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Identification of a calcium- and phospholipid-dependent protein kinase (protein kinase C) in Neurospora crassa
Plant Science, 49 (1987) 15--21 Elsevier Scientific Publishers Ireland Ltd.
15
IDENTIFICATION OF A CALCIUM- AND PHOSPHOLIPID-DEPENDENT PROTEIN KINASE (PROTEIN KINASE C) IN NEUROSPORA CRASSA
BERTRAND
FAVRE
and G I L B E R T
TURIAN
Laboratoire de Microbiologie gdndrale, D~parternent de Biologie v~g#Jtale, Universit~ de Gen~ve, 3, Place de l'Universitd, CH-1211 Gen~ve 4 (Switzerland) (Received August 25th, 1986) (Revision received November 12th, 1986) (Accepted November 12th, 1986) Protein kinase C was partially purified by DEAE-cellulose chromatography from the soluble fraction of mycelium of Neurospora crassa. In the presence of calcium phosphatidylserine was found to be the best activator among the various lipids tested. Diacylglycerol, under our experimental conditions, affected neither the K m of the enzyme for calcium nor the basal or m a x i m u m enzymatic activity. T w o endogenous substrates were copurified with the enzyme and were better phosphate acceptor than other exogenous basic or acidic proteins tested. The largest phosphorylated band observed on sodium dodecylsulfate-polyacrylamide gel had an apparent molecular weight of 85 000 daltons. The M r of the other was smaller than 14 000.
Key words: protein kinase C; calcium-dependent enzyme; phospholipid-dependent enzyme; phosphorylation; Neurospora crassa
Introduction Protein phosphorylation is of crucial importance in the complex processes involved in cellular regulation and is usually linked or coupled to the transduction of extracellular stimuli [1], many of which induce an increase in the intracellular concentration of calcium. In the early eighties it was established in animal cells that the hormonally stimulated breakdown of phosphatidylinositol 4,5biphosphate (PtIns 4,5-P:) was a prerequisite for calcium mobilization. Hydrolysis of PtIns 4,5-P2 catalyzed by a specific phospholipase C is characterized by the immediate production of two second messengers, D-myoinositol 1,4,5-triphosphate and 1,2-sn~liacylglycerol [2]. The former is responsible for the mobilization of cytoplasmic calcium from
intracellular stores and the latter is the activator of protein kinase C, which appears to be involved in the control of an increasing number of cellular functions [3]. Polyphosphoinositides have been known for many years in fungi [4], but the significance of their presence in these microorganisms has only lately been investigated [5--8]. In plants, both protein kinase C [9--11] and polyphosphoinositides [ 12] have very recently been identified. The present demonstration of protein kinase C in Neurospora crassa underlies the ubiquity of this enzyme among the eukaryores, and suggests, with previous discoveries in the same organism of calmodulin [13] and calmodulin~ependent enzymes [14--16], that calcium has an important regulatory role in the development of this fungus. Materials and methods
Abbreviations: 2-ME, 2-mercaptoethanol; Ptlns 4,5-P2, phosphatidylinositol 4,5-biphosphate; PtSer, phosphatidylserine; SDS, sodium dodecyl sulfate; TPA, 12-O-tetradecanoylphorbol-13-acetate.
All lipids and basic protein substrates were products of Sigma. Casein was from MERCK and was partially dephosphorylated accord-
0618-9452/87/$03.50 ©1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
16 ing to Ref. 17. (~,-32P)ATP was purchased from Amersham. Diethylaminoethyl-cellulose (DE52) and phosphocellulose paper (P-81) were obtained from Whatman. Electrophoresis molecular weight calibration kit was from Pharmacia. Erlenmeyer flasks (2.5 1) containing I 1 of Vogel's minimal medium [18] were inoculated with 109 macroconidia of wild t y p e N. crassa (strain STA4). Cultures were agitated in a rotatory shaker (250 rev./min) in the dark at 25°C for 24 h. Mycelium was harvested b y filtration and washed with deminerali~ed water on a Buchner funnel. Mycelial pads were cut with scissors, then frozen in liquid nitrogen and stored at - 8 0 ° C until use. Frozen mycelium ( ~ 1 5 0 g wet wt.) was disrupted in a Moulinex blender (type 643) for 2 min. The powder was suspended in 2 vols. of buffer A: 20 mM Tris--HC1 (pH 7.6), 10 mM EGTA, 2 mM EDTA, 50 mM 2-mercaptoethanol (2-ME), 0.25 M sucrose, 0.3 mM NAN3, 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 0.25 pg/ml pepstatin, 10 #g/ml leupeptin and homogenized again for 2 min. The pH was adjusted to 7.6 with 1 M Tris base. The homogenate was centrifuged at 30 000 × g for 20 min. The pellet was resuspended in 1 vol. of buffer A and reextracted as above. Pooled supernatants were further centrifuged at 100 000 × g for 1 h. The supernatant was collected through glass wool and solid (NH4)2SO4 was added to a final level of 50% saturation. The precipitate was recovered by centrifugation at 37 000 × g for 20 min and resuspended in 50 ml of buffer B: 20 mM Tris--HC1 (pH 7.6), 1 mM EGTA, 1 mM EDTA, 50 mM 2-ME and dialysed extensively against the same buffer. After another centrifugation at 37 000 × g the solution was applied to a DE52-cellulose column (5 ml of swollen resin/0.1 g of protein) preequilibrated with buffer B. The column was extensively washed with the same buffer. Adsorbed proteins were eluted with a linear gradient of NaC1 (0--0.4 M) (tot. vol. 18 × the volume of the column) added in buffer B.
