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Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein
Biochimica et Biophysica Acta, 1134(1992) 73-80
73
© 1992ElsevierScience PublishersB.V. All rights reserved0167-4889/92./$05.00
BBAMCR 13107
Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A 2 and activation by a 70 kDa protein W o l f g a n g G r o s s t, W a n n i a n Y a n g a n d W e n d y F. B o s s Botany Department, North Carolina State Unh'ersity, Raleigh, NC (U.S.A.)
(R~eeived 16July 1991) (Revisedmanuscriptreceived 15 October 1991)
Key words: Phosphatidylinositolkinase; PhospholipaseA..; Phosphatidylinositol: Phosphatidylinosito]monophosphate
Plasma membranes were isolated from carrot (Daucus carota L) cells grown in suspension culture and treated with phospholipase A 2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinasc or phosphatidylim~itol monophesphate kinase activity released from the membranes after the phospholipase A 2 treatment. Treating the plasma membranes with phospholipaso C or D did not release PI kinase activity. The phespholipase A -released Pl kinasc was activated over 2-fold by a beat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the V,,~, but does not affect the K , of the phospholipase A2-released Pl kinase.
Introduction Whether phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP 2) are precursors of the second messengers dia~lglycerol and inositol 1,4,5-trisphosphate (IP 3) [1,2], or directly affect membrane enzymes [3,4], regulation of the biosynthesis of these lipids is critical. Although PIP- and PIP2-specifie phospholipase C have been well characterized with regard to the activation by Ca 2+ in higher plants [5-8] and by Ca 2+ and GTP in animals [9,10], very little is known about the regulation of the phosphatidylinositol 4-kinase [11]. Rapid changes in
t Present address: Instimt far Pflanzenphysiolollieund Mikrobiolo8ie. Fmie Universit'ttBedin, 1000Berlin 33, Germany. Abbreviations: lP3, inceitol 1,4,.%.trisplmsphate; LPI, lysophos* phatidylia~itol; PC, phesphalidykholine;Pl, plmsphaticb/linositol; PLA2, phmpbolipase A2; PLC, phospholipase C; PLD, phospholipa~ D; PIP, phosphatidylino~itol 4-monophosphate; PIP2, phmphatidylinesitol4,5.bisplmsphate;SDS, sodium dode~l sulfate; PAGE, polyacrylamide gel electmphoresis. Co~nce: W.F. Boss, Botany Department, North Carolina State University,Ra'eigh, NC 27695,U.S.A.
the activity of P! kinasc and PIP kinase occur in response to external stimuli in higher plants [12,13]. Recently, Lassing and Lindherg [14] reported that in" stimulated platelets, the levels of PIP and PIP2 increased prior to activation of PIP2-phospholipase C. These data suggest an important role for the in~itol phospholipid kinases in signal transduction. Our focus was to characterize the plasma membrane P! kinase from carrot cells grown in suspension culture. It is well accepted that some membra'~e surface glycoproteins are anchored to the membrane with lipid anchors which can he released by phospholipase C (PLC) [15] and D (PLD) [16]. We reasoned that intracellular facing proteins on the plasma membrane also might be released from the membrane by phospholipases. We report here that phospholipase A 2 (PLA 2) released Pl kinase activity from isolated plasma membranes but PLC and PLD did not. The release of PI kinase by P L ~ z appeared not to result from nonspecific loss of the membrane lipids as was reported for the opiate receptor [17]. In addition, we observed that a heat stable, soluble 70 kDa protein activated the PLA2-released P! kinase. The release and activation of the plasma membrane PI kinase are two separate phenomena which may together or separately play a role in regulating the P! kinase activity in vivo.
74 Materials and Methods
Materials Wild carrot (Daucus carota L.) cells were maintained in suspension culture according to Chen and Boss [13]. Ceils were transferred serially every 7 days and Used for experiments at day 4. PLA 2 from Crotalus darissus terrificus venom, PLA 2 from Naja mocambique mocambique, PLA 2 from Apis mellifera venom, PLC from CIostridium perfringens Type XIV, PLD from peanut Type Ill, melittin, trypsin I, trypsin inhibitor type IV-O (chicken egg white), lysophosphatidylcholinc (LPC) and phosphatidylcholine (PC) were purchased from Sigma. [3,-32p]ATP (7000 Ci/mmol) was purchased from ICN and [mC]eboline (58.5 /.tCi/mmol) was purchased from New England Nuclear.
