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Production and function of lipid second messengers in proliferating and differentiated neuroblastoma cells
Jot~P~t of
LIPID MEDIATORS ~U~OCI~LLSIGNALLING
ELSEVIER
J. Lipid Mediators Cell Signalling 14 (1996) 349 359
Production and function of lipid second messengers in proliferating and differentiated neuroblastoma cells D. Lang a, J.N. Kanfer b, G. Goracci c, L. Freysz a aLaboratoire de Neurobiologie Molbculaire des Interactions Cellulaires, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France bUniversity of Manitoba, Department of Biochemistry and Molecular Biology, Winnipeg, Manitoba R3EOW3, Canada ~Institute of Biochemistry and Medical Chemistry, University of Perugia, 06100 Perugia, Italy
Abstract
Multiple cellular responses are regulated through the generation of lipid second messengers upon activation of phospholipases. One such response concerns the activity of a class of kinase constituting the protein kinase C family. The production of specific molecular species of lipid second messengers may be therefore of prime importance in the activation of a member of the PKC isoforms. Prompted by this possibility we investigated the production of 1,2 diacyl-sn-glycerol (DAG) and phosphatidic acid (PtdOH) in LA-N-1 neuroblastoma cells under various physiological states. 12-0-Tetradecanoylphorbol 13-acetate (TPA) stimulation activated a phospholipase D (PLD) specific for phosphatidylcholine (PtdCho) in proliferating cells and a phospholipase C (PLC) specific for phosphatidylethanolamine (PtdEtn) in retinoic acid (RA) differentiated cells. These separate activations produced different molecular species of D A G or PtdOH. PtdOH was able to stimulate the Ca 2 + dependent protein kinase C (PKC) by a mechanism which differed from the action of DAG. PtdOH did not induce the translocation of the PKC to the membrane. Moreover PtdOH, in contrast to DAG, prevented PKC degradation by inhibiting the enzymatic hydrolysis by m-calpain. These observations suggest that the stimulation of cells by agonists elicited the production of Abbreviations: Cho, choline; DAG, 1,2 diacyl-sn-glycerol; Etm, ethanolamine; FFA, free fatty acids; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdOH, phosphatidic acid; PtdSer, phosphatidylserine; PCho, phosphorylcholine; PEtm, phosphorylethanolamine; PKC, protein kinase C; PLAz, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; RA, retinoic acid; TPA, 12-0-tetradecanoylphorbol 13-acetate.
0929-7855/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved Pll S0929-7855(96)00544-5
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specific molecular species of lipid second messengers depending on the physiological status of the cells, and probably on the nature of the stimulus. It seems therefore likely that the generation of specific lipid second messengers may activate specific PKC isoforms resulting in a specific cellular response.
Keywords: 1,2-diacylglycerol; Phosphatidic acid; Protein kinase C; Calpain; Phorbol ester; LA-N-I neuroblastoma cells
1. Introduction
Many cellular processes are regulated by the binding of agonists such as hormones, neurotransmitters, growth factors and tumor promoters to specific receptors located in the plasma membrane. The initiation of information for multiple cellular responses is carried out by the activation of machinery responsible for the production of second messengers, allowing a rapid diffusion of the signal throughout the cells. These second messengers modulate various physiological and biochemical reactions (Berridge, 1988). However, the multiplicity of cellular responses upon specific stimulations cannot be explained with the present knowledge of existing second messengers. Numerous agonists transduce their signal through the activation of phospholipases, catalyzing the breakdown of membrane lipids, leading to the production of lipid second messengers (Liscovitch and Cantley, 1994). In this respect the role of diacylglycerol (DAG) as a lipid second messenger is well established (Nishizuka, 1992). DAG is generated by the activation of phospholipase C (PLC) catalyzing the hydrolysis of inositol phospholipids and plays an important role as effector molecule in the activation of protein kinase C (PKC). In the last decade it has become evident that other phospholipases like phospholipase A2 (PLA2) and phospholipase D (PLD) are activated upon cellular stimulation (Dennis et al., 1991). The activation of PLA2 and PLD results in the production of free fatty acids (FFA), lysophospholipids and phosphatidic acid (PtdOH) respectively. These lipids have been shown to modulate the activity of PKCs (McPhail et al., 1984; E1 Touny et al., 1990; Ohara et al., 1994; Stasek et al., 1993; Limatola et al., 1994). Moreover, recent studies showed that the D A G generated upon cellular stimulation does not only originate from inositol phospholipids but also from other phospholipids like phosphatidylcholine (PtdCho) (Exton, 1990) and phosphatidylethanolamine (PtdEtn) (Kiss, 1992). The various classes of phospholipid differ in their fatty acid composition (Leray et al., 1990; Lee and Hajra, 1991) and, therefore, the molecular species of the lipid second messengers produced depends on the phospholipases activated and the target phospholipids hydrolyzed. With the discovery of multiple forms of PKC (Nishizuka, 1988; Azzi et al., 1992) that vary in their tissue and subcellular distribution, substrate specificity, translocation and regulation, it seems likely that various molecular species of DAG, F F A and PtdOH may act differently on the various PKC's. The result of the formation of a diversity of messengers could be
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responsible for the multiplicity of cellular responses. Recent studies showed that the lipid second messengers individually modulate the activity of the various PKC isoforms (Liscovitch and Cantley, 1994). It is, therefore, of prime importance to determine the nature of the lipid second messenger molecular species produced following cellular stimulation and the mechanism by which they selectively regulate specific PKC isoforms activation.
2. Generation and origin of DAG molecular species by 12-0-tetradecanoylphorbol 13-acetate (TPA)-stimulation of LA-N-I neuroblastoma cells The analysis of molecular species of DAG appearing as a result of agonist binding has been investigated in different cell lines (Pessin and Raben, 1989; Pessin et al., 1990; Lee et al., 1991). These studies have shown that the nature of the molecular species produced depends on the nature of the stimuli, the type of cells in culture and the duration of the applied stimulation. This suggests that the activation of a specific PKC may be regulated by specific molecular species of DAG whose generation depends on the nature of the stimulus and the physiological state of the cell. This was confirmed by the analysis of the DAG molecular species produced on phorbol ester stimulation of the neuroblastoma cell line LA-N-1 either in the proliferative stage or differentiated by retinoic acid (RA). TPA has a wide range of effects on cellular activities, a majority of which are mediated by PKC (Kiss, 1990). The TPA treatment of proliferating or differentiated LA-N-1 cells elicited a biphasic increase in the total amount of DAG (Lang et al., 1995a). Analysis of the molecular species of DAG showed that mainly saturated/monounsaturated (16:0/18:1, 18:0/18:1) and saturated/saturated (16:0/16:0, 16:0/18:0) speTable 1 Molecular species of D A G produced on T P A stimulation in proliferating and in R A differentiated LA-N-1 cells Molecular species 16:1 2 2 : 6 n - 3 18:2 18:2 n-6 16:0 20:4 n-6 18:0-22:6 n-3 16:0 22:4 n-6 18:0 20:4 n-6 18:1-18:1 n-9 16:0 18:1 n-9 16:0 16:0 18:0 18:1 n-9 16:0-18:0
Proliferating cells (mol %) 1.8 1.1 6.7
1.4 30.3 27.0 19.6 7.4
R.A. differentiated cells (mol %) 17.4 5.5 30.2 11.8 2.1 5.4 22.9
Molecular species composition of the D A G generated in proliferating and R A differentiated LA-N-1 cells after 5 min T P A stimulation. Cells were treated as described previously (Lang et al., 1995a). D A G s were purified and separated by reverse-phase H P L C as reported (Lang et al., 1995b).
