partial characterization of protein kinase c from an insect cell line

210 Biochimica et Biophysica Acta, 1203 (1993) 210-214 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00

BBAPRO 34624

Partial characterization of protein kinase C from an insect cell line

Jyothi Gupta 1 and Roger G.H. Downer *

Department of Biology, University of Waterloo, Waterloo, Ont. N2L 3G1 (Canada)

(Received 6 April 1993)

Key words: Protein kinase C; Insect cell line; Phorbol ester; Diacylglycerol; Phospholipid; H-7; (C. fumiferana)

The characteristics of protein kinase C (EC 2.7.1.37) from an insect cell line (Choristoneura furniferana) have been described. DEAE-cellulose chromatography produced a major peak of activity which eluted at 0.04-0.055 M NaC1. The enzyme was sensitive to phosphatidylserine in the presence of calcium. Phorbol 12-myristate 13-acetate (PMA) in nanomolar concentrations stimulated protein kinase C activity 8-fold over basal levels and reduced the enzymes requirement for Ca 2÷. The enzyme had a K a of 10 nM for PMA. Diacylglycerols tested included diolein, dilinolein, diarachidonin, oleoyl-acetyl-glycerol, dioctonoyl-sn- glycerol, dipalmitin and distearin. A 2.5- to 3-fold activation was obtained in the presence of 26 /xM diolein, 40 /zM oleoyl-acetyl-glycerol and 46 /zM dioctonoyl-sn-glycerol. The enzyme activity was sensitive to the inhibitor H-7 and 50% inhibition was achieved at a concentration of 52 /zM H-7. Phosphatidylinositol enhanced enzyme activity in the absence of phosphatidylserine but phosphatidylethanolamine had no effect.

Introduction

Protein phosphorylation through cyclic AMP or calcium- and phospholipid-dependent kinases is an intracellular event leading to physiological responses after receptor activation. Some hormones and neurotrans- mitters stimulate metabolism of membrane phosphoinositides to generate at least two intracellular second messengers: inositol tr isphosphate (IP 3) and diacylglycerol (DAG) [1,2]. IP 3 mobilizes calcium from intracellular stores, whereas diacylglycerol stimulates a calcium-activated, phospholipid-dependent enzyme, protein kinase C (PKC).

PKC is widely distributed in tissues with brain and platelets exhibiting the highest activity [3]. The mecha- nism of PKC activation involves the formation on the membrane surface of a complex of PKC, phosphatidylserine (PS), calcium (Ca 2÷) and D A G [4,5]. Tumor- promoting phorbol diesters acting directly on PKC bypass the step of phospholipase C activation in the I P 3 / D A G signalling pathway [6,7]. It is now accepted that PKC is the intracellular phorbol diester receptor [8]. The presence of numerous but specific substrates for the enzyme in discrete subcellular locations aug-

* Corresponding author. Fax: + 1 (519) 7474168. 1 Present address: Department of Biochemistry, McMaster Univer-

sity, Hamilton, Ont. L8N 3Z5, Canada.

ments its pivotal role in cellular function and regula- tions [9-11]. PKC is no longer considered as a single entity, but rather a complex family of related molecular forms [12,13]. Current knowledge of PKC has been obtained primarily by studies using mammalian tissues (for Reviews see Refs. 14-17) and there is a dearth of similar information for invertebrate systems.

Studies on blowfly salivary gland clearly demon- strate hormone-mediated hydrolysis of membrane phosphoinositides in insects [18-21]. Preliminary studies in an intact cell line indicate octopamine-mediated increases in intracellular calcium levels [22]. PKC has been identified in the ventral nerve cord, ganglia and whole head preparat ion of cockroach, Periplaneta [3]. Knipper and Breer have shown the presence of PKC and the existence of high affinity binding sites for phorbol esters in locust ganglia [23]. In an earlier study [25], using an insect cell line, we have shown the modulation of octopamine-mediated cyclic AMP pro- duction by PKC. Here, we further characterize PKC in an insect cell line in order to bet ter understand enzyme regulation in insect systems.

Materials and Methods

Cell culture Cell lines of Choristoneura fumiferana (IBRI-CF1)

were obtained from Dr. Sohi, Forest Pest Management Institute at Sault Saint Marie, Ontario, and reared in

211

Grace's Insect Culture Medium (pH 6.2) supplemented with 0.25% tryptose broth, 10% fetal bovine serum and 0.0025% penicillin/streptomycin (5000 units and 5 mg/ml) in 75 cm 2 flasks at 28°C. Cultures were ini- tially seeded at 2.105 cells/ml and subcultured every 5 days as previously described [25].