Fractions of 10 ml were collected and assayed for protein kinase activity. Active fractions were pooled and dialysed against 50 mM Tris--HC1 (pH 7.6), 1 mM EGTA, 50 mM 2-ME and 1 ~g/ml leupeptin. All procedures were performed at 4°C. Protein kinase was assayed as described [19] in a total volume of 40 ul in a medium consisting of 50 mM Tris--HC1 (pH 7.6), 10 mM MgCI2, 75 ~M ATP containing a b o u t 2.5 × l 0 s cpm (7-32P)ATP, 0.5 mM EGTA, and when stated 0.6 mM CaC12, 100 ~g/ml phosphatidylserine (PtSer). Sample volume was always 20 pl. Exogenous substrates were added to a final concentration of 0.25 mg/ml for basic proteins and 2 mg/ml for casein. For the measurement of the calcium dependence, the concentration of EGTA was raised to 3 mM and appropriate amounts of calcium were added to give a free calcium concentration in the range of 10-8--10 -s M. The free calcium concentration was calculated with a computer program kindly provided by Dr. Jos A. Cox. The reaction was initiated by the addition of MgATP. After 10 min at 30°C, 0.4 pl of 7.5 M H3PO4 was added, 30 ~1 of the solution were spotted onto Whatman P-81 phosphocellulose paper (1 × 2.5 cm). Papers were washed three times in 75 mM H3PO4 (10 ml/paper), then put into tubes to which 3 ml water were added before radioactivity was counted. Protein concentration was determined as in Ref. 20 using bovine serum albumin as standard. All phospholipid concentrations were determined as published [21]. Lipids, dissolved in CHC13--MeOH (95 : 5), were dried under a strong nitrogen stream and resuspended in 50 mM Tris--HC1 (pH 7.6) by sonication. When the effect of diolein (mixture of 1,3-(85%) and 1,2-(15%) isomer) was tested, it was solubilized separately in the same way, then mixed with PtSer by vigorous vortexing prior to their addition in the reaction mixture. Phosphorylated proteins were separated by electrophoresis on a linear gradient (7.5-15%) sodium dodecyl sulfate (SDS)-polyacrylamide gel prepared as described in [22]. The
17 gel was stained with Coomassie brilliant blue, dried and exposed on a Kodak X-Omat SO 282 film with an amplifier screen. Our results are based upon 3 experiments, one of which is shown. Results
Chromatography on DEAE-cellulose has proven to be useful for the identification and purification of protein kinase C from crude extracts, particularly when its enzymatic activity is low in comparison with other protein kinase activities [23]. As we were unsuccessful in demonstrating the presence of a calcium- and phospholipid-dependent protein kinase activity in homogenates of mycelium of N. crassa, the extracts fractionated with (NH4)2SO4 to 50% saturation were loaded onto a DE52-column. A typical elution profile is shown in Fig. 1. The pattern of protein kinase activities, revealed by adding protamine-sulfate, histone H1 (type III-S)or casein as exogenous substrates, was more complex than those described by others [24,25]. Cyclic AMP-dependent protein kinase corresponded to the first peak of basic protein kinase. With histone H1 as a substrate, a major calcium- and phospholipid-sensitive protein kinase activity eluted between 0.17 and 0.21 M NaC1. In addition, similar, apparently minor activities seemed to elute above 0.3 M NaC1 (Fig. 1B). The possible existence of more than one protein kinase C-like activity in N. crassa is now under investigation. The following experiments questioned only the less acidic, major calcium- and phospholipid-dependent protein kinase activity eluting below 0.3 M NaC1. Interestingly, this peak could not be exactly superimposed upon any other expressed in the absence of both activators (Fig. 1B). Figure 2A shows the substrate specificity of pooled and dialysed active fractions {92-96). Endogenous phosphorylation was predominant. Relatively high levels of both protamine-sulfate and casein kinase activities expressed in the absence of calcium and PtSer
could probably be explained by contamination of protein kinase C by other protein kinases (see Fig. 1) though it was reported that animal protein kinase C could phosphorylate protamine-sulfate in a calciumand phospholipid-independent manner [23]. Dilution of the sample did not increase the level of exogenous phosphorylation. The endogenous substrates of protein kinase C coeluting with the enzyme were analysed by SDS-polyacrylamide gel electrophoresis followed by autoradiography of the gel (Fig. 2B). Two new bands clearly appeared on the autoradiograph when calcium and PtSer were added. The Mr of the largest corresponded to ~ 85 kDa. The other highly phosphorylated band was of very low molecular weight as it migrated with the front dye. Some