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Table 2 Release of choline and ethanolamine water-soluble metabolites by TPA stimulation of proliferating and RA differentiated LA-N-1 cells Metabolites
Proliferatingcells (% of control values)
PCho Cho PEtn Etn
67 162* 99 95
RA differentiated cells (% of control values) l 17 156" 106
[14C]Choline or [14C]Ethanolamine prelabeled LA-N-1 cells were scraped into Leibowitz's medium, allowed to equilibrate for 1 h and stimulated with TPA (100 nM) for 30 min. Headgroup metabolites were collected and analyzed as reported by Kiss (1991). The data are representative of 2 different experiments each performed in triplicate. SD< 10%. *p<0.01. cies were generated in proliferating cells. In the case of R A differentiated cells the increase of D A G corresponded mainly to the production of saturated/ polyunsaturated (18:0/22:6, 16:0/20:4, 18:0/20:4) and monounsaturated/ monounsaturated (18:1 / 18:1) species (Table 1). These observations raised the question on the origin of these D A G . A comparison of the D A G molecular species composition with that of the various membrane phospholipids suggest that T P A activates the hydrolysis of PtdCho in proliferating cells and PtdEtn in R A differentiated cells (Lang et al., 1995a). This was confirmed by the analysis of water-soluble metabolites released from prelabeled phospholipids upon cellular stimulation. T P A stimulated the release of choline from PtdCho in proliferating cells, but in R A differentiated cells it stimulated the release of phosphoethanolamine from PtdEtn (Table 2). These results suggest that in LA-N-1 cells different hydrolytic pathways were activated by T P A leading to the generation of the various molecular species of D A G , depending on the physiological status of the cell. In proliferating cells PtdCho was the substrate for a P L D coupled to a phosphatidate phosphatase but in R A differentiated cells PtdEtn served as substrate for a PLC. Since ethanolamine phospholipids contain about 30% plasmalogens it should be noted that in these cells the activation of PLC m a y also produce alkenyl-acylglycerol which has been reported to affect P K C activity (Ford et al., 1989) Whether this mechanism operates in LA-N-1 cells has not been established. Thus it became evident that different D A G molecular species can be generated by modulating different pathways. Since these molecules participate in the regulation of P K C activities, it is possible that different P K C isoforms are targets for various D A G molecules resulting in different cellular responses. This hypothesis is consistent with the observation that the activity of various P K C isoforms are affected differently by various lipid second messengers (Liscovitch and Cantley, 1994; N a k a m u r a and Nishizuka, 1994) and that specific P K C s may be involved in the mechanism leading to specific cellular responses (Shinomura et al., 1991).
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3. Role of phosphatidic acid (PA) in signal transduction The activation of P L D by TPA in proliferating LA-N-1 cells produces in a first step P t d O H which is then dephosphorylated to DAG. PtdOH itself is increasingly recognized as a candidate lipid second messenger, modulating multiple cellular responses (Limatola et al., 1994; Kroll et al., 1989). Since some of the cellular responses triggered by PtdOH are also mediated by PKC it has been suggested that PtdOH can modulate the activity of this enzyme. This prompted us to investigate the effect of P t d O H on the activity of PKC in proliferating LA-N- 1 cells. Confluent LA-N-1 cells were treated with PtdOH (200/zM) or TPA (100 nM). The measurement of cytosolic and membrane bound PKC activities revealed that P K C was translocated and activated in a dogmatic manner in the presence of TPA but PA transiently stimulated the cytosolic enzyme without enzyme translocation to the membrane (Fig. 1). 200
A 150
100
so w
200
I
_.= o
I
I
I
I
B
==
150
100
50
0
I
I
5
10
I
I
15 20 T i m e (re.in)
I
I
25
30
35
Fig. 1. In vivo effect of PtdOH and T P A on P K C activity in LA-N-1 cells. The cells were incubated in Leibovitz medium containing either 1 0 - 7 M T P A (A) or 200 # M PtdOH (B) for the indicated times. Cells were harvested, homogenized and the activities of cytosolic and particulate P K C assayed as reported by Block et al. (1992). The standard P K C assay medium contained 20 m M T r i s - H C 1 pH 7.5, 1 m M E G T A , 1,0 m M CaCI2, 5.0 m M MgCI2, 2.0 m M PMSF, 230 p M PS, 20 # g histone, 20 ,uM ~'-[32P]ATP (specific activity: 2 × 105 cpm/nmol). The activity of cytosolic O-O-O and particulate • - • - • P K C is expressed as % of the total activity present in non-treated cells. Each value represents the mean _+ SEM of 2 different experiments in triplicate.