Plasma membrane isolation Plasma membranes were isolated by aqueous twophase partitioning as previously described [18], except that the isolated membranes were washed once with 20 mM Tris-HCI (pH 7.2), 1 mM CaCI 2, 0.01% (v/v) Triton X-100 before they were used for treatments unless indicated otherwise.
PLA z treatment of plasma membranes The isolated plasma membranes were resuspended in buffer A (20 mM Tris-HCI (pH 7.2), 1 mM CaCI 2, 0.01% (v/v)Triton X-100) and phospholipase in buffer A or buffer A alone was added to give a final concentration of 30/~g membrane protein/20 p.I buffer. The samples were incubated for 10 rain at room temperature under constant shaking (200 rpm). At the end of incubation time, EGTA was added to give a final concentration of 1.5 raM. The samples were centrifuged for 30 min at 45000 × g and the supernatant and the membrane pellet were collected. The resuspended pellet and the supernatant were assayed immediately for phosph,~lipid kinase activity.
Enzyme assays PI kinase activity was assayed using the following procedure: 20 p.I enzyme preparation (released from up to 30 p.g of membrane protein) was added to 30 p.I reaction mixture to give a final concentration of 50 mM Tris-HCl (pH 7.0), 20 mM MgCI 2, 0.6 mM PI (prepared as mixed micelles using 3% (v/v) Triton X-100), 0.2 mM sodium molybdate, 0.25% (v/v) Triton X-100 and 0.6 mM AT, ~ containing [3,-32p]ATP (0.1 tzCi/nmol). For studies of the trypsin sensitivity of the 70 kDa activator and of the effect of increasing activator concentration, the pbosphorylation mixture contained 30 mM Tris-HCI (pH 7.2), 7.5 mM MgCI2, 0.6 mM el, 1 mM sodium molybdate, 0.3% (v/v) Triton X-100 and 0.9 mM ATe. To measure PIP and diacyl-
glycerol kinase activity, either PIP or dipalmitoylglycerol was added instead of el. The reaction mixture was incubated for 10 min with constant shaking. The reaction was stopped by the addition of 1.5 ml of ice-cold CHCI3/MeOH (1:2, v/v). HC[ (0.5 ml of 2.4 M) was added and the lipids were extracted and chromatographed on thin-layer plates as previously described [13] except that TLC plates were developed in C H C I 3 / M e O H / 1 5 M N H 4 O H / H 2 0 (86:76:6:18, v/v). Radioactivity was quantified with a Bioscan System 500 Imaging Scanner. Protein was determined using the bicinchoninic acid method as described by Smith et al. [19].
Metabolism of phosphatidylcholine in isolated plasma membranes Cells were labeled with [Inc]choline (1.4 p.Ci/0.3 to 0.4 g fresh wt.) for 45 h and harvested by filtration on Whatman No. 1 filter paper. Plasma membranes were isolated by aqueous two-phase partitioning [18], resuspended in Tris-HCI buffer (pH 7.2) and treated for 10 min with PLA 2, calcium, EGTA and melittin as indicated. After treatment, the reaction was stopped by adding 5 mM EGTA. The membrane lipids were extracted in C H C I 3 / M e O H / 2 . 4 M HCI (1:2:1, v/v) as previously described [13]. The 14C recovered in both the organic and aqueous phases was quantified by scintillation counting in a Beckman LS 7000 scintillation counter using ScintiVerse (Fisher Scientific). LPC and PC in the organic phase were separated by thinlayer chromatography using LK-5 plates (Whatman) and C H C i 3 / M e O H / N H 4 O H (65:25:5, v / v ) as a solvent. [mC]PC and [14C]LPC were identified by comigration with standards and quantified with a Bioscan System 500 Imaging Scanner.
Partial purification of the 70 kDa protein Cells were homogenized as described above and the homogenate was centrifuged for 60 rain at 45 000 × g. The supernatant was collected, boiled for I0 min and filtered (0.2 p.m). The filtered solution was then loaded onto a Q-Sepharose column (1.5 × 20 cm) equilibrated with buffer B (10 mM Tris-HCl (pH 7.5), 2 mM KCI, 0.2 mM EDTA, 0.2 mM MgCl2). The column was washed with 0.25 M KCI in butter I~, |oiJowed by a salt gradient from 0.25 to 0.8 M KCI. Aliquots of the fractions eluted from the column were tested for activation. For this assay, 10 /~l of PLA2-released PI kinase was incubated for 2 min with 2 to I0/zl of the column fractions and EGTA (5 mM final concentration) was added to inhibit phospholipasc A 2 activity. The PLA2-released PI kinase was incubated for 2 min with 2-10 pA of the. c~lumn fraction and the reaction was started by the addition of reaction mixture (see above). Fractions that activated PI kinase were pooled, concentrated using Centricon-30 (Amicon), and loaded
75 o n t o a Sephacryl S-300 c o l u m n (I × 55 cm). Fractions w e r e eluted with b u f f e r B a n d tested for activation. P e a k fractions w e r e p o o l e d a n d used for f u r t h e r studies.
