Synergistic activation of protein kinase C by arachidonic acid and diacylglycerols in vitro: generation of a stable membrane-bound, cofactor-independent state of protein kinase C activity

ELSEVIER

Biochimica et Biophysica Acta 1291 (1996) 167-176

Biochi~ic~a et BiophysicaA~ta

Synergistic activation of protein kinase C by arachidonic acid and diacylglycerols in vitro: generation of a stable membrane-bound, cofactor-independent state of protein kinase C activity Joel B. Schachter a,*, David S. Lester b, Daniel L. Alkon a a

Laboratory of Adaptive Systems, National Institute for Neurological Disorders and Stroke, Bethesda, MD 20892, USA b Division of Research and Testing, CDER, FDA, Laurel, MD 20708, USA

Received 30 January 1996; revised 2 May 1996; accepted 22 May 1996

Abstract The present study examines the synergistic activation of PKC by arachidonic acid and diacylglycerols in phospholipid vesicles and demonstrates that this combination of activators leads to the formation of a constitutively active, phospholipid-bound form of the enzyme. Activation of PKC was almost entirely calcium-dependent with vesicles containing dioleoylglycerol alone. In contrast, considerable calcium-independent activity was observed when vesicles contained both a diacylglycerol and free arachidonic acid. High-affinity association of enzyme activity with diacylglycerol-containing vesicles was calcium dependent and reversible. However, addition of arachidonic acid to diacylglycerol-containing vesicles resulted in irreversible PKC binding in the absence of calcium. Immunoblot analysis indicated that the calcium-independent binding was not isozyme-specific. The activity of the vesicle-associated PKC, bound to vesicles in the absence of calcium, was predominantly calcium-dependent. On the other hand, when the binding and isolation of vesicle-bound enzyme was conducted in the presence of calcium, the subsequent activity was almost entirely resistant to calcium chelation. This vesicle-associated form of the enzyme, when detergent extracted and recombined with phospholipid vesicles, maintained significant 'constitutive' activity (activity in the absence of both diacylglycerol and calcium). The data from this in vitro system provide the basis for a model of the physiological regulation of PKC in which the combined actions of arachidonate and diacylglycerol facilitate the stable formation of a tightly membrane-associated, intrinsically active form of PKC. Keywords: Kinase C; Diacylglycerol; Arachidonic acid; Calcium; Lipid; Vesicle

1. Introduction Previous studies have suggested that a persistent activation of protein kinase C might be involved in sustained changes in neuronal activity (see [1,2]). While the importance of protein kinase C (PKC) as a mediator of a variety of cellular responses is widely recognized, there is still an incomplete understanding o f how physiological processes regulate the activity of this enzyme (see [3,4]). The protein has long been described as a calcium and phospholipid-de-

Abbreviations: AA, arachidonic acid; DAG, diacylglycerol; DOG, 1,2-dioleoylglycerol; DPG, 1,2-dipalmitoylglycerol; DPPS, dipalmitoylphosphatidylserine; OArG, 1-oleoyl-2-arachidonoylglycerol; PC, phosphatidylcholine; PKC, protein kinase C; PS, phosphatidylserine; SAG, 1-stearoyl-2-arachidonoylglycerol;TBS, Tris-buffered saline. * Corresponding author. Present address: Department of Pharmacology, Univ. of North Carolina, Chapel Hill, NC 27599-7365, USA. Fax: + 1 (919) 9665640.

pendent enzyme which is activated by the products of phospholipase C action on phosphatidylinositol. Most models for this activation process involve conversion of the protein from an inactive, soluble form to an active form which binds to membranes and to phospholipid-containing vesicles or micelles in a calcium-dependent, reversible manner [5-9]. The soluble form of the enzyme is typically observed following tissue disruption in an EGTA-containing buffer. However, PKC is also found in a membrane-associated form which is not released by calcium chelation [10,11]. In neuronal tissue almost 50% of PKC occurs as the chelator-resistant, membrane-bound form (see [12]). Long-lasting changes in the ratio of soluble to membrane-bound PKC have been reported in response to physiological events such as electrophysiological and behavioral training paradigms [13-15]. F r o m where, then, does the population of chelator resistent, membrane bound PKC derive? Existing models of the activation

0304-4165/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0304-4165(96)00063- 3

168

J.B. Schachteret al. / Biochimica et Biophysica Acta 1291 (1996) 167-176

process do not provide an explanation for the formation of this long lasting, membrane-bound form during normal physiological processes. Neither can this form be attributed to specific isozymes having greater membrane affinity since the soluble and membrane-bound forms possess essentially the same distribution of isozymes [16,17]. The pharmacological generation of this form by phorbol esters or high calcium concentrations has been examined in some detail [18-20]. However several groups have shown that diacylglycerol, the physiological activator of PKC, does not promote the formation of the irreversibly bound form (e.g., [21-24]). The inability of diacylglycerol to generate a membrane-inserted form of the enzyme may indicate that other messengers are involved in the production of this species. A number of reports have appeared of PKC activation by cis-unsaturated free fatty acids (CUFA) in solution [25-30], however this activity requires rather high concentrations (50 to 300 IxM) of the fatty acids. Interestingly, CUFA have been reported to potentiate DAG-dependent PKC activity at more physiologic concentrations [1,31-34]. This synergism has also been demonstrated in two established neuronal systems where long term PKC activation has been implicated in sustained electrophysiologic changes [1,2]. Synergistic activation of PKC by arachidonate and diacylglycerol has now been further characterized in a well-defined model membrane bilayer system with the objective of identifying and characterizing relationships between membrane association and enzyme activity states. This model system is intended to represent the environment of a cellular membrane bilayer during the activation process. The stimulation of both phospholipases A 2 and C during cellular activation releases free fatty acids and various diglyceride species within the cellular membrane bilayer which ultimately undergo further metabolism/reacylation. The model membrane system used here creates a static bilayer composition containing phospholipids, diglycerides and fatty acid species. There are no 'soluble' fatty acids in this system. This is an important distinction from other studies of fatty acid actions on PKC activity. Additionally, this study has utilized a preparation of PKC activity purified from brain tissue, rather than isozymes from a baculovirus expression system, since notable differences have been reported in the activator and substrate properties of enzymes from these two sources [35].