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Table 3 In vitro effect of PtdOH and TPA on LA-N-1 PKC activity Addition
Specific activity pmol/min per mg protein
None PtdSer 230/~M
524 _+21 l 2541 _+316
PtdSer 230/~M TPA 0.1 ~tM
3557 + 350
PtdOH 300 /~M
1794 _ 491
PtdOH 300 ,uM TPA 0.1 /~M
3445 +479
PtdSer 230 /~M PtdOH 300 /~M
3897 _+699
Partially purified PKC from LA-N-I was obtained as described previously (Block et al., 1992) and the effect of PtdOH on its activity as reported in Fig. 1. The specific activity represents the mean + SEM of 2 experiments in triplicate. The basal PKC assay medium contained 20 mM Tris HCI pH 7.5, 1 mM EGTA, 1,0 mM CaCI2, 5.0 mM MgCI2, 2.0 mM PMSF, 20 /~g histone, 20 /~M g-[32P]ATP (specific activity 2 x 105 cpm/nmol). To obtain information on the mechanism o f activation o f P K C by P t d O H , partially purified enzyme f r o m L A - N - 1 was prepared and the effect o f P t d O H on its activity was investigated. Table 3 shows that P t d O H directly activated the Ca ÷ ÷ dependent P K C obtained f r o m LA-N-1 cells. N o effect o f P t d O H on P K C activity was observed in the absence o f Ca + +. In the presence o f P t d O H the addition o f T P A increased the P K C activity as observed with phosphatidylserine (PtdSer)/TPA. However, P t d O H did not interact with the T P A binding site as attested by the lack o f inhibition o f [3H]PDBu binding to the enzyme by P t d O H (Stasek et al., 1993; L a n g et al., 1995b). P t d O H also further activated P K C in the presence o f optimal concentration o f PtdSer. These results indicate that P A can substitute for T P A or D A G directly for increased enzyme activity. P t d O H seems to activate calcium dependent P K C by a mechanism distinct from that o f P t d S e r / T P A and suggests that P t d O H m a y have two sites o f interaction with P K C . One would be a site interacting with acidic lipids, since P t d O H mimics the requirement o f PtdSer and acts in synergy with TPA. The observation that P t d O H increased the P K C activity in the presence o f PtdSer in the same m a n n e r as that o f T P A , suggests that the enzyme m a y possess an unidentified site where P t d O H m a y intervene. P t d O H activated preferentially the cytosolic P K C and did not trigger any translocation o f the enzyme from the cytosol to the m e m b r a n e indicating that P t d O H m a y have a physiological role in the activation o f cytosolic P K C . This statement is in agreement with the observation that in the liver and the heart homogenates, P t d O H provided markedly different profiles o f protein p h o s p h o r y l a t i o n as c o m p a r e d with PtdSer plus D A G (Bocckino et al., 1991). It is tempting to suggest that P t d O H m a y cause a redistribution o f P K C from the m e m b r a n e to the cytosol and activates the cytosolic enzyme which would be a direct effect o f P t d O H . However, it c a n n o t be excluded that in vivo P t d O H m a y activate phospholipase A (Ueda et al., 1993) which in turn generates F F A and lysophospholipid and, thereby stimulating cytosolic P K C (Sasaki et al., 1993).
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4. Effect of lipids on the PKC proteolysis mediated by calpain Although there are many observations regarding the mechanism involved in the activation of PKC, little is known about the factors which control the inactivation of these kinases, once they have been activated. Several mechanisms can be suggested: (1) the dissociation of PKC and the cofactors (Ca + +, PtdSer and DAG) leading to the translocation of the membrane bound form back to the cytosol in an inactive form, to our knowledge such a mechanism has not been described; (2) The activation of D A G kinase or/and D A G lipase lowering the D A G level (Kanoh et al., 1990); and (3) The degradation of PKC by proteolysis. Several proteases using PKC as substrate have been described. Among these calpains have attracted increased interest in recent years (Sorimachi et al., 1994). These enzymes are neutral cysteine endopeptidases, have an absolute Ca ÷ + dependence for activity and form a family of at least 6 distinct members (Saido et al., 1992; Saido et al., 1994). The /~-and m-calpain have a ubiquitous distribution in animal cells. Calpains catalyse the cleavage of the hinge region of the catalytic and the regulatory subunits of PKC generating a soluble Ca + +, phospholipid independent form termed PKM. This reaction has been considered as an irreversible activation of PKC (Murray et al., 1987). However, recent studies revealed that P K M is degraded by m-calpain at a faster rate than PKC (Shea et al., 1994) and that it did not accumulate in the cells suggesting that the role of calpains in the regulation of PKC activity may be limited to its inactivation. The inactivation of the PKC activity would depend, therefore, on the activation of the calpains and their interaction with the substrate. PKC is activated by PtdSer and various lipid second messengers and the question arises whether lipids modulate the calpain activities. Recent studies have shown that Ft-calpain was activated exclusively by the acidic phospholipids phosphatidylinositol (PtdIns) and Ptdlns(4,5)P2 by lowering the required Ca ÷ ÷ concentration for it (Saido et al., 1992). In contrast, the m-calpain activity was modulated differently by various lipids. Indeed ~PKC was completely degraded in about 30 min when incubated with m-calpain in the absence of lipids. The addition of various phospholipids PtdSer, PtdCho, PtdIns, and PtdOH produced a partial inhibition of the proteolytic activity. More than 30% of the initial amount of the substrates were still present after 30 min (Fig. 2). D A G and a mixture of D A G plus PtdSer did not significantly affect the m-calpain activity, questioning the sensitivity of PKC, in the active or inactive state, towards m-calpain. In order to get some insights into the mechanisms leading to the inhibition of c~PKC proteolysis the effect of the lipids on the hydrolysis of azocasein by m-calpain has been investigated. With the exception of PtdOH the various phospholipids tested, D A G and the mixture D A G + PtdSer did not affect the proteolysis of azocasein (Table 4). These results indicate that these lipids did not act on m-calpain. Therefore the inhibition of e P K C proteolysis by PtdSer, PtdIns, and PtdCho may be due to the modification of the lipid environment of the substrate creating a steric hindrance for the interaction with the enzyme, preventing its inactivation. In contrast, PtdOH inhibited the hydrolysis of azocasein (Table 4). With the incubation medium containing 11 m M Ca ÷ ÷ and 0.14 m M PtdOH, the
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60
I,
40
ii
ig o
C
I:tdSer
Prier 4DAG
0AG
P1dQH P~dCho Ptdlns
Fig. 2. Effect of lipids on ePKC proteolysis by m-calpain, aPKC (80 ng) was in 25 /~1 of 50 mM Tris-HC1, pH 7.4, 5 mM EDTA, 2 mM PMSF containing 5.2 ng m-calpain (specific activity: 0.857/~g azocasein//~g m-calpain per min) and various lipids (20/~g). The reaction was started by the addition of CaCI 2, final concentration 7 mM. Incubation was for 30 min at 30°C and stopped by adding 10/~1 of Laemmli buffer for subsequent SDS 7% polyacrylamide gels electrophoresis. After transfer to nitrocellulose, the strips were incubated with a commercial monoclonal antibody that recognizes the catalytic domain of PKCe (UBI) and alkaline phosphatase staining as described (Shea et al., 1993). Nitrocellulose replicas were scanned using a Scan Jet IIp scanner and compared with the Scan Analysis densitometric program. q u e s t i o n o f C a + + c h e l a t i n g b y t h e a c i d i c p h o s p h o l i p i d is r u l e d o u t f o r its effect o n c a l p a i n a c t i v i t y s u g g e s t i n g t h a t P t d O H is a p o t e n t d i r e c t i n h i b i t o r o f m - c a l p a i n . Since P t d O H a c t i v a t e s , in v i v o , c y t o s o l i c P K C t h e result s u g g e s t s t h a t this p h o s p h o lipid m a y essentially be i n v o l v e d in t h e r e g u l a t i o n o f c y t o s o l i c p r o t e i n p h o s p h o r y l a tion whereas the other phospholipids may favour membrane protein phosphorylation. Table 4 Effect of lipids on the activity of m-calpain towards azocasein Addition
Relative % of hydrolysis
Control PtdSer PtdSer + DAG DAG PtdOH PtdCho PtdIns
100 102 123 96 53* 128 84
0.1 nag [14C] azocasein (specific activity 500 000 cpm/mg) was dissolved in 50 mM Tris-HCl, pH 7.4, 2.3 mM DTT containing 2.7/zg m-calpain and various lipids (20/~g). The reaction was started by addition of CaC12 (11 mM final concentration) and incubation (final volume 200/tl) was performed for 30 min at 30°C. The reaction was terminated by addition of 100/~1 azocasein (5 mg/ml) and 300/tl of 10% ice cold TCA. After centrifugation the supernatant was removed and counted. * p<0.01.