Enzyme preparation Cells were washed and suspended in ice-cold 25 mM

Tris-HC1 (pH 7.5)/0.25 M sucrose/2.5 mM MGC12/2.5 mM EGTA/50 mM 2-mercaptoethanol [3]. The cells were then centrifuged for 5 min at 300 × g to remove cell debris, and the resulting supernatant centrifuged at 100 000 × g to yield cytosolic fractions. The cytosolic fraction was applied to DEAE-cellulose columns previously equilibrated with 20 mM Tris-HCl (pH 7.5)/0.5 mM EGTA/2 mM EDTA/2 mM phenylmethylsulfo- nyl fluoride (PMSF) [26]. After sample application the column was washed with 15 ml of the buffer and then eluted with a step-gradient of 0 to 0.1 M NaC1. Frac- tions of approx. 1 ml were collected and 50/xl aliquots of each fraction were assayed for PKC activity in the presence of phorbol 12-myristate 13-acetate (PMA), 0.65 /zM, and 20 /zg of phosphatidylserine (0.1 mM final concentration) or in the presence of 1 mM EGTA.

Assay of protein kinase C PKC was assayed by measuring the incorporation of

32p into histone from [y-32p]ATP using a slight modifi- cation of the method of Kishimoto et al. [27]. All reagents and buffers used in tissue preparation and PKC determinations were prepared with Chelex-100 treated water (Sigma Chemical Company, St. Louis, MO) to remove Ca 2÷ [28]. Briefly, the reaction mixture (0.25 ml) contained 5 /~mol Tris-HC1 (pH 7.5), 1.25 /zmol MgCI2, 2.5 /zmol NaF, 50 /zg histone and 2.5 nmol [y-32p]ATP ((5-10)" 104 cpm/nmol). Where indicated, Ca 2÷ (final concentration 0.5 mM) and phosphatidylserine (0.1 mM) were added to the reaction mixture. This preparation was incubated for 5 min at 30°C in a shaking water bath and then a 25/zl aliquot was spotted at the origin (pretreated with 75/xl 20% trichloroacetic acid) of a 1.5 × 13 cm chromatographic strip (Whatman, 31-ETCHR). Ascending chromatography was performed as described by Huang and Robinson [29]. Subsequently the filters were dried and approx. 3 cm, containing the precipitated histone, was placed in scintillation vials, 10 ml ScintiVerse E (Fisher Scientific) added and radioactivity counted in a scintillation counter. Sample protein was determined using the Bio-Rad protein assay [30].

Basal activity was deducted from Ca2+/phospho - lipid stimulated activity. The difference, indicative of PKC activity, is expressed as pmol PO 4 transferred/ min per mg protein.

Chemicals [y-32p]ATP (10 to 25 Ci/mmol)was obtained from

ICN Biomedicals, Canada. Phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, distearin, dipalmitin, dilinolein and diarachidonin were obtained from Serdary Research Laboratories, London, Ontario. All other chemicals used were from Sigma, St. Louis, MO.

Data presentation Data from representative experiments have been

presented as means of triplicate incubations which usually did not vary by more than 10%. All experiments were performed at least three times.

R e s u l t s a n d D i s c u s s i o n

The PKC enzyme preparation was stable for nearly 2 months when stored at -70°C in the presence of 20% glycerol. However, results reported in this paper were obtained with enzyme preparations that had been stored no more than 2 weeks. Fig. 1 shows the presence of PKC activity in the soluble fraction of CF1 cells. Calcium, PS, diolein and PMA alone failed to increase PKC activity. Phosphatidylserine (0.1 mM needed for maximal activation) in the presence of 0.5 mM Ca 2+ caused a 2-fold increase in enzyme activity whereas, in the presence of PS and 15/~M diolein, the enzyme activity was only marginally stimulated. On the other hand, the phorbol diester PMA, at 0.65 ~M and 6.5 /xM concentrations, activated the enzyme 4-fold over Ca2+/ps stimulated activity and 8-fold over basal activity.

A major peak of PKC activity was obtained follow- ing DEAE-cellulose chromatography (Fig. 2). The enzyme eluted at 0.04 M to 0.055 M NaCI, with a yield of approx. 40% and a 10-fold purification. The response of PKC to Ca 2÷ concentration is influenced by the lipid composition of membranes [27]. Calcium ions are

Activity 2 (xlO -~)

0 EGTA Ca Ca DG PMA PMA

PS (l) ~o) Fig. 1. Stimulation of protein kinase C activity in the cytosol of CF-I cells by 0.5 mM Ca 2+, 0.1 mMPS, 16 ~M diolein, 0.65 ~M and 6.5 ~M PMA. EGTA concentration was 0.5 raM. The enzyme activity is defined as pmol 32po~- from [I--32p]ATP incorporated into his-

tone/min per mg protein.