properties of the endogenous phosphorylation were characterized. First, it was checked that the activation of the reaction by calcium and PtSer did not result from the proteolysis of an unidentified inactive protein kinase catalyzed by a calcium-activated neutral protease which could also be stimulated by certain phospholipids [26]. The stimulation elicited by calcium, in the presence of PtSer, was completely reversible (Fig. 2A). This result and the initial linear kinetics of the phosphorylation reaction do not support the generation by proteolysis of a calcium-independent protein kinase. The slow decrease in the radioactivity, observed after the chelation of calcium by an excess of EGTA, could arise from a slight contaminating protein phosphatase activity. Among the various lipids tested, PtSer was the best activator (Table I and Fig. 3B). Neither cyclic nucleotides {10 uM) nor calcium plus bovine brain calmodulin (1 pM) had any effect on the basal enzyme activity. The calcium dependence of the phosphorylation reaction in the presence of optimum concentration of PtSer is shown in Fig. 3C. Diolein {10 ug/ml), under the same conditions, had no significant effect on the Km-value of the enzyme for calcium nor on the basal or maximum enzymatic activity (Fig. 3C). Other concentrations of
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Fig. 1. E l u t i o n profile o f protein kinase activities from a D E 5 2 - c o l u m n (3.1 cm diameter, 14 cm long); 2.1 g o f protein were loaded o n t o the c o l u m n . T h e f l o w rate was 60 m l / h and the fraction v o l u m e 10 ml. Every fifth fraction was assayed for protein kinase activity under standard c o n d i t i o n s w i t h various substrates. ( A ) casein (~ D); protamine-sulfate (= - -=); A2s0n m ( - - - - ) . (B) histone H 1 (type III-S) ( / i); histone H I (III-S) + calcium + PtSer ( • v), NaCI concentration (-- -- --).
19
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EGTA Fig. 2. Substrate specificity of pooled and dialyzed fractions (92--96) of protein kinase C (prot. conc. 1 rag/ ml). (A) F r o m left to right, endogenous, protamine-sulfate, protamine free base, histone H1 (type III-S), hmtone H2B (type VII-S), mixture of histones (type II-A) and casein, respectively. ~, EGTA, B, c a l c i u m + PtSer; standard conditions. (B) Autoradiograph of endogenously phosphorylated substrates separated on a linear gradient (7.5--15%) SDS-polyacrylamide gel. Phosphorylation reactions were carried on for 60 min in the presence or absence of calcium and PtSer (standard conditions). Protein (40 ~g) was loaded onto the gel. The dried film was exposed at --80°C with an amplifier screen for 4 h. Table I. Lipid specificity with or without calcium. Endogenous phosphorylation, standard conditions. Lipid added a
Protein kinase activity (%)b _ C a 2+
+ C a ~÷
Phosphatidylserine Phosphatidylinositol Phosphatidylethanolamine Phosphatidylcholine Phosphatidic acid
16 9 16 6 18
100 33 10 6 15
Diolein
13
10
Oleic acid
15
29
a All lipids were tested at a concentration of 0.13 raM, corresponding to 0.1 mg/ml PtSer. b Relative to the activity obtained with PtSer in the presence of calcium (2.1 pmol P/min).
diacylglycerol and also of TPA (up to 1 ~M) were tested at a suboptimum cortcentration of calcium without more success. Discussion
The presence of polyphosphoinositides in microorganisms suggests that the main features of the model of signal transduction, in which calcium plays a pivotal role, proposed for animal cells also applies to lower eukaryotes. Some recent studies devoted to this subject [5--8], with the present demonstration of the existence of at least one protein kinase C-like activity in a fungus, strengthen this hypothesis. Some of the pro-
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Fig. 3. A c t i v a t i o n of protein kinase C by calcium and PtSer (pooled fractions 92--96, e n d o g e n o u s phosphorylation). (A) Time course of e n z y m a t i c activity, calcium (~ =); calcium + PtSer (c. = ). A f t e r 12 min of incubation (arrow), part of the reaction m i x t u r e was transferred to a n o t h e r tube containing an adequate v o l u m e of c o n c e n t r a t e d E G T A (buffered at pH 7.6 with Tris base) to give a final c o n c e n t r a t i o n of 5 mM and incubated still m o r e for 20 rain (v,). (B) D e p e n d e n c e on PtSer c o n c e n t r a t i o n in the presence of calcium. (C) D e p e n d e n c e on calcium c o n c e n t r a t i o n in the presence o f PtSer (+ +), or PtSer + diolein (10 ~ g / m l ) ( o - - - - - o ) . Other conditions were similar to those described u n d e r Materials and methods.