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5. Conclusion D u r i n g the last few y e a r s c o n s i d e r a b l e p r o g r e s s has been m a d e in u n d e r s t a n d i n g the role a n d f u n c t i o n o f lipid s e c o n d messengers in signal t r a n s d u c t i o n . O f p a r t i c u l a r i m p o r t a n c e has been the c h a r a c t e r i z a t i o n o f new lipid s e c o n d messengers a n d their effect o n P K C activities (Liscovitch a n d Cantley, 1994). P K C c a n be c o n s i d e r e d as a cellular r e g u l a t o r a n d t h e r e f o r e the i n t e r a c t i o n with specific m o l e c u l a r species o f lipid s e c o n d messengers m a y induce specific cellular responses. T h e increasing n u m b e r o f v a r i o u s P K C i s o f o r m s (Azzi et al., 1992) a n d the o b s e r v a t i o n t h a t different m o l e c u l a r specie o f D A G o r P t d O H are g e n e r a t e d in p r o l i f e r a t i n g a n d R A d i f f e r e n t i a t e d L A - N 1 cells on T P A s t i m u l a t i o n are consistent with this hypothesis. M o r e o v e r , the d a t a s h o w i n g t h a t P t d O H d i d n o t o n l y m o d u l a t e the C a + + d e p e n d e n t P K C activity b u t was also able to inhibit its p r o t e o l y s i s b y m - c a l p a i n suggest t h a t in p r o l i f e r a t i n g cells the d u r a t i o n o f P K C a c t i v a t i o n was increased b y P t d O H . These results p r o v i d e evidence t h a t the T P A t r e a t m e n t o f the L A - N - 1 n e u r o b l a s t o m a cells stimulates the p r o d u c t i o n o f different m o l e c u l a r species o f D A G a n d P t d O H t h r o u g h the h y d r o l y s i s o f different p h o s p h o lipids b y the a c t i v a t i o n o f specific p h o s p h o l i p a s e s C o r D d e p e n d i n g on the p h y s i o l o g i c a l status o f the cells in question. I f we a s s u m e t h a t the different P K C ' s activities are m o d u l a t e d by specific lipid s e c o n d messengers it is likely t h a t the a c t i v a t i o n o f specific p h o s p h o l i p a s e s (PLA2, P L C , P L D ) u p o n r e c e p t o r a g o n i s t i n t e r a c t i o n w o u l d be one o f the first steps i n v o l v e d in the i n d u c t i o n o f specific cellular responses. T h e i s o l a t i o n a n d c h a r a c t e r i z a t i o n o f increasing n u m b e r s o f p h o s p h o l i p a s e i s o e n z y m e s (Liscovitch a n d Cantley, 1994) argue in f a v o r o f such a hypothesis.
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Kiss Z. (1991) Determination of phospholipase D mediates hydrolysis of phosphatidyl-ethanolamine. Lipids 26, 777 780. Kiss Z. (1992) The long-term combined sitmulatory effects of ethanol and phorbol ester on phosphatidylethanolamine hydrolysis are mediated by a phospholipase C and prevented by overexpressed alpha-protein kinase C in fibroblasts. Eur. J. Biochem. 209, 467-473. Kroll M.H., Zavoico G.B. and Schafer A.I. (1989) Second messenger function of phosphatidic acid in platelet activation. J. Cell Physiol. 139, 558 564. Lang D., Leray C., Mestre R., Massarelli R., Dreyfus H. and Freysz L. (1995a) Molecular species analysis of 1,2 diglycerides on phorbol ester stimulation of LA-N-1 neuroblastoma cells during proliferation and differentiation. J. Neurochem. 65, 810 817. Lang D., Malviya A.N., Hubsch A., Kanfer J.N. and Freysz L. (1995b) Phosphatidic acid activation of protein kinase C in LA-N-l-neuroblastoma cells. Neurosci. Lett. 201, 199 202. Lee C., Fisher S.K., Agranoff B.W. and Hajra A.K. (1991) Quantitative analysis of molecular species of diacylglycerol and phosphatidate formed upon muscarinic receptor activation of human SK-N-SH neuroblastoma cells. J. Biol. Chem. 266, 22 837 22 846. Lee C. and Hajra A.K. (1991) Molecular species of diacylglycerols and phosphoglycerides and the post mortem changes in the molecular species of diacylglycerols in rat brains. J. Neurochem. 56, 370-379. Leray C., Pelletier A., Massarelli R., Dreyfus H. and Freysz L. (1990) Molecular species of choline and ethanolamine phospholipids in rat cerebellum during development. J. Neurochem. 54, 1677-1681. Limatola C., Schaap D., Moolenaar W.H. and Van Blitterswijk W.J. (1994) Phosphatidic acid activation of protein kinase C-s is overexpressed in COS cells: a comparison with other protein kinase C isotopes and other acidic lipids. Biochem. J. 304, 1001-1008. Liscovitch M. and Cantley L.C. (1994) Lipid second messengers. Cell 77, 329-334. McPhail L.C., Clayton C.C. and Snyderman R. (1984) A potential second messenger role for unsaturated fatty acids: activation of Ca 2 +-dependent protein kinase. Science 224, 622 625. Murray A.W., Fournier A. and Hardy S.J. (1987) Proteolytic activation of protein kinase C: a physiological reaction? TIBS 12, 53 54. Nakamura S. and Nishizuka Y. (1994) Lipid mediators and protein kinase C activation for the intracellular signaling network. J. Biochem. 