Determination of Mr T h e native M r o f the activator w a s estimated by gel filtration o n Sephacryl S-300 (1 × 80 cm) using the following Me s t a n d a r d s : 7 - g l u t a m y l t r a n s p e p t i d a s e (80000), bovine s e r u m albumin (66000), peroxidase (40000) a n d c h y m o t r y p s i n (25 000).
F o r protein p h o s p h o r y l a t i o n 4 /.LCi of [ y - 3 2 p ] A T P (3.5 / . t C i / n m o l ) was a d d e d to 80 p l m e m b r a n e s in b u f f e r A. A f t e r 10 rain at r o o m t e m p e r a t u r e , the s a m p l e w a s c e n t r i f u g e d 90 min at 4 5 0 0 0 × g a n d 80 #1 o f S D S - P A G E (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) s a m p l e b u f f e r (62.5 m M Tris-HCI ( p H 6.8), 10% glycerol, 2 % SDS, 5 % /3-mercaptoe t h a n o l ) w a s a d d e d to the s u p e r n a t a n t . S D S - P A G E a n d a u t o r a d i o g r a p h y w e r e d o n e as d e s c r i b e d by Blowers et al. [20]. Results
Release of phosphatidylinositol kinase activity by phospholipase A z treatment W o r k i n g with c a r r o t suspension cell cultures, we f o c u s e d o n the c h a r a c t e r i z a t i o n o f a p l a s m a m e m b r a n e - a s s o c i a t e d Pl kinase. W h e n w a s h e d p l a s m a m e m b r a n e s f r o m c a r r o t cells w e r e i n c u b a t e d in 0.01% T r i t o n X-100 a n d 1 m M CaCI 2 f o r 10 m i n at r o o m t e m p e r a t u r e , a b o u t 1% o f the total P l ~ n a s e activity w a s r e c o v e r e d in the soluble fraction. A d d i t i o n o f P L A 2 to t h e i n c u b a t i o n m e d i u m g a v e a 5- t o 1 0 - f o l d i n c r e a s e o f Pl kinase activity in t h e soluble fraction ( P L A - f r a c t i o n ) (Table I), w h i c h w a s a c c o m p a n i e d by a loss o f activity in t h e t r e a t e d p l a s m a m e m b r a n e s . T h e P L A 2 itself c o n t a i n e d n o kinase activity a n d only back-
All t~ealmcnt~",~'.src~,nth~ prese~e of ! mM CaCI: and the calcium was removed by adding EGTA prior to phospho~lation as described in Materials and Methods. Data are the meaas of four values from two experiments. The [32p]PlP recovered in the control was 3150 dpm. The enzyme activity is e:~prasscdper ml PLA-fraction and not per mg protein, because the addition of the PLA 2 preparation made it difficult to extrapolate the amount of released iffotein in the fraction. However. for each treatment the same amount of starting material (plasma membrane protein)was used. PLA2-released PI kinase activity [32P]Plp
none PLC 20 U/ml PLD 20 U/ml Pt.A 2 a 20 U / m l PLA2" (0~ C) PI-A2~* + 5 mg BSA/ml PLA2 h PLA2b +0.2 mg melittin/ml Melittin 0.2 m~/ml Melittin 0.(Mmg/ml Proteinase inhibitors ~ PLA2 a + proteina~ inhibitors c PLA2;' +20 ttM ATP PLA2 ~ +alkaline p;,osphatase d PLA2 a + 0.2 mM Na2Mo 4
~'u ~
'
.,'~
' ,,'o ' A~(,mi~=.~)
pmol/min /ml
% of control
21.7 14.5 28.6 270.2 Q.8 171.9 127.8 69.2 6.2 48.4 7.4 212.7 256.1 261.5 284.3
100:[: 2 67+ 14 132+ 8 1245 -+I I 45± 7 792=1:12 589+ 14 319± 9 27-1:6 223 d:22 34:t: I I 980_+13 I 180+ 12 1205+ 15 1310± 4
a !,L~ 2 was from Crotalus dun~sus teniflcus. PLA 2 was used at 20 U/ml in all instances. b PLA2 was from Apis mellifera venom. I mM phenylmethylsolfowJl fluoride, 1O0 Fg pepstatin A, 2 /~g leupeptin, 20 p.g trypsin inhibiter from chicken egg white. d Alkaline phosphatase attached to agarose beads was used and removed by centrifugation after incubation.