21
perties revealed so far by the fungal enzyme resemble those of animal [23] and plant protein kinase C [9--11]. These include the observations that: (a) the enzyme was acidic, (b) its activity required calcium in the micromolar range, and (c) PtSer was the best activator among the various lipids tested. The main difference with the animal enzyme was the apparent lack of effect of diacylglycerol or of the tumor promoter TPA on cinetical parameters of the phosphorylation reaction. Interestingly, Elliott and Skinner [10] were unable to show a specific binding of phorbol ester on the plant protein kinase C, even though Ol~uh and Kiss [11] reported a stimulatory effect of diolein or TPA on the wheat enzyme, and Portillo and MazSn [7] demonstrated an increase in the phosphorylation level of a membrane protein in response to the addition of TPA to yeast cells. Further investigations are needed to resolve this apparent disagreement between studies. The substrate specificity of the enzyme is unclear because of the strong endogenous phosphorylation. The identity of both substmtes is unknown. Surprisingly, they copurified with the enzymatic activity through three different chromatography columns {unpublished}. Along with further biochemical characterization of fungal protein kinase C, the study of its role in the development and differentiation of fungi could help to resolve its mode of action in cell proliferation. Two important questions are now to be answered in microorganisms, (1) what external stimuli activate protein kinase C? and (2) what proteins serve as substrates in vivo for the enzyme?
Acknowledgments We are grateful to Dr. Mukti Ojha and Mrs. Arlette Cattaneo for both scientific and technical advise.
References 1 P. Cohen, Eur. J. Biochem,, 151 (1985) 439. 2 M.J. Berridge and R.F. Irvine, Nature, 312 (1984) 315. 3 Y. Nishizuka, Science, 233 (1986) 305. 4 R.H. Michell, Biochim. Biophys. Acta, 415 (1975) 81. 5 P.J.M. Van Haastert, R.J.W. De Wit and M.M. Van Lookeren Campagne, Biochem. Biophys. Res. Commun., 128 (1985) 185. 6 G.N. Europe-Firmer and P.C. Newel|, Biochem. Biophys. Res. Commun., 130 (1985) 1115. 7 F. Portillo and M.J. MazAn, FEBS Lett., 192 (1985) 95. 8 J.S. Dahl and C.E. Dahl, Biochem. Biophys. Res. Commun., 133 (1985) 844. 9 A. Schaefer, F. Bygrave, S. Matzenauer and D. Matin6, FEBS Lett., 187 {1985) 25. 10 D.C. Elliot and J.D. Skinner, Phytochemistry, 25 (1986) 39. 11 Z. Ol~ih and Z. Kiss, FEBS Lett., 195 (1986) 33. 12 W.F. Boss and M.O. Massel, Biochem. Biophys. Res. Commun., 132 (1985) 1018. 13 R. Ortega Perez, D. Van Tuinen, D. Matin6 and G. Turian, FEBS Lett., 133 (1981) 205. 14 R. Ortega Perez, D. Van Tuinen, D. Marm~ and G. Turian, Biochim. Biophys. Acta, 758 (1983) 84. 15 D. Van Tuinen, R. Ortega Perez, D. Marm6 and G. Turian, FEBS Lett., 176 (1984) 317. 16 J.A. Reig, M.T. T~llez-InSn, M.M. Flawi~ and H.N. Tortes, Biochem. J., 221 (1984) 541. 17 E.M. Reimann, D.A. Walsh and E.G. Krebs, J. Biol. Chem., 246 (1971) 1986. 18 H.J. Vogel, Microbiol. Genet. Bull., 13 (1956) 42. 19 R. Roskoski, Hormone Action, in: J.D. Corbin and J.G. Hardmann (Eds.), Methods Enzymol., Vol. 99, Academic Press, New York, London, 1983, p. 3. 20 T. Spector, Anal. Biochem., 86 (1978) 142. 21 B.N. Ames and D.T. Dubin, J. Biol. Chem., 235 (1960) 769. 22 U,K. Laemmli, Nature, 227 (1970) 680. 23 U. Kikkawa, R. Minakuchi, Y. Takai and Y. Nishizuka, Hormone Action, in: J.D. Corbin and J.G. Hardman (Eds.), Methods Enzymol., Vol. 99, Academic Press, New York, London, 1983, p. 288. 24 P.A. Powers and M.L. Pall, Biochem. Biophys. Res. Commun., 95 (1980) 701. 25 N.D. Judewicz, G.C. Glikin and H.N. Torres, Arch. Biochem. Biophys., 206 (1981) 87. 26 S.A, Coloican and D.R. Hathaway, J. Biol. Chem., 259 (1984) 11627.