115, 1024-1034. Nishizuka Y. (1988) The molecular heterogeneity of protein kinase C and its implication for cellular regulation. Nature 334, 661 665. Nishizuka Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258, 607-614. Ohara Y., Peterson T.E., Zheng B., Kuo J.F. and Harrison D.G. (1994) Lysophosphatidylcholine increases vascular superoxide anion production via protein kinase C activation. Arterioscler. Thromb. 14, 1007-1013. Pessin M.S., Baldassare J.J. and Raben D.M. (1990) Molecular species analysis of mitogen-stimulated 1,2 diglycerides in fibroblasts. J. Biol. Chem. 265, 7959-7966. Pessin M.S. and Raben D.M. (1989) Molecular species analysis of 1,2 diglycerides stimulated by c~-thrombin in cultured fibroblasts. J. Biol. Chem. 264, 8729 8738. Saido T.C., Shibata M., Takenawa T., Murofushi H. and Suzuki K. (1992) Positive regulation of #-calpain action by polyphosphoinositides; J. Biol. Chem. 267, 24 585-24 590. Saido T.C., Sorimachi H. and Suzuki K. (1994) Calpain: new perspectives in molecular diversity and physiological-pahtological involvement. FASEB J. 8, 814-822. Sasaki Y., Anaola Y. and Nishizuka Y. (1993) Potentiation of diacylglycerol-induced activation of protein kinase C by lysophospholipids: subspecies difference. FEBS Lett. 320, 47 51. Shea T.B., Beermann M.L., Griffin W.R. and Leli U. (1994) Degradation of protein kinase Ca and its free catalytic subunit, protein kinase M in intact human neuroblastoma cells and under cell-free conditions. FEBS Lett. 350, 223 229. Shea T.B., Paskevitch P.A. and Beerman M.L. (1993) The protein phosphatase inhibitor okadaic acid increases axonal neurofilaments and neurite caliber, and decreases axonal microtubules in NB2a/dl cells. J. Neurosci. Res, 35, 507 52l. Shinomura T., Mishama H., Matsushima S., Aasaoka Y. and Nishizuka Y. (1991) Degradation of phospholipids and protein kinase C activation for the control of neuronal functions. In: Bazan N.G.,
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Murphy M.G. and Toffano G. (Eds.), Neurobiology of Essential Fatty Acids, Plenum, New York, pp. 361 373. Sorimachi H., Said T.C. and Suzuki K. (1994) New era of calpain research: discovery of tissue-specific calpains. FEBS Lett. 343, 1 5. Stasek J.E., Natarajan V. and Garcia J.G.N. (1993) Phosphatidic acid directly activates endothelial cell protein kinase C. Biochem. Biophys. Res. Commun. 191, 134 141. Ueda H., Kobayashi T., Kishimoto M., Tsutsumi T. and Okuyama H. (1993) A possible pathway of phosphoinositide metabolism through EDTA-insensitive phospholipase A1 followed by lysophosphoinositide-specific phospholipase C in rat brain. J. Neurochem. 61, 1874 1881.
LIPID MEDIATORS ~U~OCI~LLSIGNALLING
ELSEVIER
J. Lipid Mediators Cell Signalling 14 (1996) 349 359
Production and function of lipid second messengers in proliferating and differentiated neuroblastoma cells D. Lang a, J.N. Kanfer b, G. Goracci c, L. Freysz a aLaboratoire de Neurobiologie Molbculaire des Interactions Cellulaires, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France bUniversity of Manitoba, Department of Biochemistry and Molecular Biology, Winnipeg, Manitoba R3EOW3, Canada ~Institute of Biochemistry and Medical Chemistry, University of Perugia, 06100 Perugia, Italy
Abstract
Multiple cellular responses are regulated through the generation of lipid second messengers upon activation of phospholipases. One such response concerns the activity of a class of kinase constituting the protein kinase C family. The production of specific molecular species of lipid second messengers may be therefore of prime importance in the activation of a member of the PKC isoforms. Prompted by this possibility we investigated the production of 1,2 diacyl-sn-glycerol (DAG) and phosphatidic acid (PtdOH) in LA-N-1 neuroblastoma cells under various physiological states. 12-0-Tetradecanoylphorbol 13-acetate (TPA) stimulation activated a phospholipase D (PLD) specific for phosphatidylcholine (PtdCho) in proliferating cells and a phospholipase C (PLC) specific for phosphatidylethanolamine (PtdEtn) in retinoic acid (RA) differentiated cells. These separate activations produced different molecular species of D A G or PtdOH. PtdOH was able to stimulate the Ca 2 + dependent protein kinase C (PKC) by a mechanism which differed from the action of DAG. PtdOH did not induce the translocation of the PKC to the membrane. Moreover PtdOH, in contrast to DAG, prevented PKC degradation by inhibiting the enzymatic hydrolysis by m-calpain. These observations suggest that the stimulation of cells by agonists elicited the production of Abbreviations: Cho, choline; DAG, 1,2 diacyl-sn-glycerol; Etm, ethanolamine; FFA, free fatty acids; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdOH, phosphatidic acid; PtdSer, phosphatidylserine; PCho, phosphorylcholine; PEtm, phosphorylethanolamine; PKC, protein kinase C; PLAz, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; RA, retinoic acid; TPA, 12-0-tetradecanoylphorbol 13-acetate.
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specific molecular species of lipid second messengers depending on the physiological status of the cells, and probably on the nature of the stimulus. It seems therefore likely that the generation of specific lipid second messengers may activate specific PKC isoforms resulting in a specific cellular response.