212

• 02 .04 .05 .06 .08 0.1M

CPM (xlO -2)

14

10

• ~ / o . °e°°°-o'°O°e*o'°'°'*e°°~- O"-wO,~ooOoo0 ooiO" 00"000000000.~0000000~000000~

• • | I I | I

0 5 10 15 20 25 30 35 40 45 Fraction Number

Fig. 2. DEAE-cellulose chromatography of protein kinase C. The high speed soluble fraction was applied onto a DEAE-cellulose column previously equilibrated with 20 mM Tris-HC1 (pH 7.5) containing 0.5 mM EGTA, 2 mM EDTA and 2 rnM PMSF. The column was washed with 15 ml of the buffer and eluted with a 0-0.1 M NaC1 step gradient. Fractions of approximately 1 ml were collected and 50/zl aliquot of each

fraction was assayed for PKC activity as described under Materials and Methods. (o) Basal activity and (o) PMA-PS.

required for the expression of enzyme activity in the enzyme assay. However, in the presence of PMA, the calcium dose response curve exhibits a shift to the left indicating that PMA greatly increased the apparent affinity of PKC for Ca 2÷ (Fig. 3). Therefore, in the presence of PMA the enzyme is activated at much lower concentrations of Ca 2+ which is in agreement with the characteristic of PKC from other tissues [32,36]. PMA alone was not able to activate PKC, but could do so in the presence of PS. The enzyme at- tained maximum activation in the presence of PMA at 10 ~M Ca 2+ and equilibrated up to a concentration of 1 mM Ca 2+, after which the activity rapidly dropped. In the presence of PS alone, maximal activity was reached at 500/zM Ca 2÷.

PMA activates the PKC of CF1 ceils both in vivo [24] and in the in vitro assay employed in this study. Kinetic analysis of the rat brain enzyme indicated that PMA, at nanomolar concentrations, behaves like DAG at micromolar concentrations by directly activating PKC

[6]. Fig. 4 shows the concentration-dependent enhancement of PKC activity in the presence of varying concentration of PMA. The enzyme was maximally activated at 650 nM PMA concentration and had a K a of 10 nM. The ability of PMA to enhance PKC activity in CF1 cells is consistent with studies using synaptosomal preparations of locust ganglia [24]. However, further comparisons between our study and [24] are difficult, as the latter does not report basal incorporation with Ca 2÷ and PS in the absence of PMA.

A potent inhibitor of PKC in vitro is 1-(5-isoquino- linesulfonyl)-2-methylpiperazine (H-7) [33]. Fig. 5 de- picts the effect of varying concentration of H-7 on PKC activity. A 50% inhibition of enzyme activity occurred at a concentration of approx. 52/xM H-7.

Diacylglycerols may serve as natural intracellular activators of PKC. PKC from various mammalian tissues [34] is greatly stimulated by addition of diacylglycerol at less than 5% (w/w) of the concentration of phospholipids. The effect is greatest for DAG which

Activity (xlO -2)

5

4

3

2

I

0 0.00

| | | | |

0.01 0.10 0.50 1.00 10.00 C a C i 2 (mM)

Fig. 3. Effect of varying Ca 2+ concentrations on PKC activity in the presence of PS (0) and PMA-PS (e). The final concentration of PS

was 0.1 mM and PMA was 6.5/~M.

100

s o J

Activation (%) 40 60 / ~

20

0 s t | | | s

10 35 150 350 650 1000 P M A (nM)

Fig. 4. Effect of varying PMA concentrations on PKC activity. The values correspond to percent activation over basal activity.

80

PKC(%) 60

Ac t iv i ty 40

20

0 0 20 40 60 80 100 120

H-7 (gM) Fig. 5. Effect of inhibitor H-7 on PKC activity. The enzyme was assayed in the presence of 0.5 mM Ca 2÷ and 0.1 mM PS and various

concentrations of H-7.