15
IDENTIFICATION OF A CALCIUM- AND PHOSPHOLIPID-DEPENDENT PROTEIN KINASE (PROTEIN KINASE C) IN NEUROSPORA CRASSA
BERTRAND
FAVRE
and G I L B E R T
TURIAN
Laboratoire de Microbiologie gdndrale, D~parternent de Biologie v~g#Jtale, Universit~ de Gen~ve, 3, Place de l'Universitd, CH-1211 Gen~ve 4 (Switzerland) (Received August 25th, 1986) (Revision received November 12th, 1986) (Accepted November 12th, 1986) Protein kinase C was partially purified by DEAE-cellulose chromatography from the soluble fraction of mycelium of Neurospora crassa. In the presence of calcium phosphatidylserine was found to be the best activator among the various lipids tested. Diacylglycerol, under our experimental conditions, affected neither the K m of the enzyme for calcium nor the basal or m a x i m u m enzymatic activity. T w o endogenous substrates were copurified with the enzyme and were better phosphate acceptor than other exogenous basic or acidic proteins tested. The largest phosphorylated band observed on sodium dodecylsulfate-polyacrylamide gel had an apparent molecular weight of 85 000 daltons. The M r of the other was smaller than 14 000.
Key words: protein kinase C; calcium-dependent enzyme; phospholipid-dependent enzyme; phosphorylation; Neurospora crassa
Introduction Protein phosphorylation is of crucial importance in the complex processes involved in cellular regulation and is usually linked or coupled to the transduction of extracellular stimuli [1], many of which induce an increase in the intracellular concentration of calcium. In the early eighties it was established in animal cells that the hormonally stimulated breakdown of phosphatidylinositol 4,5biphosphate (PtIns 4,5-P:) was a prerequisite for calcium mobilization. Hydrolysis of PtIns 4,5-P2 catalyzed by a specific phospholipase C is characterized by the immediate production of two second messengers, D-myoinositol 1,4,5-triphosphate and 1,2-sn~liacylglycerol [2]. The former is responsible for the mobilization of cytoplasmic calcium from
intracellular stores and the latter is the activator of protein kinase C, which appears to be involved in the control of an increasing number of cellular functions [3]. Polyphosphoinositides have been known for many years in fungi [4], but the significance of their presence in these microorganisms has only lately been investigated [5--8]. In plants, both protein kinase C [9--11] and polyphosphoinositides [ 12] have very recently been identified. The present demonstration of protein kinase C in Neurospora crassa underlies the ubiquity of this enzyme among the eukaryores, and suggests, with previous discoveries in the same organism of calmodulin [13] and calmodulin~ependent enzymes [14--16], that calcium has an important regulatory role in the development of this fungus. Materials and methods
Abbreviations: 2-ME, 2-mercaptoethanol; Ptlns 4,5-P2, phosphatidylinositol 4,5-biphosphate; PtSer, phosphatidylserine; SDS, sodium dodecyl sulfate; TPA, 12-O-tetradecanoylphorbol-13-acetate.
All lipids and basic protein substrates were products of Sigma. Casein was from MERCK and was partially dephosphorylated accord-
0618-9452/87/$03.50 ©1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
16 ing to Ref. 17. (~,-32P)ATP was purchased from Amersham. Diethylaminoethyl-cellulose (DE52) and phosphocellulose paper (P-81) were obtained from Whatman. Electrophoresis molecular weight calibration kit was from Pharmacia. Erlenmeyer flasks (2.5 1) containing I 1 of Vogel's minimal medium [18] were inoculated with 109 macroconidia of wild t y p e N. crassa (strain STA4). Cultures were agitated in a rotatory shaker (250 rev./min) in the dark at 25°C for 24 h. Mycelium was harvested b y filtration and washed with deminerali~ed water on a Buchner funnel. Mycelial pads were cut with scissors, then frozen in liquid nitrogen and stored at - 8 0 ° C until use. Frozen mycelium ( ~ 1 5 0 g wet wt.) was disrupted in a Moulinex blender (type 643) for 2 min. The powder was suspended in 2 vols. of buffer A: 20 mM Tris--HC1 (pH 7.6), 10 mM EGTA, 2 mM EDTA, 50 mM 2-mercaptoethanol (2-ME), 0.25 M sucrose, 0.3 mM NAN3, 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 0.25 pg/ml pepstatin, 10 #g/ml leupeptin and homogenized again for 2 min. The pH was adjusted to 7.6 with 1 M Tris base. The homogenate was centrifuged at 30 000 × g for 20 min. The pellet was resuspended in 1 vol. of buffer A and reextracted as above. Pooled supernatants were further centrifuged at 100 000 × g for 1 h. The supernatant was collected through glass wool and solid (NH4)2SO4 was added to a final level of 50% saturation. The precipitate was recovered by centrifugation at 37 000 × g for 20 min and resuspended in 50 ml of buffer B: 20 mM Tris--HC1 (pH 7.6), 1 mM EGTA, 1 mM EDTA, 50 mM 2-ME and dialysed extensively against the same buffer. After another centrifugation at 37 000 × g the solution was applied to a DE52-cellulose column (5 ml of swollen resin/0.1 g of protein) preequilibrated with buffer B. The column was extensively washed with the same buffer. Adsorbed proteins were eluted with a linear gradient of NaC1 (0--0.4 M) (tot. vol. 18 × the volume of the column) added in buffer B.