Keywords: 1,2-diacylglycerol; Phosphatidic acid; Protein kinase C; Calpain; Phorbol ester; LA-N-I neuroblastoma cells
1. Introduction
Many cellular processes are regulated by the binding of agonists such as hormones, neurotransmitters, growth factors and tumor promoters to specific receptors located in the plasma membrane. The initiation of information for multiple cellular responses is carried out by the activation of machinery responsible for the production of second messengers, allowing a rapid diffusion of the signal throughout the cells. These second messengers modulate various physiological and biochemical reactions (Berridge, 1988). However, the multiplicity of cellular responses upon specific stimulations cannot be explained with the present knowledge of existing second messengers. Numerous agonists transduce their signal through the activation of phospholipases, catalyzing the breakdown of membrane lipids, leading to the production of lipid second messengers (Liscovitch and Cantley, 1994). In this respect the role of diacylglycerol (DAG) as a lipid second messenger is well established (Nishizuka, 1992). DAG is generated by the activation of phospholipase C (PLC) catalyzing the hydrolysis of inositol phospholipids and plays an important role as effector molecule in the activation of protein kinase C (PKC). In the last decade it has become evident that other phospholipases like phospholipase A2 (PLA2) and phospholipase D (PLD) are activated upon cellular stimulation (Dennis et al., 1991). The activation of PLA2 and PLD results in the production of free fatty acids (FFA), lysophospholipids and phosphatidic acid (PtdOH) respectively. These lipids have been shown to modulate the activity of PKCs (McPhail et al., 1984; E1 Touny et al., 1990; Ohara et al., 1994; Stasek et al., 1993; Limatola et al., 1994). Moreover, recent studies showed that the D A G generated upon cellular stimulation does not only originate from inositol phospholipids but also from other phospholipids like phosphatidylcholine (PtdCho) (Exton, 1990) and phosphatidylethanolamine (PtdEtn) (Kiss, 1992). The various classes of phospholipid differ in their fatty acid composition (Leray et al., 1990; Lee and Hajra, 1991) and, therefore, the molecular species of the lipid second messengers produced depends on the phospholipases activated and the target phospholipids hydrolyzed. With the discovery of multiple forms of PKC (Nishizuka, 1988; Azzi et al., 1992) that vary in their tissue and subcellular distribution, substrate specificity, translocation and regulation, it seems likely that various molecular species of DAG, F F A and PtdOH may act differently on the various PKC's. The result of the formation of a diversity of messengers could be
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responsible for the multiplicity of cellular responses. Recent studies showed that the lipid second messengers individually modulate the activity of the various PKC isoforms (Liscovitch and Cantley, 1994). It is, therefore, of prime importance to determine the nature of the lipid second messenger molecular species produced following cellular stimulation and the mechanism by which they selectively regulate specific PKC isoforms activation.
2. Generation and origin of DAG molecular species by 12-0-tetradecanoylphorbol 13-acetate (TPA)-stimulation of LA-N-I neuroblastoma cells The analysis of molecular species of DAG appearing as a result of agonist binding has been investigated in different cell lines (Pessin and Raben, 1989; Pessin et al., 1990; Lee et al., 1991). These studies have shown that the nature of the molecular species produced depends on the nature of the stimuli, the type of cells in culture and the duration of the applied stimulation. This suggests that the activation of a specific PKC may be regulated by specific molecular species of DAG whose generation depends on the nature of the stimulus and the physiological state of the cell. This was confirmed by the analysis of the DAG molecular species produced on phorbol ester stimulation of the neuroblastoma cell line LA-N-1 either in the proliferative stage or differentiated by retinoic acid (RA). TPA has a wide range of effects on cellular activities, a majority of which are mediated by PKC (Kiss, 1990). The TPA treatment of proliferating or differentiated LA-N-1 cells elicited a biphasic increase in the total amount of DAG (Lang et al., 1995a). Analysis of the molecular species of DAG showed that mainly saturated/monounsaturated (16:0/18:1, 18:0/18:1) and saturated/saturated (16:0/16:0, 16:0/18:0) speTable 1 Molecular species of D A G produced on T P A stimulation in proliferating and in R A differentiated LA-N-1 cells Molecular species 16:1 2 2 : 6 n - 3 18:2 18:2 n-6 16:0 20:4 n-6 18:0-22:6 n-3 16:0 22:4 n-6 18:0 20:4 n-6 18:1-18:1 n-9 16:0 18:1 n-9 16:0 16:0 18:0 18:1 n-9 16:0-18:0
Proliferating cells (mol %) 1.8 1.1 6.7
1.4 30.3 27.0 19.6 7.4
R.A. differentiated cells (mol %) 17.4 5.5 30.2 11.8 2.1 5.4 22.9
Molecular species composition of the D A G generated in proliferating and R A differentiated LA-N-1 cells after 5 min T P A stimulation. Cells were treated as described previously (Lang et al., 1995a). D A G s were purified and separated by reverse-phase H P L C as reported (Lang et al., 1995b).
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Table 2 Release of choline and ethanolamine water-soluble metabolites by TPA stimulation of proliferating and RA differentiated LA-N-1 cells Metabolites
Proliferatingcells (% of control values)
PCho Cho PEtn Etn
67 162* 99 95
RA differentiated cells (% of control values) l 17 156" 106
[14C]Choline or [14C]Ethanolamine prelabeled LA-N-1 cells were scraped into Leibowitz's medium, allowed to equilibrate for 1 h and stimulated with TPA (100 nM) for 30 min. Headgroup metabolites were collected and analyzed as reported by Kiss (1991). The data are representative of 2 different experiments each performed in triplicate. SD< 10%. *p<0.01. cies were generated in proliferating cells. In the case of R A differentiated cells the increase of D A G corresponded mainly to the production of saturated/ polyunsaturated (18:0/22:6, 16:0/20:4, 18:0/20:4) and monounsaturated/ monounsaturated (18:1 / 18:1) species (Table 1). These observations raised the question on the origin of these D A G . A comparison of the D A G molecular species composition with that of the various membrane phospholipids suggest that T P A activates the hydrolysis of PtdCho in proliferating cells and PtdEtn in R A differentiated cells (Lang et al., 1995a). This was confirmed by the analysis of water-soluble metabolites released from prelabeled phospholipids upon cellular stimulation. T P A stimulated the release of choline from PtdCho in proliferating cells, but in R A differentiated cells it stimulated the release of phosphoethanolamine from PtdEtn (Table 2). These results suggest that in LA-N-1 cells different hydrolytic pathways were activated by T P A leading to the generation of the various molecular species of D A G , depending on the physiological status of the cell. In proliferating cells PtdCho was the substrate for a P L D coupled to a phosphatidate phosphatase but in R A differentiated cells PtdEtn served as substrate for a PLC. Since ethanolamine phospholipids contain about 30% plasmalogens it should be noted that in these cells the activation of PLC m a y also produce alkenyl-acylglycerol which has been reported to affect P K C activity (Ford et al., 1989) Whether this mechanism operates in LA-N-1 cells has not been established. Thus it became evident that different D A G molecular species can be generated by modulating different pathways. Since these molecules participate in the regulation of P K C activities, it is possible that different P K C isoforms are targets for various D A G molecules resulting in different cellular responses. This hypothesis is consistent with the observation that the activity of various P K C isoforms are affected differently by various lipid second messengers (Liscovitch and Cantley, 1994; N a k a m u r a and Nishizuka, 1994) and that specific P K C s may be involved in the mechanism leading to specific cellular responses (Shinomura et al., 1991).