2. Materials and methods 2.1. Materials

Lipids were obtained from Avanti Polar Lipids (Birmingham, AL), arachidonic acid from Nu-Check Prep (St. Elysius, MN). Immunologic reagents and detergents were from Pierce (Rockford, IL). All other reagents were from

Sigma (St. Louis, MO). All experiments were performed with the same batch of histone (Lot #82H8020) as very significant variation in results was noted for different lots. 2.2. P K C purification and assay

PKC was purified from the cytosolic fraction of rat brain, essentially as described by Lester [33]. Brains of twenty-five Wistar rats (42 days old) were homogenized in ice-cold buffer containing the following: 20 mM Tris (pH 7.5), 200 mM sucrose, 10 mM EGTA (pH 7.5), 2 mM EDTA (pH 7.5), 0.3% mercaptoethanol, 1 mM PMSF, and 10 ~xg/ml leupeptin. Following centrifugation at 100000 x g for 1 hour, the supernatant was applied to a DEAE Sepharose column (2.5 x 12 cm) which was washed and eluted with a 0-300 mM NaC1 gradient in 20 mM Tris (pH 7.5), 0.5 mM EGTA, 0.5 mM EDTA, 0.3% mercaptoethanol, 1 mM PMSF (Buffer A). The active fractions from the eluent were pooled at a KC1 concentration of 1.5 M, clarified by centrifugation, and applied to a phenyl Sepharose column (1.25 X 15 cm). This was eluted with a reverse KCI gradient (1.5 M to 0). The active fractions were concentrated, made to 10% with glycerol and run over an ACA 34 gel-exclusion column (2 X 180 cm) equilibrated with 10% glycerol in buffer A. The active fractions were pooled and snap-frozen as aliquots in liquid nitrogen. SDS-PAGE revealed one major band at approx. 80 000 daltons. Activity was assayed in a 250 txl volume containing Buffer A adjusted to 0.1 mM free calcium, 5 mM MgCI 2, 1 mM dithiothreitol, 20 IxM [,,/_32P]ATP (1 ~Ci/ml), 100 IxM lipid vesicles and 25 Fg Histone type III-S. Assays without calcium utilized the same mixture, minus calcium and with 0.51 mM EGTA. The reaction was carded out for 4 min at 30°C and was terminated with 1 ml of ice cold 10% trichloroacetic acid. After 30 min at 4°C the assays were filtered over 0.45 micron nitrocellulose, washed 4times with 5% TCA, and assessed by Cerenkov counting. 2.3. Preparation o f vesicles

Multilamellar vesicles consisting of PS:PC (1:4) were prepared by evaporating chloroform stock solutions under nitrogen and drying under vacuum, followed by vortexing and brief sonication in 20 mM Tris (pH 7.5), 0.5 mM EGTA, 0.5 mM EDTA, calcium-free buffer. Both diacylglycerols and arachidonic acid were added as chloroform stocks before the evaporation in order to assure their homogeneous incorporation into the lipid bilayers. Stock solutions (20-fold concentrated) containing 2 mM phospholipids (0.4 mM PS, 1.6 mM PC, plus up to 0.1 mM DAG a n d / o r 0.2 mM AA) were prepared fresh daily. The rest of the assay mixture was not added until immediately before initiation of the assay. This was to ensure that added Ca 2+ would not have to time to bind and form Ca 2+-PS complexes. The addition of fatty acids and DAGs

J.B. Schachteret al. / Biochimica et Biophysica Acta 1291 (1996) 167-176

were done in conjunction with a decrease in the PC content so as to keep the PS and total lipid concentration constant. 2.4. Binding of PKC to vesicles

PKC binding to vesicles was assessed by following PKC activity eluting from AcA-44 gel columns. Enzyme (5 tzg protein) and vesicles (80 txg phospholipid) in 20 mM Tris, 5 mM MgC12, 0.51 mM EGTA, and with or without 0.62 mM CaCI: (giving 0.1 mM free calcium) were preincubated 5 min at 30°C in a volume of 300 Ixl. They were then run over AcA-44 columns (1 × 50 cm), preequilibrated with the same buffer, supplemented with 2% glycerol and 0.1 m g / m l BSA. The eluents were assessed for activity in the presence of either the same vesicles used for the preincubation or, in the case of vesicles containing no diacylglycerol, with vesicles and 100 nM phorbol dibutyrate. The columns were calibrated with blue dextran and phenol red and ran identically and reproducibly. Free enzyme ran within the included volume of the columns, while vesicles eluted in the void volume. 2.5. Detergent extraction of bound PKC and recombination with lipid vesicles

Enzyme (500 ng protein) and vesicles (2 Izg phospholipid) in a volume of 20 ixl were preincubated at 30°C for various times in the presence (0.1 mM free Ca 2+) or absence (0.5 mM EGTA) of calcium. The incubation was terminated by the addition of 1% NP-40 (at a molar ratio of 170:1, detergent to phospholipid) and extraction was allowed to proceed for 60 min on ice. Subsequently, a portion of the mixture (3.5%) was diluted (700-fold) into the assay solution containing 100 IxM (20 Ixg) phospholipid. 2.6. lmmunoblotting of vesicle-associated PKC isozymes

Samples (100 Izl) were taken from the combined fractions (6-11) of AcA-44 column eluates from the following lipid incubations; DPG + Ca 2÷, DPG + AA, DPG + AA + Ca 2÷, OArG, OArG + Ca 2÷, OArG + AA. These conditions were considered to be representative of the various states of the enzymes with the different lipid and Ca 2÷ mixtures tested. The samples were diluted with 200 pA of Tris-HCl, 20 mM (pH 7.5), EDTA 0.5 mM, loaded into a Bio-Rad multiwell dot blot apparatus, and slowly filtered through nitrocellulose paper (Scheicher and Schuell, 0.22 mm). The nitrocellulose was cut into strips and then treated for 1 h with SuperBlock (Pierce, Rockford, IL) followed by three rinses in wash buffer (Tris-buffered saline [TBS] plus 0.1% bovine serum albumin and 0.05% Tween-20) at room temperature. Primary antibodies were either purchased from Gibco (anti-or and -13), or were generously provided as a gift by Dr. David Burns at