Fractions of 10 ml were collected and assayed for protein kinase activity. Active fractions were pooled and dialysed against 50 mM Tris--HC1 (pH 7.6), 1 mM EGTA, 50 mM 2-ME and 1 ~g/ml leupeptin. All procedures were performed at 4°C. Protein kinase was assayed as described [19] in a total volume of 40 ul in a medium consisting of 50 mM Tris--HC1 (pH 7.6), 10 mM MgCI2, 75 ~M ATP containing a b o u t 2.5 × l 0 s cpm (7-32P)ATP, 0.5 mM EGTA, and when stated 0.6 mM CaC12, 100 ~g/ml phosphatidylserine (PtSer). Sample volume was always 20 pl. Exogenous substrates were added to a final concentration of 0.25 mg/ml for basic proteins and 2 mg/ml for casein. For the measurement of the calcium dependence, the concentration of EGTA was raised to 3 mM and appropriate amounts of calcium were added to give a free calcium concentration in the range of 10-8--10 -s M. The free calcium concentration was calculated with a computer program kindly provided by Dr. Jos A. Cox. The reaction was initiated by the addition of MgATP. After 10 min at 30°C, 0.4 pl of 7.5 M H3PO4 was added, 30 ~1 of the solution were spotted onto Whatman P-81 phosphocellulose paper (1 × 2.5 cm). Papers were washed three times in 75 mM H3PO4 (10 ml/paper), then put into tubes to which 3 ml water were added before radioactivity was counted. Protein concentration was determined as in Ref. 20 using bovine serum albumin as standard. All phospholipid concentrations were determined as published [21]. Lipids, dissolved in CHC13--MeOH (95 : 5), were dried under a strong nitrogen stream and resuspended in 50 mM Tris--HC1 (pH 7.6) by sonication. When the effect of diolein (mixture of 1,3-(85%) and 1,2-(15%) isomer) was tested, it was solubilized separately in the same way, then mixed with PtSer by vigorous vortexing prior to their addition in the reaction mixture. Phosphorylated proteins were separated by electrophoresis on a linear gradient (7.5-15%) sodium dodecyl sulfate (SDS)-polyacrylamide gel prepared as described in [22]. The
17 gel was stained with Coomassie brilliant blue, dried and exposed on a Kodak X-Omat SO 282 film with an amplifier screen. Our results are based upon 3 experiments, one of which is shown. Results
Chromatography on DEAE-cellulose has proven to be useful for the identification and purification of protein kinase C from crude extracts, particularly when its enzymatic activity is low in comparison with other protein kinase activities [23]. As we were unsuccessful in demonstrating the presence of a calcium- and phospholipid-dependent protein kinase activity in homogenates of mycelium of N. crassa, the extracts fractionated with (NH4)2SO4 to 50% saturation were loaded onto a DE52-column. A typical elution profile is shown in Fig. 1. The pattern of protein kinase activities, revealed by adding protamine-sulfate, histone H1 (type III-S)or casein as exogenous substrates, was more complex than those described by others [24,25]. Cyclic AMP-dependent protein kinase corresponded to the first peak of basic protein kinase. With histone H1 as a substrate, a major calcium- and phospholipid-sensitive protein kinase activity eluted between 0.17 and 0.21 M NaC1. In addition, similar, apparently minor activities seemed to elute above 0.3 M NaC1 (Fig. 1B). The possible existence of more than one protein kinase C-like activity in N. crassa is now under investigation. The following experiments questioned only the less acidic, major calcium- and phospholipid-dependent protein kinase activity eluting below 0.3 M NaC1. Interestingly, this peak could not be exactly superimposed upon any other expressed in the absence of both activators (Fig. 1B). Figure 2A shows the substrate specificity of pooled and dialysed active fractions {92-96). Endogenous phosphorylation was predominant. Relatively high levels of both protamine-sulfate and casein kinase activities expressed in the absence of calcium and PtSer
could probably be explained by contamination of protein kinase C by other protein kinases (see Fig. 1) though it was reported that animal protein kinase C could phosphorylate protamine-sulfate in a calciumand phospholipid-independent manner [23]. Dilution of the sample did not increase the level of exogenous phosphorylation. The endogenous substrates of protein kinase C coeluting with the enzyme were analysed by SDS-polyacrylamide gel electrophoresis followed by autoradiography of the gel (Fig. 2B). Two new bands clearly appeared on the autoradiograph when calcium and PtSer were added. The Mr of the largest corresponded to ~ 85 kDa. The other highly phosphorylated band was of very low molecular weight as it migrated with the front dye. Some properties of the endogenous phosphorylation were characterized. First, it was checked that the activation of the reaction by calcium and PtSer did not result from the proteolysis of an unidentified inactive protein kinase catalyzed by a calcium-activated neutral protease which could also be stimulated by certain phospholipids [26]. The stimulation elicited by calcium, in the presence of PtSer, was completely reversible (Fig. 2A). This result and the initial linear kinetics of the