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3. Role of phosphatidic acid (PA) in signal transduction The activation of P L D by TPA in proliferating LA-N-1 cells produces in a first step P t d O H which is then dephosphorylated to DAG. PtdOH itself is increasingly recognized as a candidate lipid second messenger, modulating multiple cellular responses (Limatola et al., 1994; Kroll et al., 1989). Since some of the cellular responses triggered by PtdOH are also mediated by PKC it has been suggested that PtdOH can modulate the activity of this enzyme. This prompted us to investigate the effect of P t d O H on the activity of PKC in proliferating LA-N- 1 cells. Confluent LA-N-1 cells were treated with PtdOH (200/zM) or TPA (100 nM). The measurement of cytosolic and membrane bound PKC activities revealed that P K C was translocated and activated in a dogmatic manner in the presence of TPA but PA transiently stimulated the cytosolic enzyme without enzyme translocation to the membrane (Fig. 1). 200
A 150
100
so w
200
I
_.= o
I
I
I
I
B
==
150
100
50
0
I
I
5
10
I
I
15 20 T i m e (re.in)
I
I
25
30
35
Fig. 1. In vivo effect of PtdOH and T P A on P K C activity in LA-N-1 cells. The cells were incubated in Leibovitz medium containing either 1 0 - 7 M T P A (A) or 200 # M PtdOH (B) for the indicated times. Cells were harvested, homogenized and the activities of cytosolic and particulate P K C assayed as reported by Block et al. (1992). The standard P K C assay medium contained 20 m M T r i s - H C 1 pH 7.5, 1 m M E G T A , 1,0 m M CaCI2, 5.0 m M MgCI2, 2.0 m M PMSF, 230 p M PS, 20 # g histone, 20 ,uM ~'-[32P]ATP (specific activity: 2 × 105 cpm/nmol). The activity of cytosolic O-O-O and particulate • - • - • P K C is expressed as % of the total activity present in non-treated cells. Each value represents the mean _+ SEM of 2 different experiments in triplicate.
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Table 3 In vitro effect of PtdOH and TPA on LA-N-1 PKC activity Addition
Specific activity pmol/min per mg protein
None PtdSer 230/~M
524 _+21 l 2541 _+316
PtdSer 230/~M TPA 0.1 ~tM
3557 + 350
PtdOH 300 /~M
1794 _ 491
PtdOH 300 ,uM TPA 0.1 /~M
3445 +479
PtdSer 230 /~M PtdOH 300 /~M
3897 _+699
Partially purified PKC from LA-N-I was obtained as described previously (Block et al., 1992) and the effect of PtdOH on its activity as reported in Fig. 1. The specific activity represents the mean + SEM of 2 experiments in triplicate. The basal PKC assay medium contained 20 mM Tris HCI pH 7.5, 1 mM EGTA, 1,0 mM CaCI2, 5.0 mM MgCI2, 2.0 mM PMSF, 20 /~g histone, 20 /~M g-[32P]ATP (specific activity 2 x 105 cpm/nmol). To obtain information on the mechanism o f activation o f P K C by P t d O H , partially purified enzyme f r o m L A - N - 1 was prepared and the effect o f P t d O H on its activity was investigated. Table 3 shows that P t d O H directly activated the Ca ÷ ÷ dependent P K C obtained f r o m LA-N-1 cells. N o effect o f P t d O H on P K C activity was observed in the absence o f Ca + +. In the presence o f P t d O H the addition o f T P A increased the P K C activity as observed with phosphatidylserine (PtdSer)/TPA. However, P t d O H did not interact with the T P A binding site as attested by the lack o f inhibition o f [3H]PDBu binding to the enzyme by P t d O H (Stasek et al., 1993; L a n g et al., 1995b). P t d O H also further activated P K C in the presence o f optimal concentration o f PtdSer. These results indicate that P A can substitute for T P A or D A G directly for increased enzyme activity. P t d O H seems to activate calcium dependent P K C by a mechanism distinct from that o f P t d S e r / T P A and suggests that P t d O H m a y have two sites o f interaction with P K C . One would be a site interacting with acidic lipids, since P t d O H mimics the requirement o f PtdSer and acts in synergy with TPA. The observation that P t d O H increased the P K C activity in the presence o f PtdSer in the same m a n n e r as that o f T P A , suggests that the enzyme m a y possess an unidentified site where P t d O H m a y intervene. P t d O H activated preferentially the cytosolic P K C and did not trigger any translocation o f the enzyme from the cytosol to the m e m b r a n e indicating that P t d O H m a y have a physiological role in the activation o f cytosolic P K C . This statement is in agreement with the observation that in the liver and the heart homogenates, P t d O H provided markedly different profiles o f protein p h o s p h o r y l a t i o n as c o m p a r e d with PtdSer plus D A G (Bocckino et al., 1991). It is tempting to suggest that P t d O H m a y cause a redistribution o f P K C from the m e m b r a n e to the cytosol and activates the cytosolic enzyme which would be a direct effect o f P t d O H . However, it c a n n o t be excluded that in vivo P t d O H m a y activate phospholipase A (Ueda et al., 1993) which in turn generates F F A and lysophospholipid and, thereby stimulating cytosolic P K C (Sasaki et al., 1993).