169

Sphinx Pharmaceuticals in Durham, NC (anti-y, -& -~ and -~). These antibodies were diluted (l:400) in wash buffer and incubated with the nitrocellulose strips for 1 h at room temperature. The strips were rinsed 5-times with wash buffer and placed in secondary antibody (biotinylated goat anti-rabbit for 1 h as above. Five rinses were repeated. Avidin-biotin conjugated alkaline phosphatase (Pierce, Rockford, IL) was added for 30 min as per the instructions supplied and the strips were again rinsed 5-times. The color reagent, NBT-BCIP was added and the reaction was allowed to proceed for 20 min before rinsing and storing in deionized water. 2.7. Quantitative analysis of dot immunoblots

Strip blots were digitally scanned at 300 dpi into NIH Image (public domain software) and stored as TIFF images (tagged image file format). These TIFF images were transferred to an M1 Imaging System (Imaging Research, St. Catherines, Ont., Canada) and analyzed using graphic gel analysis transept bars. Briefly, images were inverted to scale blots appropriately on an 8 bit image scale. Transept bars were placed over the strip blot, so as to encompass the blots in their entirety. Baseline was substracted automatically using a point of inflection algorithm. Baseline-subtracted plots were then produced of the entire strip, such that average blot intensities could be compared within individual strips.

3. Results 3.1. Synergism between arachidonate and diacylglycerol: role of the diglyceride fatty acid content

In the vesicle system employed here, PKC activity was entirely dependent upon the presence of phospholipid, diacylglycerol, and calcium [36]. Consistent with a previous report using this model system [1] arachidonic acid at concentrations of 5 - 2 0 IxM had no effect alone (not shown), but caused a twofold enhancement of dioleoylglycerol-dependent PKC activity (Fig. 1A). This effect was also seen with the polyunsaturated linoleic acid, but not with the fully saturated stearic or palmitic acids [33]. To define the role of the diglyceride acyl chain composition in the synergy, four diacylglycerols with varying degrees of acyl chain unsaturation were compared. In the absence of free arachidonic acid, the calcium-dependent responses to diacylglycerols varied with the acyl chain composition (Fig. 1). Both of the polyunsaturated diacylglycerols 1stearoyl-2-arachidonoylglycerol (SAG, ECs0 -- 0.5 t~M and Vmax = 0.72 ixmol/min per mg) and 1-oleoyl-2arachidonoylglycerol (OArG, ECs0 = 0.2 I~M and Vmax = 0.68 ixmol/min per mg) were more potent and more efficacious than diolein (DOG, EC50 = 1.3 p~M and Vmax = 0.48 Ixmol/min per mg), whereas the fully saturated dipalmitin (DPG, ECs0 = 1.8 IxM and Vrnax = 0.39

J.B. Schachter et al. / Biochimica et Biophysica Acta 1291 (1996) 167-176

170

A

B 1.2-

E ~., t,,F=

1.2 E

1.O, 0.8

E

1.0

rE

-5

0.8

E

-'I v

=

0.6

v

r-

0

0.6

0 0.4

•~

0.4

0 r-

~- 0.2

0.2

.m

~-

0.0

o

~

4&

&

1'o

re,.

1

D

[DOG] uM

C

0.0

-.~

0.8

~

=E

0.6

=

0.6

0.4.

,~

0.4

0.2

-~ ~

0.2

~-

0.0

._

,~ O

x::~. or-

0.0

0

2

4

6

8

I0

[SAG] uM

2

3

4

5

[DPG] uM

0

1

2

3

4

5

[OArG] uM

Fig. 1. Comparison of diglyceride species for synergism with arachidonate. (A) Dose-response curves to 1,2-dioleoyl-sn-glycerol (18:1, 18:] DOG) without (circles), or with (squares), I0 IJ-M AA and in the absence (0.5 mM EGTA, open symbols), or presence (closed symbo]s), of I00 I~M free calcium.

(B) Same as in (A) but with 1,2-dipalmitoyl-sn-glycerol(16:0, 16:0 DPG) as the diglyceride. (C) As above with 1-stearoyl-2-arachidonoyl-sn-glycerol (18:0, 20:4 SAG). (D) With 1-oleoyl-2-arachidonoyl-sn-glycerol(18:1, 20:40ArG).

ixmol/min per mg) was the least potent and least efficacious diacylglycerol tested. As in the case of diolein, dipalmitin's activity was almost completely calcium-dependent and only a small degree of calcium independent activity was observed upon the addition of free arachidonate (Fig. 1B). In contrast, the arachidonoyl-containing diglycerides exhibited considerable calcium-independent activity in the absence of free arachidonate (up to 0.45 ixmol/min per mg for OArG). With the further addition of free arachidonate, the calcium-independent activation by these diglycerides (0.8 izmol/min per mg, Fig. 1C and D) was nearly as high as the maximal activity observed in the presence of arachidonate and 100 IxM calcium (0.95 ixmol/min per mg). Because the addition of arachidonate does not alter the cofactor requirements of the enzyme (the K m values for calcium and ATP are unchanged, data not shown) this 'ceiling effect' for activation may indicate that, in the presence of a polyunsaturated diacylglycerol, arachidonic acid promotes activation of nearly the entire population of enzyme in the absence of calcium. Thus, unsaturation in both the diglyceride acyl chains and in the free fatty acid

eliminate the calcium requirement for activation of soluble PKC without affecting the K m for calcium.

3.2. Effects of calcium, diacylglycerol, and arachidonate on PKC binding to phospholipid vesicles In an attempt to differentiate whether the observed synergism was due to enhanced catalytic activity or to enhanced enzyme-lipid interactions, the binding of PKC to vesicles was assessed by determining the distribution of enzyme activity after gel-exclusion chromatography (see Fig. 2). In the absence of vesicles the enzyme ran in the included, volume of the column (fractions 12-18, not shown), whereas in the presence of vesicles containing activators, a n d / o r calcium (100 IxM), a significant portion of the enzyme activity eluted in the void volume, indicating enzyme-vesicle binding. By assessing the activity of the enzyme in the void and included volumes under identical conditions the relative amounts of bound and free enzyme could be quantified, regardless of the efficacy of the activating conditions. Table 1 shows the results of a number of such experiments. Overall, enzyme binding to

J.B. Schachter et al. / Biochimica et Biophysica Acta 1291 (1996) 167-176 15000

A

• I~

OArG + Calcium OArG ÷ EGTA

]

I

Table 1 Distribution of enzyme activity after chromatography through AcA 44 Vesicle

EGTA Peak I:Peak I1

Calcium Peak l:Peak II

PS/PC PS/PC/AA PS/PC/DPG PS/PC/DPG/AA PS/PC/OArG PS/PC/OArG/AA

2:98 4:96 7:93 33:67 32:68 66:34

28:72 23:77 69:31 63:37 68:32 76:24

10000 a. O

5000

i ....