phosphorylation reaction do not support the generation by proteolysis of a calcium-independent protein kinase. The slow decrease in the radioactivity, observed after the chelation of calcium by an excess of EGTA, could arise from a slight contaminating protein phosphatase activity. Among the various lipids tested, PtSer was the best activator (Table I and Fig. 3B). Neither cyclic nucleotides {10 uM) nor calcium plus bovine brain calmodulin (1 pM) had any effect on the basal enzyme activity. The calcium dependence of the phosphorylation reaction in the presence of optimum concentration of PtSer is shown in Fig. 3C. Diolein {10 ug/ml), under the same conditions, had no significant effect on the Km-value of the enzyme for calcium nor on the basal or maximum enzymatic activity (Fig. 3C). Other concentrations of
18
2.0
1.5
~I
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o
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2
/
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x
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r
'
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=
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v
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s
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0
0.2
U o
Z 0.5
0.1
s
s
s
s
d
s
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0 0'
510
1 0 '0
Fraction
Number
1 5 ,0
Fig. 1. E l u t i o n profile o f protein kinase activities from a D E 5 2 - c o l u m n (3.1 cm diameter, 14 cm long); 2.1 g o f protein were loaded o n t o the c o l u m n . T h e f l o w rate was 60 m l / h and the fraction v o l u m e 10 ml. Every fifth fraction was assayed for protein kinase activity under standard c o n d i t i o n s w i t h various substrates. ( A ) casein (~ D); protamine-sulfate (= - -=); A2s0n m ( - - - - ) . (B) histone H 1 (type III-S) ( / i); histone H I (III-S) + calcium + PtSer ( • v), NaCI concentration (-- -- --).
19
A
endo
4
protS
prot
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H2B
-H-
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>, 4=°~
> o~ ,4=-
u
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~.
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MW
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94 I
220
I
67
60 I I
43 I
36 I
30 I
20.1
I
14.4
I
front
I
Co2++ PtSer
EGTA Fig. 2. Substrate specificity of pooled and dialyzed fractions (92--96) of protein kinase C (prot. conc. 1 rag/ ml). (A) F r o m left to right, endogenous, protamine-sulfate, protamine free base, histone H1 (type III-S), hmtone H2B (type VII-S), mixture of histones (type II-A) and casein, respectively. ~, EGTA, B, c a l c i u m + PtSer; standard conditions. (B) Autoradiograph of endogenously phosphorylated substrates separated on a linear gradient (7.5--15%) SDS-polyacrylamide gel. Phosphorylation reactions were carried on for 60 min in the presence or absence of calcium and PtSer (standard conditions). Protein (40 ~g) was loaded onto the gel. The dried film was exposed at --80°C with an amplifier screen for 4 h. Table I. Lipid specificity with or without calcium. Endogenous phosphorylation, standard conditions. Lipid added a
Protein kinase activity (%)b _ C a 2+
+ C a ~÷
Phosphatidylserine Phosphatidylinositol Phosphatidylethanolamine Phosphatidylcholine Phosphatidic acid
16 9 16 6 18
100 33 10 6 15
Diolein
13
10
Oleic acid
15
29
a All lipids were tested at a concentration of 0.13 raM, corresponding to 0.1 mg/ml PtSer. b Relative to the activity obtained with PtSer in the presence of calcium (2.1 pmol P/min).
diacylglycerol and also of TPA (up to 1 ~M) were tested at a suboptimum cortcentration of calcium without more success. Discussion
The presence of polyphosphoinositides in microorganisms suggests that the main features of the model of signal transduction, in which calcium plays a pivotal role, proposed for animal cells also applies to lower eukaryotes. Some recent studies devoted to this subject [5--8], with the present demonstration of the existence of at least one protein kinase C-like activity in a fungus, strengthen this hypothesis. Some of the pro-
20
A
100
75 o..
5O E
o..
25
.i--
°--
15
30
0
Time
r-
45
60
(min)
.~
v
B
C °~
C
(D 0
0
t"--
E
2
\
o
E
Q.
1
v
3
2
1
0
- l o g PtSer ( m g / m l )
8
7
6
5
- l o g Ca2+(M)
Fig. 3. A c t i v a t i o n of protein kinase C by calcium and PtSer (pooled fractions 92--96, e n d o g e n o u s phosphorylation). (A) Time course of e n z y m a t i c activity, calcium (~ =); calcium + PtSer (c. = ). A f t e r 12 min of incubation (arrow), part of the reaction m i x t u r e was transferred to a n o t h e r tube containing an adequate v o l u m e of c o n c e n t r a t e d E G T A (buffered at pH 7.6 with Tris base) to give a final c o n c e n t r a t i o n of 5 mM and incubated still m o r e for 20 rain (v,). (B) D e p e n d e n c e on PtSer c o n c e n t r a t i o n in the presence of calcium. (C) D e p e n d e n c e on calcium c o n c e n t r a t i o n in the presence o f PtSer (+ +), or PtSer + diolein (10 ~ g / m l ) ( o - - - - - o ) . Other conditions were similar to those described u n d e r Materials and methods.