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4. Effect of lipids on the PKC proteolysis mediated by calpain Although there are many observations regarding the mechanism involved in the activation of PKC, little is known about the factors which control the inactivation of these kinases, once they have been activated. Several mechanisms can be suggested: (1) the dissociation of PKC and the cofactors (Ca + +, PtdSer and DAG) leading to the translocation of the membrane bound form back to the cytosol in an inactive form, to our knowledge such a mechanism has not been described; (2) The activation of D A G kinase or/and D A G lipase lowering the D A G level (Kanoh et al., 1990); and (3) The degradation of PKC by proteolysis. Several proteases using PKC as substrate have been described. Among these calpains have attracted increased interest in recent years (Sorimachi et al., 1994). These enzymes are neutral cysteine endopeptidases, have an absolute Ca ÷ + dependence for activity and form a family of at least 6 distinct members (Saido et al., 1992; Saido et al., 1994). The /~-and m-calpain have a ubiquitous distribution in animal cells. Calpains catalyse the cleavage of the hinge region of the catalytic and the regulatory subunits of PKC generating a soluble Ca + +, phospholipid independent form termed PKM. This reaction has been considered as an irreversible activation of PKC (Murray et al., 1987). However, recent studies revealed that P K M is degraded by m-calpain at a faster rate than PKC (Shea et al., 1994) and that it did not accumulate in the cells suggesting that the role of calpains in the regulation of PKC activity may be limited to its inactivation. The inactivation of the PKC activity would depend, therefore, on the activation of the calpains and their interaction with the substrate. PKC is activated by PtdSer and various lipid second messengers and the question arises whether lipids modulate the calpain activities. Recent studies have shown that Ft-calpain was activated exclusively by the acidic phospholipids phosphatidylinositol (PtdIns) and Ptdlns(4,5)P2 by lowering the required Ca ÷ ÷ concentration for it (Saido et al., 1992). In contrast, the m-calpain activity was modulated differently by various lipids. Indeed ~PKC was completely degraded in about 30 min when incubated with m-calpain in the absence of lipids. The addition of various phospholipids PtdSer, PtdCho, PtdIns, and PtdOH produced a partial inhibition of the proteolytic activity. More than 30% of the initial amount of the substrates were still present after 30 min (Fig. 2). D A G and a mixture of D A G plus PtdSer did not significantly affect the m-calpain activity, questioning the sensitivity of PKC, in the active or inactive state, towards m-calpain. In order to get some insights into the mechanisms leading to the inhibition of c~PKC proteolysis the effect of the lipids on the hydrolysis of azocasein by m-calpain has been investigated. With the exception of PtdOH the various phospholipids tested, D A G and the mixture D A G + PtdSer did not affect the proteolysis of azocasein (Table 4). These results indicate that these lipids did not act on m-calpain. Therefore the inhibition of e P K C proteolysis by PtdSer, PtdIns, and PtdCho may be due to the modification of the lipid environment of the substrate creating a steric hindrance for the interaction with the enzyme, preventing its inactivation. In contrast, PtdOH inhibited the hydrolysis of azocasein (Table 4). With the incubation medium containing 11 m M Ca ÷ ÷ and 0.14 m M PtdOH, the
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60
I,
40
ii
ig o
C
I:tdSer
Prier 4DAG
0AG
P1dQH P~dCho Ptdlns
Fig. 2. Effect of lipids on ePKC proteolysis by m-calpain, aPKC (80 ng) was in 25 /~1 of 50 mM Tris-HC1, pH 7.4, 5 mM EDTA, 2 mM PMSF containing 5.2 ng m-calpain (specific activity: 0.857/~g azocasein//~g m-calpain per min) and various lipids (20/~g). The reaction was started by the addition of CaCI 2, final concentration 7 mM. Incubation was for 30 min at 30°C and stopped by adding 10/~1 of Laemmli buffer for subsequent SDS 7% polyacrylamide gels electrophoresis. After transfer to nitrocellulose, the strips were incubated with a commercial monoclonal antibody that recognizes the catalytic domain of PKCe (UBI) and alkaline phosphatase staining as described (Shea et al., 1993). Nitrocellulose replicas were scanned using a Scan Jet IIp scanner and compared with the Scan Analysis densitometric program. q u e s t i o n o f C a + + c h e l a t i n g b y t h e a c i d i c p h o s p h o l i p i d is r u l e d o u t f o r its effect o n c a l p a i n a c t i v i t y s u g g e s t i n g t h a t P t d O H is a p o t e n t d i r e c t i n h i b i t o r o f m - c a l p a i n . Since P t d O H a c t i v a t e s , in v i v o , c y t o s o l i c P K C t h e result s u g g e s t s t h a t this p h o s p h o lipid m a y essentially be i n v o l v e d in t h e r e g u l a t i o n o f c y t o s o l i c p r o t e i n p h o s p h o r y l a tion whereas the other phospholipids may favour membrane protein phosphorylation. Table 4 Effect of lipids on the activity of m-calpain towards azocasein Addition
Relative % of hydrolysis
Control PtdSer PtdSer + DAG DAG PtdOH PtdCho PtdIns
100 102 123 96 53* 128 84
0.1 nag [14C] azocasein (specific activity 500 000 cpm/mg) was dissolved in 50 mM Tris-HCl, pH 7.4, 2.3 mM DTT containing 2.7/zg m-calpain and various lipids (20/~g). The reaction was started by addition of CaC12 (11 mM final concentration) and incubation (final volume 200/tl) was performed for 30 min at 30°C. The reaction was terminated by addition of 100/~1 azocasein (5 mg/ml) and 300/tl of 10% ice cold TCA. After centrifugation the supernatant was removed and counted. * p<0.01.
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5. Conclusion D u r i n g the last few y e a r s c o n s i d e r a b l e p r o g r e s s has been m a d e in u n d e r s t a n d i n g the role a n d f u n c t i o n o f lipid s e c o n d messengers in signal t r a n s d u c t i o n . O f p a r t i c u l a r i m p o r t a n c e has been the c h a r a c t e r i z a t i o n o f new lipid s e c o n d messengers a n d their effect o n P K C activities (Liscovitch a n d Cantley, 1994). P K C c a n be c o n s i d e r e d as a cellular r e g u l a t o r a n d t h e r e f o r e the i n t e r a c t i o n with specific m o l e c u l a r species o f lipid s e c o n d messengers m a y induce specific cellular responses. T h e increasing n u m b e r o f v a r i o u s P K C i s o f o r m s (Azzi et al., 1992) a n d the o b s e r v a t i o n t h a t different m o l e c u l a r specie o f D A G o r P t d O H are g e n e r a t e d in p r o l i f e r a t i n g a n d R A d i f f e r e n t i a t e d L A - N 1 cells on T P A s t i m u l a t i o n are consistent with this hypothesis. M o r e o v e r , the d a t a s h o w i n g t h a t P t d O H d i d n o t o n l y m o d u l a t e the C a + + d e p e n d e n t P K C activity b u t was also able to inhibit its p r o t e o l y s i s b y m - c a l p a i n suggest t h a t in p r o l i f e r a t i n g cells the d u r a t i o n o f P K C a c t i v a t i o n was increased b y P t d O H . These results p r o v i d e evidence t h a t the T P A t r e a t m e n t o f the L A - N - 1 n e u r o b l a s t o m a cells stimulates the p r o d u c t i o n o f different m o l e c u l a r species o f D A G a n d P t d O H t h r o u g h the h y d r o l y s i s o f different p h o s p h o lipids b y the a c t i v a t i o n o f specific p h o s p h o l i p a s e s C o r D d e p e n d i n g on the p h y s i o l o g i c a l status o f the cells in question. I f we a s s u m e t h a t the different P K C ' s activities are m o d u l a t e d by specific lipid s e c o n d messengers it is likely t h a t the a c t i v a t i o n o f specific p h o s p h o l i p a s e s (PLA2, P L C , P L D ) u p o n r e c e p t o r a g o n i s t i n t e r a c t i o n w o u l d be one o f the first steps i n v o l v e d in the i n d u c t i o n o f specific cellular responses. T h e i s o l a t i o n a n d c h a r a c t e r i z a t i o n o f increasing n u m b e r s o f p h o s p h o l i p a s e i s o e n z y m e s (Liscovitch a n d Cantley, 1994) argue in f a v o r o f such a hypothesis.
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