contains unsaturated fatty acid at least at position 2. The enzyme from rat cerebral cytosol which has been extensively studied exhibits a 3-fold activation in the presence of DAG (0.8 /~g/ml or 1 /xM) [27]. The effect of various diacylglycerols on PKC activity was studied (Table I). The final concentration of the diacylglycerols in the reaction mixture was 5 times greater than that used with mammalian PKC [27]. The results indicate that, at this concentration, insect PKC is not sensitive to any of the compounds tested. Since diolein at a concentration of 15 /~M (Fig. 1) marginally enhanced PKC activity, its effect at increasing concentrations was tested (Fig. 6) along with the effect of oleoyl-acetyl-glycerol (OAG) and dioctonoyl-sn-glycerol (DOG). The latter two were selected as they are known to activate PKC in other systems and are also more readily soluble than diolein. When 4 /~g of OAG (40 ~M final concentration) and 4 /zg of DOG (46 ~M final concentration) were used, a 3-fold activation over Ca2+/ps activity was observed, whereas 4 /xg of diolein (26/zM final concentration) enhanced PKC activity 2-fold. The magnitude of activation is similar to

TABLE I

Effect of various diacylglycerols on protein kinase C

PKC was assayed as described under Materials and Methods. After the chloroform, methanol or hexane was evaporated under nitrogen, the diacylglycerols were suspended in Tris-HCl (pH 7.5), by sonica- tion for 5 min at 0°C. The reaction mixture contained phosphatidylserine (20/zg) and the diglycerol indicated (1 /zg).

Treatment PKC activity

None 123.6 Diolein (6/zM) 130.0 Dilinolein (7/zM) 127.6 Diarachidonin (6/zM) 130.6 Oleoyl-acetyl glycerol (10/zM) 126.6 Dioctonoyl-sn-glycerol (11/.LM) 125.8 Dipalmitin (7/zM) 133.6 Distearin (6/zM) 101.0

213

mammalian PKC [27], but the concentration of diacylglycerols needed to achieve this is excessively high.

The lack of sensitivity of the enzyme preparation to DAG is unlikely to be due to solubility problems. The effect of DAG was extensively studied over a wide range of concentrations and in the presence and absence of Triton X-100. The experiments were also performed in the presence of varying concentrations of PS and Ca 2 ÷ (data not shown). Under all of the above conditions PKC did not appear to be greatly stimulated by DAG at concentrations used on mammalian PKC. The maximal activation (2.5-fold) was obtained at a high concentration of 26 /~M. In addition, PKC from rat brain was isolated and its response to DAG under our assay conditions was in agreement with that reported [27] (data not shown). However, the CF1-PKC enzyme was consistently sensitive to PMA, exhibiting a 4-fold activation over that obtained with Ca2+/PS. PKC is the phorbol diester receptor and DAG and phorbol esters interact at a common site on the receptor [6,7,37]. The ability of PMA to activate CF1-PKC, both in vitro and in vivo, and to translocate the enzyme from the cytosol to the particulate fraction [24] indi- cates the presence of a binding site(s) for phorbol esters. It may be reasonable to conclude that PKC from CF1 has a low affinity for DAG.

Although phosphatidylserine is the sole phospholipid effective for the activation of PKC [38] other species of phospholipids modulate the activation of the enzyme considerably [39]. The effect of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) (approx. 25, 50 and 100 /xM) on PKC was tested (Table II). PE alone did not activate PKC and when PS was supplemented with PE no enhancement in enzyme activity was observed. PI, appeared to interact directly and activate the enzyme at final concentrations of 50 /~M and 100/zM. However, when PI was added as a supplement to PS, no additive effect was seen. Kaibuchi et al. [39] reported enhancement of rat brain PKC

Activity 2 (x1°-2)

0 " " " ' " '

0 0.8 1.6 2.4 4

~g

Fig. 6. Effect of varying concentrations of diolein ( [] ), OAG ([]) and DOG (D) in the presence of 0.1 mMPS and 0.5 mM Ca 2+ on PKC

activity.

214

TABLE II

Specificity of phospholipid for activation of protein kinase C

PKC activity in the presence of 0.5 mM Ca 2+ was 38.4 and in the presence of 20 /zg (0.1 raM) phosphatidylserine (PS) was 102.4. Varying concentrations of phosphatidylethanolamine and phosphatidylinositol were tested in the presence and absence of PS.

Treatment Amount added - PS + PS ~g (/zM)

Phosphatidylethanolamine 5 (25) 40.8 103.6 10 (50) 38.0 107.0 20 (100) 38.6 105.6

Phosphatidylinositol 5 (25) 79.8 115.2 10 (50) 100.4 120.2 20 (100) 120.6 138.4

activity when PE was a supplement to PS but that PI had no effect at micromolar concentrations of Ca 2 +.

A key difference between insect PKC and the mammalian enzyme is that the former appears to have a low affinity for DAG. The CF1 cells used in the present study are embryonic and have been shown previously to have an adenylate cyclase that does not respond to forskolin [25]. Therefore, the PKC from this source may not be representative of all insect PKC.

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partial characterization of protein kinase c from an insect cell line

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