21
perties revealed so far by the fungal enzyme resemble those of animal [23] and plant protein kinase C [9--11]. These include the observations that: (a) the enzyme was acidic, (b) its activity required calcium in the micromolar range, and (c) PtSer was the best activator among the various lipids tested. The main difference with the animal enzyme was the apparent lack of effect of diacylglycerol or of the tumor promoter TPA on cinetical parameters of the phosphorylation reaction. Interestingly, Elliott and Skinner [10] were unable to show a specific binding of phorbol ester on the plant protein kinase C, even though Ol~uh and Kiss [11] reported a stimulatory effect of diolein or TPA on the wheat enzyme, and Portillo and MazSn [7] demonstrated an increase in the phosphorylation level of a membrane protein in response to the addition of TPA to yeast cells. Further investigations are needed to resolve this apparent disagreement between studies. The substrate specificity of the enzyme is unclear because of the strong endogenous phosphorylation. The identity of both substmtes is unknown. Surprisingly, they copurified with the enzymatic activity through three different chromatography columns {unpublished}. Along with further biochemical characterization of fungal protein kinase C, the study of its role in the development and differentiation of fungi could help to resolve its mode of action in cell proliferation. Two important questions are now to be answered in microorganisms, (1) what external stimuli activate protein kinase C? and (2) what proteins serve as substrates in vivo for the enzyme?
Acknowledgments We are grateful to Dr. Mukti Ojha and Mrs. Arlette Cattaneo for both scientific and technical advise.
References 1 P. Cohen, Eur. J. Biochem,, 151 (1985) 439. 2 M.J. Berridge and R.F. Irvine, Nature, 312 (1984) 315. 3 Y. Nishizuka, Science, 233 (1986) 305. 4 R.H. Michell, Biochim. Biophys. Acta, 415 (1975) 81. 5 P.J.M. Van Haastert, R.J.W. De Wit and M.M. Van Lookeren Campagne, Biochem. Biophys. Res. Commun., 128 (1985) 185. 6 G.N. Europe-Firmer and P.C. Newel|, Biochem. Biophys. Res. Commun., 130 (1985) 1115. 7 F. Portillo and M.J. MazAn, FEBS Lett., 192 (1985) 95. 8 J.S. Dahl and C.E. Dahl, Biochem. Biophys. Res. Commun., 133 (1985) 844. 9 A. Schaefer, F. Bygrave, S. Matzenauer and D. Matin6, FEBS Lett., 187 {1985) 25. 10 D.C. Elliot and J.D. Skinner, Phytochemistry, 25 (1986) 39. 11 Z. Ol~ih and Z. Kiss, FEBS Lett., 195 (1986) 33. 12 W.F. Boss and M.O. Massel, Biochem. Biophys. Res. Commun., 132 (1985) 1018. 13 R. Ortega Perez, D. Van Tuinen, D. Matin6 and G. Turian, FEBS Lett., 133 (1981) 205. 14 R. Ortega Perez, D. Van Tuinen, D. Marm~ and G. Turian, Biochim. Biophys. Acta, 758 (1983) 84. 15 D. Van Tuinen, R. Ortega Perez, D. Marm6 and G. Turian, FEBS Lett., 176 (1984) 317. 16 J.A. Reig, M.T. T~llez-InSn, M.M. Flawi~ and H.N. Tortes, Biochem. J., 221 (1984) 541. 17 E.M. Reimann, D.A. Walsh and E.G. Krebs, J. Biol. Chem., 246 (1971) 1986. 18 H.J. Vogel, Microbiol. Genet. Bull., 13 (1956) 42. 19 R. Roskoski, Hormone Action, in: J.D. Corbin and J.G. Hardmann (Eds.), Methods Enzymol., Vol. 99, Academic Press, New York, London, 1983, p. 3. 20 T. Spector, Anal. Biochem., 86 (1978) 142. 21 B.N. Ames and D.T. Dubin, J. Biol. Chem., 235 (1960) 769. 22 U,K. Laemmli, Nature, 227 (1970) 680. 23 U. Kikkawa, R. Minakuchi, Y. Takai and Y. Nishizuka, Hormone Action, in: J.D. Corbin and J.G. Hardman (Eds.), Methods Enzymol., Vol. 99, Academic Press, New York, London, 1983, p. 288. 24 P.A. Powers and M.L. Pall, Biochem. Biophys. Res. Commun., 95 (1980) 701. 25 N.D. Judewicz, G.C. Glikin and H.N. Torres, Arch. Biochem. Biophys., 206 (1981) 87. 26 S.A, Coloican and D.R. Hathaway, J. Biol. Chem., 259 (1984) 11627.