, W. and Papahadjopoulos, D. (1979) Biochemistry 18, 780-790. Rebecchi, M.J. and Gershengorn, M.C. (1983) B&hem. J. 216, 287-294. Schettini, G., Cronin. M.J., Hewlett, E.L., Thorner, M.O. and Macleod, R.M. (1984) Endocrinology 115, 1308-1314. Smith, M.A. and Vale, W.W. (1981) Endocrinology 108, 752-759. Snyder, G.D. and Bleasdale, J.E. (1982) Mol. Cell. Endocrinol. 28, 55-63.
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Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara. Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. Tapley, P.M. and Murray, A.W. (1984) B&hem. Biophys. Res. Commun. 122, 158-164. Turgeon, J.L., Ashcroft, S.J.H.. Waring, D.W., Milewski, M.A. and Walsh, D.A. (1984) Mol. Cell. Endocrinol. 34, 107-112.
Uratsuji, Y., Nakanishi, H., Takeyama, Y., Kishimoto, A. and Nishizuka, Y. (1985) Biochem. Biophys. Res. Commun. 130, 654-661. Wolf, M.. Levine, III, H., May, Jr., W.S., Cuatrecasas, P. and Sahyoun. N. (1985) Nature 317, 546-549.