0 0

5

10

15

Fraction

30000

B

[

20

25

,

30

Number

~.

OArG/AA + Calcium

T~

OArG/AA + EGTA

20000' 5 O. O

171

Enzyme binding to vesicles of varying compositions was assessed as described in Fig. 2 and in methods. Activity eluting from ACA 44 columns was determined with 100 I~M calcium either in the presence of PS:PC vesicles plus 100 nM phorbol dibutyrate (for assay of enzyme bound to PS:PC and PS:PC/AA vesicles) or in the presence of vesicles of the same composition as for the binding preincubation. Relative peak ratios (% of total activity measured) are reported for the enzyme activity from fractions 7 through 10 (Peak I, void volume) versus that in fractions 12 through 18 (Peak II, included volume). Data shown are averages of three column runs each. The ranges for the values shown are less than 5%.

10000

0'

' , ....

0

5

10 Fraction

15

20

25

,

30

Number

Fig. 2. Effects of calcium and arachidonic acid on the elution profiles of PKC from ACA 44 gel-exclusion columns following preincubation with phospholipid vesicles. PKC (5 ~g) was preincubated at 30°C for 5 minutes in the presence or absence of calcium with PS:PC vesicles containing OArG plus or minus AA. The various preincubation mixtures were then run over one of two 1 X 50 cm columns of ACA 44, equilibrated in the absence (0.5 mM EGTA, open squares) or presence (closed circles) of 100 IxM free calcium. Free enzyme ran in the included volume (fractions 12-18, not shown), while vesicles eluted in the void volume (fractions 7-10). Data shown are from representative column runs using PS:PC vesicles containing 5 mol.% OArG made in the absence (A) or the presence (B) of 10 mol.% AA.

v e s i c l e s w a s p r o m o t e d b y c a l c i u m a n d w a s f u r t h e r stabil i z e d b y d i a c y l g l y c e r o l . A l t h o u g h the free fatty a c i d d i d n o t p r o m o t e b i n d i n g b y itself, a d d i t i o n o f a r a c h i d o n i c acid to d i p a l m i t o y l g l y c e r o l - c o n t a i n i n g v e s i c l e s i n c r e a s e d calc i u m - i n d e p e n d e n t b i n d i n g . C o m b i n a t i o n o f free a r a c h i d o n a t e w i t h the a r a c h i d o n y l - c o n t a i n i n g d i g l y c e r i d e , O A r G , r e s u l t e d in the m a j o r i t y o f the e n z y m a t i c a c t i v i t y e l u t i n g in the v o i d v o l u m e in the a b s e n c e o f c a l c i u m (see Fig. 2B, o p e n squares). T h i s s u g g e s t s t h a t the c o m b i n a t i o n o f free arachidonate with an arachidonyl-containing diglyceride leads to the f o r m a t i o n o f a t i g h t - b i n d i n g c o m p l e x b e t w e e n these vesicles and PKC which no longer requires calcium for stabilization.

3.3. Actiuity o f b o u n d enzyme: role o f calcium A s c a l c i u m is n o t r e q u i r e d for P K C b i n d i n g to v e s i c l e s c o n t a i n i n g O A r G + A A , the r e l a t i v e roles o f c a l c i u m in

b i n d i n g v e r s u s t h a t in a c t i v i t y m i g h t b e d i s c r i m i n a t e d . T a b l e 2 ( p a r t A ) s h o w s a p a r t i a l c h a r a c t e r i z a t i o n o f the a c t i v i t y e l u t e d in the v o i d v o l u m e f r o m c o l u m n r u n s o f vesicles containing l-oleoyl-2-arachidonoylglycerol ( O A r G ) a n d a r a c h i d o n i c acid. L e s s total a c t i v i t y w a s m e a s u r e d w h e n the initial i n c u b a t i o n a n d the c o l u m n e l u t i o n w a s c o n d u c t e d in the a b s e n c e o f c a l c i u m (top r o w o f T a b l e 2). T h i s a c t i v i t y w a s e n h a n c e d 2 . 5 - f o l d b y i n c l u s i o n o f c a l c i u m in the a s s a y m i x t u r e . W h e n c a l c i u m ( 1 0 0 IxM) w a s i n c l u d e d in the initial i n c u b a t i o n ( b e f o r e r u n n i n g the s a m p l e o v e r the c o l u m n ) as well as in the e l u t i o n buffer, the a c t i v i t y in the v o i d v o l u m e w a s i n d e p e n d e n t o f a d d e d

Table 2 Calcium dependence of PKC activity after preincubation with vesicles containing OArG plus AA Pretreatment

Assay

A. Column

no calcium

with calcium

No calcium With calcium

(cpm× 10 -3 ) 8.2 (1.3) 57.9 (3.0)

20.0 (1.1) 59.9 (3.4)

B. Preincubation

no calcium

with calcium

No calcium With calcium

(pmol) 7.7 (0.8) 44.5 (5.3)

66.4 (1.5) 75.3 (2.3)

A. Activity eluting from the columns used to generate the data for Fig. 2, panel B, was assessed for its dependence on calcium by assaying the peak fractions in the presence of calcium or of excess EGTA. Because the protein content of these fractions could not be measured, the activity was expressed as cpm for a 30 minute assay. Errors are shown in parentheses. B. Activity assessed after preincubation with lipids, but without passing the sample through the column. Following preincubation for 15 min at 30°C, a portion of the mixture (17.5 ng of enzyme) was diluted into the assay (15 min).

172

J.B. Schachter et al. / Biochirnica et Biophysica Acta 1291 (1996) 167-176

Table 3 Cofactor dependence of activity following preincubation with OArG 4- AA vesicles and detergent solubilization/dilution Preincubaton

Assay

lipid/calcium

PS:PC ( - )

PS:PC ( + )

OArG/AA ( - )

OArG/AA ( + )

No/Yes Yes/No Yes/Yes

(pmol) 3.8 (1.4) 4.1 (2.0) 44.8 (4.7)

6.2 (2.2) 16.9 (2.7) 48.9 (3.3)

101.1 (6.9) 101.5 (5.1) 105.8 (8.2)

235.2 (5.1) 243.8 (10.6) 223.5 (12.8)

PKC was preincubated without or with vesicles containing 2 IxM OArG and 10 IxM AA for 15 min at 30°C in the absence and presence of calcium. The preincubation was stopped by the addition of 1% NP-40 for 1 h on ice. The extract was then diluted 700-fold into assay mixtures containing vesicles of the indicated compositions without ( - ) or with ( + ) calcium. Data shown are from 3 experiments. Standard errors are indicated in parentheses.

calcium or of excess EGTA (bottom row of Table 2A). This appears to be a specific property of the vesicle-bound portion of enzyme isolated by gel filtration, since a simple preincubation of enzyme with lipid (with no gel filtration step) does not generate a completely calcium-independent enzyme population (Table 2, part B).

3.4. Characterization of stabilized activity states by detergent extraction and dilution with PS:PC vesicles The data in Tables 1 and 2 suggested that in the presence of OArG and AA, PKC binds tightly to phospholipid vesicles in two different fashions which are influenced by the calcium availability at the time of binding. One state, formed in the absence of calcium, has reduced overall activity, but is sensitive to added calcium. The other state, formed in the presence of calcium, has a high level of activity which is independent of the continued presence of free calcium.

The examination of vesicle-bound enzyme does not allow a determination of the role of the lipid activators in maintaining these states because the activity was assessed in the continued presence of the activating vesicle composition. To circumvent this, PKC was first allowed to bind to vesicles containing OArG + AA and then was solubilized from these vesicles with an excess of detergent. The solubilized enzyme was then added back to intact PS:PC vesicles (containing neither diacylglycerol nor arachidonic acid) in such a manner as to dilute the detergent below its critical micelle concentration (0.018%), and to dilute the OArG and AA well below their effective levels (about 50 nM and 1 IxM, respectively). Table 3 contains data from experiments utilizing the extraction/dilution technique. When the binding of PKC to OArG + AA vesicles was conducted in the presence of calcium [Preincubation: Lipid-yes, Calcium-yes], the subsequently extracted enzyme showed significant activity following dilution with PS:PC vesicles in the absence of calcium (excess EGTA)

80

nB

J 0

E e-

0

>. L 0 e" c'a g~ 0

OArG + A A OArG 2 uM

60-

40

"-

DPG + A A

•

PS:PC + A A

20 tO

~

---o~

DPG 10 uM PS:PC

U'I

~

o 0

S

10

15

t

1 5 " + 3'

Preincubation Time (minutes) Fig. 3. Time-course for the development of cofactor-independent activity as assessed by detergent extraction and dilution with PS:PC vesicles, Enzyme was preincubated with the indicated lipids for various times in the presence of 100 I,LM free calcium (see Section 2). 1% NP-40 was then added to solubilize the vesicles. The solubilized DAGs were diluted below their effective concentrations and the detergent was reduced below its CMC by a 700-fold dilution in the presence of PS:PC vesicles. Enzyme activity was immediately assessed in the absence of calcium (0.5 mM EGTA). Preincubations were for 2, 5, 10, and 15 min at 30°C and an additional preincubation was carried out for 15 min at 30°C followed by 3 h at 4°C (15" + 3'). Preincubations were conducted with vesicles composed of PS:PC alone (circles), or of PS:PC with 10 tzM DPG (triangles) or 2 txM OArG (squares). Vesicles were prepared in the absence (open symbols) or presence (closed symbols) of 10 tzM AA.

J.B. Schachteret al. / Biochimica et BiophysicaActa 1291 (1996) 167-176 [Assay: PS:PC ( - ) ] . The activity measured in PS:PC vesicles was relatively unaffected by the addition of calcium [Assay: PS:PC ( + ) ] unless the assay vesicles were supplemented with OArG + AA. Conversely, when the preincubation was carried out in the absence of calcium (excess EGTA) [Preincubation: Lipid-yes, Calcium-no], the extracted/reconstituted enzyme showed minimal activity in the absence of activators (less than 10% of that seen when the preincubation contained calcium). This preincubation did not cause enzyme inactivation since preincubated enzyme was still capable of full activity. Dilution with OArG + A A vesicles fully stimulated PKC activity to the level seen for enzyme exposed to a sham procedure (where phospholipid vesicles were not added until after the detergent [Preincubation: Lipid-no, Calcium-yes]). In addition to being dependent upon the presence of calcium during the preincubation, the development of the cofactorindependent activity was dependent both upon the time of preincubation with vesicles and upon the lipid composition of the vesicles (Fig. 3). Arachidonic acid enhanced the kinetics of this process, particularly when the fatty acid was esterified to the diacylglycerol. The time dependence and calcium requirement during the preincubation phase of this protocol suggests that the activity, observed after dilution with PS:PC vesicles in EGTA, is not merely due to the carryover of small amounts of OArG (about 3 nM) into the assay. Rather, the data suggests that a long-lasting change has occurred in the enzyme. Alternatively, a portion of the enzyme may have bound OArG so tightly that the protein-lipid interaction cannot be effectively solubilized with 1% NP-40. This latter interpretation is, however, not supported by the fact that the same lasting increase of

Table 4 Densitometric analysis of immunoblotting for different isozyme species in the vesicle-associatedpools PKC isozyme Condition DPG +Ca 2+ DPG + AA DPG+AA+Cae+ OArG OArG+Ca2+ OArG + AA

10 5 25 12 110 32

5 8 7 15 140 80

6 9 14 30 100 80

2 2 9 3 6 4

4 5 10 8 11 10

4 6 9 8 10 10

Dot immunoblots of the vesicle-boundPKC isozymes were conducted by sampling the void volumes of the AcA-44 column runs described in Fig. 2 and Table 1. Six sets of column fractions were tested representing different combinations of lipids and calcium. Images of the blots were scanned and quantified as described in Section 2.7. Numerical values represent relative staining intensities (relative optical density units). Data shown are averages of three series of immunoblots made from three separate runs for each of the lipid and Ca2+ conditions.The control value (from a column run with vesicles but without enzyme) has been set to 0.0, as its intensity was equivalent to background. The value for the 3, isozyme associated with OArG+ Ca2+ vesicles has been arbitrarily set to 100. All other values are relative to this one. Standard errors were all less than 10% of the values shown.

173

activity was observed when the enzyme was preincubated for a more prolonged time (15 min at 30°C followed by 3 h at 4°C) with PS:PC vesicles containing no diglyceride (Fig. 3, open circles). 3.5. Isozyme immunoblotting of the vesicle-associated PKC Three calcium-dependent (ot,13,~/) and three calcium-independent (~,¢,~) isozyme species were examined by immunoblots of the vesicle-bound fractions eluting from the gel exclusion columns. All of these isozymes were readily detectable in the enzyme preparation that was used for this study. However, a comparison of the immunostain intensities of the various isozymes associated with the vesicles to those in the stock enzyme preparation, suggested that under the conditions examined the Ca2+-independent species (~,~,~) may be associated with the vesicles in significantly lower concentrations than those of the Ca 2+dependent forms. While the calcium-dependent forms showed large changes in their vesicle association under various conditions, the calcium-independent forms were generally less affected (see below). This would be consistent with the idea that the fatty acid-diglyceride induced Ca2+-independent activity was not due to selective activation of Ca2+-independent isozymes (see Section 4). In the presence of diglyceride alone (OArG), vesicle binding of the oL, 13, and ~/ isozymes was prominently enhanced by calcium (see Table 4). The addition of free arachidonic acid dramatically enhanced binding of the et,13, and ~/ isozymes in a calcium-independent manner. Interestingly, arachidonic acid also increased binding of the 8, e, and ~ isozymes to DPG vesicles in the presence of calcium. Detailed examination of the data in Table 4 suggests that the formation of a stable, membrane-bound form of PKC may occur for all isozyme species in a manner which differs in the specific composition of the activators for each isozyme. Due to the relative amounts of the various isozymes associated with the vesicles, it appears that most of the calcium-independent activity observed in the presence of OArG (with or without arachidonate) may be attributed to the membrane association and activation of the so-called Ca2+-dependent isozymes (ct, 13 and -y). Definitive proof of this assertion awaits a detailed examination of each of the cloned isozymes.

4. Discussion 4.1. Role of calcium in the synergistic activation of PKC by arachidonic acid and diglycerides This investigation into the properties of arachidonic acid potentiation of diacylglycerol-stimulated PKC activity suggests that a reevaluation of the commonly accepted models of PKC activation is necessary. According to the existing models [5-9] calcium binding to the enzyme

174

J.B. Schachter et al. / Biochimica et Biophysica Acta 1291 (1996) 167-176

results in a reversible conformational change that promotes membrane association, as a complex of enzyme, calcium, and PS, and subsequently allows enzyme activation by DAG within the membrane. Thus, although enzyme interaction with DAG is thought to reduce the calcium requirement for activation, calcium is still considered to be an essential cofactor for both membrane binding and catalytic activity. In the present studies we have demonstrated that the addition of arachidonic acid to diacylglycerol-containing vesicles resulted in calcium-independence for both membrane-binding (see Table 1) and histone phosphorylating activity (see Fig. 1). Calcium independence was observed regardless of whether the arachidonate was esterifled to the diacylglycerol or was present in the unesterified (free fatty acid) form, and was greatest when present together in both forms. In the present study, calcium-independent activation was elicited by physiologically relevant agents which were totally partitioned into phospholipid bilayer structures. Thus the enzyme interacts with the bilayer, even in the absence of calcium. Properties of the bilayer must, therefore, play a role in enzyme binding and activation. This obviates the requirement for a calcium-induced conformational change of the soluble enzyme, leading to membrane association. Calcium merely increases the magnitude of these responses, perhaps by enhancing the affinity or kinetics of the protein-lipid interaction.

4.2. Role of calcium in PKC binding to phospholipid vesicles Two types of binding of PKC to phospholipid vesicles were observed in this study (Table 1). The first type, represented by the binding of PKC to PS:PC vesicles was of rather low affinity, based upon the small percentage of enzyme remaining bound to the vesicles after gel filtration. DPG enhanced the association as evidenced by the increased percentage of enzyme co-eluting with the vesicles in the void volume. For either of these vesicles, the binding was completely dependent on the presence of calcium and could be fully and rapidly reversed by the addition of EGTA (not shown). A second type of binding began to be evident when DAG-containing vesicles were supplemented with arachidonic acid. The free fatty acid promoted a dramatic increase in the percentage of enzyme activity co-eluting with vesicles in the presence of excess EGTA, suggesting that arachidonic acid promotes an enzyme-vesicle interaction of high affinity in the absence of calcium. In the presence of both OArG and free arachidonic acid, this high affinity type of binding became prominent, showing nearly complete calcium-independence (the majority of enzyme activity eluted in the void volume in the presence of EGTA).

4.3. Relationship between binding and activity Comparison of the data in Table 1 with that in Fig. 1 indicates that, in contrast to the low affinity, calcium-de-

pendent binding of PKC to vesicles induced by diacylglycerol alone, the combination of arachidonic acid with diacylglycerol promotes a higher affinity, calcium-independent binding of enzyme to vesicles. This binding corresponds with both a calcium-independent activity and with a synergistically increased activity in the presence of calcium. In the case of enzyme bound to vesicles containing both free and esterified arachidonate (EGTA-insensitive binding), two types of activity states were observed (Table 2). When binding occurred in the absence of calcium, the activity of the bound enzyme was predominantly calciumdependent. When binding occurred in the presence of calcium the enzyme was more active and this activity was almost entirely resistant to EGTA. Not even detergent treatment and subsequent dialysis reversed this activity. This suggests either that the vesicle-bound enzyme sequesters calcium so as to make it inaccessible to chelation, or that a stable active conformation has been established which no longer requires calcium. Support for a stable conformational change is provided by the data in Fig. 3 which demonstrates the time-dependence of the formation of a persistently activated enzyme state. The formation of this state is greatly facilitated by, but is not absolutely dependent upon, the presence of diacylglycerol and arachidonic acid. The fact that the effect is both time-dependent and dependent upon the presence of calcium in the preincubation indicates that the extracted, reconstituted activity is not simply due to carryover of trace amounts of diacylglycerol. Additionally, this activity state could be established by prolonged incubations of enzyme with vesicles containing no diglyceride. The recognition of two activation states of vesicle-bound PKC is reminiscent of two forms of PKC extracted from EGTA-treated rat brain membranes by exposure to high pressures [37]. One form extracted from the membrane behaved in the same manner as the soluble form, while the second form extracted in this manner remained tightly associated with lipids and exhibited cofactor-independent activity. We may only speculate that these two forms of PKC found in EGTA-treated biological membranes are related to the two activity states seen for the chelator-resistant binding described here in vesicles.

4.4. Role of PKC isozymes While the different isozymes of PKC may exhibit some quantitative differences in their lipid binding and activation properties, the phenomena examined in this study do not appear to be specific for individual Ca2+-dependent (eL, [3, and ~/) or Ca2+-independent (8, e, 4) isozymes. No qualitative differences between these isozymes have been shown in previous studies using this liposome system [23,38]. While we cannot rule out this possibility, it seems less likely that the calcium-independent isozymes of PKC are the major source of the activity measured here since they appear to represent only a small percentage of the

J.B. Schachter et al. / Biochimica et Biophysica Acta 1291 (1996) 167-176 PKC immunoreactivity associated with the vesicles, and since several studies have shown that histone is a poor substrate for these isozymes [39-42]. Western blot analysis confirmed that calcium-dependent isozymes of PKC remained vesicle-associated following binding and gel filtration in the continuous presence of EGTA. Calcium-independent activity has been previously reported for the isolated e~ isozyme o f PKC following phorbol ester activation [20] and for the 13 and ~/ isozymes following activation by short-chain PC micelles [43,44] or by high concentrations of free fatty acid mixed with long-chain PC vesicles [45]. As the present study utilizes a purified enzyme preparation in the presence o f protease inhibitors, the arachidonateinduced calcium-independence is not likely to be the result of a proteolytic activation process. 4.5. Potential role o f membrane biophysical effects relating to fatty acid unsaturation in the lipid activators o f PKC The activity of PKC is known to be regulated by the nature of the hydrocarbon chains of the phospholipid bilayer. The degree of unsaturation of both PS and PC [46,47] as well as that of the D A G species [48-50] regulates the activation state of PKC. Unsaturation of diglyceride hydrocarbon chains affects physical properties of bilayers such as lipid packing, lipid movement and segregation, and lateral phase separation into gel and liquid crystalline phases (see [9,51-53]). Unesterified (free) fatty acids ( U E F A ) also affect biophysical properties o f membranes (see [51]). U E F A promote protein penetration into the hydrocarbon core region of the bilayer through disruption of lipid packing and reduction of the membrane surface pressure. A similar effect is attributed to calcium binding to acidic phospholipids due to charge neutralization of the headgroups. Thus, it is reasonable to consider that free arachidonic acid may induce calcium-independent binding and activation of PKC via an alteration of the membrane properties that promotes an enhanced p r o t e i n lipid interaction (i.e., bilayer insertion). As a consequence of this enhanced p r o t e i n - l i p i d interaction it is suggested that the enzyme may undergo some form of conformational or energetic state change which is associated with a stable, constitutive activation. The data obtained using this simple model membrane system provide biochemical insight into observations from complex cellular systems where this diglyceride and fatty acid synergism has been reported [1,2,54-56]. In addition to regulating the acute activity of this enzyme, the association of free arachidonic acid with an arachidonoyl-contain° ing D A G (in the presence o f calcium) facilitates generation of a tightly membrane-associated, constitutively active state o f PKC. A recent study by Blumberg and associates has questioned whether phorbol esters actually give rise to this state in a cellular system [57]. However, the formation of this constitutively active state by physiological regulators

175

may participate in long-lasting changes in cellular activity such as the changes in electrophysiological properties of neurons following associative learning or conditioning paradigms in intact animals or brain slices (see [4,58-60]).

Acknowledgements W e would like to thank Dr. James Olds for his assistance in quantitating the immuno-dot blots.

References [1] Lester, D.S., Collin, C., Etcheberrigaray, R. and Alkon, D.L. (1991) Biochem. Biophys. Res. Commun. 179, 1522-1528. [2] Bramham, C.R., Alkon, D.L. and Lester, D.S. (1994) J. Neurosci. 60, 737-743. [3] Burns, D.J. and Bell, R.M. (1992) Protein Kinase C: Current Concepts and Future Perspectives (Lester, D.S. and Epand, R.M., eds.), pp. 25-40, Ellis Horwood, New York. [4] Lester, D.S. and Bramham, C.R. (1993) Cellul. Signal. 5, 695-708. [5] Huang, K.P. (1989) Trends Neurosci. 12, 425-432. [6] Newton, A.C. and Koshland, D.E., Jr. (1989) J. Biol. Chem. 264, 14909-14915. [7] Bell, R.M. and Burns, D.J. (1991) J. Biol. Chem. 266, 4661-4664. [8] Sando, J.J., Maurer, M.C., Bolen, E.J. and Grisham, C.M. (1992) Cell. Signal. 4, 595-609. [9] Zidovetzki, R. and Lester, D.S. (1992) Biochim. Biophys. Acta 1134, 261-272. [10] Kikkawa, U., Takai, Y., Minakuchi, R., Inohara, S. and Nishizuka, Y. (1982) J. Biol. Chem. 257, 13341-13348. [11] Lester, D.S. (1989)J. Neurochem. 52, 1950-1953. [12] Lester, D.S. (1992) Protein Kinase C: Current Concepts and Future Perspectives (Lester, D.S. and Epand, R.M., eds.), pp. 80-101, Ellis Horwood, New York. [13] Akers, R., Lovinger, D., Colley, P., Linden, D. and Routenberg, A. (1986) Science 231,587-589. [14] Bank, B., DeWeer, A., Kuzirian, A.M., Rasmussen, H. and Alkon, D.L. (1988) Proc. Natl. Acad. Sci. USA 85, 1988-1992. [15] Olds, J.L., Anderson, M.L., McPhie, D.L., Staten, L.D. and Alkon, D.L. (1989) Science 245, 866-869. [16] Shearman, M.S., Naor, Z., Kikkawa, Y. and Nishizuka, Y. (1987) Biochem. Biophys. Res. Commun. 147, 911-919. [17] Yoshida, Y., Huang, F.L., Nakabayashi, H. and Huang, K.P. (1988) J. Biol. Chem. 263, 9868-9873. [18] Bazzi, M.D. and Nelsestuen, G.L. (1988) Biochem. Biophys. Res. Commun. 152, 336-343. [19] Bazzi, M.D. and Nelsestuen, G.L. (1988) Biochemistry 27, 75897583. [20] Kazanietz, M.G., Krausz, K.W. and Blumberg, P.M. (1992) J. Biol. Chem. 267, 20878-20886. [21] Bazzi, M.D. and Nelsestuen, G.L. (1989) Biochemistry 28, 93179323. [22] Chauhan, A., Brockerhoff, H., Wishniewski, H.M. and Chauhan, V.P.S. (1991) Arch. Biochem. Biophys. 287, 382-387. [23] Epand, R.M., Stafford, A.R. and Lester, D.S. (1992) Eur. J. Biochem. 208, 327-332. [24] Mosior, M. and Epand, R.M. (1994) J. Biol. Chem. 269, 1377913805. [25] McPhail, L.C., Clayton, C.C. and Snyderman, R. (1984) Science 224, 622-625. [26] Murakami, K. and Routtenberg, A. (1985) FEBS Lett. 192, 189-193. [27] Nishikawa, M., Hidaka, H. and Shirakawa, S. (1988) Biochem. Pharmacol. 37, 3079-3089.

176

J.B. Schachter et al. // Biochimica et Biophysica Acta 1291 (1996) 167-176

[28] Sekiguchi, K., Tsukuda, M., Ogita, K., Kikkawa, U. and Nishizuka, Y. (1987) Biochem. Biophys. Res. Commun. 145, 797-802. [29] El Touny, S., Khan, W. and Hannun, Y. (1990) J. Biol. Chem. 265, 16437 - 16443. [30] Buday, L. and Farago, A. (1990) FEBS Lett, 276, 223-226. [31] Seifert, R., Schachtele, C. and Schultz, G. (1987) Biochem. Biophys. Res. Commun. 154, 20-26. [32] Verkest, V., McArthur, M. and Hamilton, S. (1988) Biochem. Biophys. Res. Commun. 152, 825-829. [33] Lester, D.S. (1990) Biochim. Biophys. Acta 1054, 297-303. [34] Shinomura, T., Asaoka, Y., Oka, M., Yoshida, K. and Nishizuka, Y. (1991) Proc. Natl. Acad. Sci. USA 88, 5149-5153. [35] Johnson, M.S., Simpson, J., MacEwan, D.J., Ison, A., Clegg, R.A., Connor, K. and Mitchell, R. (1995) Mol. Cell. Biochem. 146, 127-137. [36] Boni, L.T. and Rando, R.R. (1985) J. Biol. Chem. 260, 1081910825. [37] Orr, N., Yavin, E. and Lester, D.S. (1992) J. Neurochem. 58, 461-470. [38] Bolen, E.J. and Sando, J.J. (1992) Biochemistry 31, 5945-5951. [39] Parker, P.J. (1992) Protein Kinase C: Current Concepts and Future Perspectives (Lester, D.S. and Epand, R.M., eds.), pp. 3-24, Ellis Horwood, New York. [40] McGlynn, E., Liebetanz, J., Reutener, S., Wood, J., Lydon, N.B., Hofsteller, H., Vanek, M., Mayer, T. and Fabbro, D. (1992) J. Cell. Biochem. 49, 239-250. [41] Liyanage, M., Frith, D., Livneh, E. and Stabel, S. (1992) Biochem J. 283, 781-787. [42] Dekker, L.V., Parker, P.J. and Mclntyre, P. (1992) FEBS Lett. 312, 195-199. [43] Walker, J.M., Homan, E.C. and Sando, J.J. (1990) J. Biol. Chem. 265, 8016-8021.

[44] Maurer, M.C., Grisham, C.M. and Sando, J.J. (1992) Archs Biochem. Biophys. 298, 561-568. [45] Chen, S.G., Kulju, D., Halt, S. and Murakami, K. (1992) Biochem. J. 284, 221-226. [46] Lester, D.S., Orr, N. and Brumfeld, V. (1990) J. Protein Chem. 9, 209-220. [47] Snoek, G.T., Feijen, A., Hage, W.J., Van Rotterdam, W. and De Laat, S.W. (1988) Biochem J, 255, 629-637. [48] Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. (1980) 255, 2273-2376. [49] Cabot, M.C. and Jaken, S., (1984) Biochem. Biophys. Res. Commun. 125, 163-169. [50] Lapetina, E.G., Reep, B., Ganong, B.R. and Bell R.M. (1985) J. Biol. Chem. 260, 1358-1361. [51] Leger, C.L. (1993) Prostaglandins Leukot Essential Fatty Acids 48, 17-21. [52] Goldberg, E.M., Lester, D.S., Borchardt, D.B. and Zidovetzki, R. (1994) Biophys.J. 66, 382-393. [53] Epand, R.M. and Lester D.S. (1990) Trends Pharmacol. Sci. 11, 317-320. [54] Herrero, I., Miras-Portugal, M.T. and Sanchez-Preito, J. (1992) J. Neurochem. 59, 1574-1577. [55] Schaechter, J.D. and Benowitz, L.I. (1993) J. Neurosci. 13, 43614371. [56] Szamel, M., Rejhermann, B., Krebs, B., Kurrle, R. and Resch, K. (1989) J. Immunol. 143, 2806-2813. [57] Szallasi, Z., Smith, C.B. and Blumberg, P.M. (1994) J. Biol. Chem. 269, 27159-27162. [58] Alkon, D.L. and Rasmussen, H. (1988) Science 239, 998-1005. [59] Lester, D.S. and Alkon (1991) Prog. Brain Res. 89, 235-248. [60] Routtenberg, A. (1991) Prog. Brain Res. 89, 235-248.