g r o u n d levels o f PI kinase activity w e r e d e t e c t e d in the P L A - f r a c t i o n w h e n t h e P L A 2 t r e a t m e n t was c a r r i e d o u t o n ice (Table !). T r e a t m e n t o f m e m b r a n e s with
_*"I B < ,i
ii:i[ ,
Effect of furious phospholipa~s and compounds on the release of P! kinase from washedplasma membranes
Additions
Protein phosp.:~orytation
o;
TABLE I
---.........
go
3o "lime (mla)
Fig. I. Concentration-dependent release of PI kinase activity by PLA 2. (A) Isolated plasma membranes were incubated with increasing concentrations of PLA 2 fm 1Orain at rc~m t¢inl~aZurc. Th~ o.mpl~ wcrc centrifu~:cd and the ngmbrane-free supernatant was assayed for Pl kinase activity. (13)Isolated plasma ,membranes were treated with 20 U/ml PLA2 and the released P! kinase activity was measured over time. Values are the average of two numhets from one experiment. The effor is indicated when it is larger than the symbols. The experiment has been repeated two times and the trends were similar.
76
PLA 2 from either Apis mellifera, Naja mocambique mocambique (data not shown) or Crotalus durissus terrificus resulted in release of PI kinase activity. O f the lipid kinases tested, only PI kinase was released from the plasma membrane vesicles. No PIP kinase or diacylglycerol kinase activity was detected (data not shown). Phospholipase C or phospholipase D did not release PI kinase activity from plasma m e m b r a n e s (Table I). In fact, PLC treatment resulted in a decrease in the recovered PI kinase activity compared to the control value. As shown in Fig. IA, PI kinase activity was released from the membrane by PLA 2 in a concentration-dependent manner. Incubation with about 20 U PLA 2 per ml for 10 rain at R T gave maximum recovery of PI kinase activity in the 45 000 × g supernatant. A t higher PLA 2 concentrations the kinase activity decreased slightly. The release of PI kinase activity in the presence of 20 U P L A 2 / m l increased gradually over the 10 rain incubation time (Fig. 1B). Longer incubation periods, however, did not result in an increase in the PI kinase activity released. The decrease in PI kinase activity observed with higher P L A 2 concentrations or with increasing treatment times may have been caused by an increase in free fatty acids or lysolipids which have been shown to inhibit PI kinase [21] or by some other factor released during the treatment. In any event, these data indicate that continued treatment with PLA 2 did not facilitate the release of active PI kinase. Therefore, the routine t r e a t m e n t of plasma ;;.~tni~.,iles was with 20 U / m l P L A 2 for 10 rain. U n d e r these conditions 10 to 20% of the total PI kinase was released from the plasma membranes. It should be noted that u n d e r the assay conditions used only P1-4 kinase activity was measured. H P L C analysis of the deacylated reaction products verified that more than 99% PI-4-P and less than 1% PI-3-P was formed
plus Phosphollpese A=
~|,on
mM Calcium chloride Fig. 2. Effect of calcium on the release of PI kinase by PLA 2. Plasma membranes were treated as described, except that increasing concentrations of CaCI2 were used. Following the incubation. EGTA was added to give an excess of 0.5 mM relative to the Ca2+ concentration. The samples were centrifuged and the membrane-free supernatant was assayed for PI kinase activity. Values are the average of two numbers from one experiment. The error is indicated when it is larger than the symbols. The experiment has been repeated two times and the trends were similar.
by the PI kinase (A. Graziani and L.C. Cantley, unpublished data). Calcium was required for the PLA2-induced release of PI kinase activity (Fig. 2). The optimum calcium concentration for PLA2-induced release was between 0.5 to 1 m M CaCI 2. Increasing the calcium concentration to 2 mM inhibited the release of the active kinase in the presence of P L A 2 while calcium alone decreased the recovery of soluble PI kinase activity at all concentrations tested (Fig. 2). Note that E G T A was added at the end of the 10 rain incubation period to chelate the free calcium; therefore, the observed decrease in activity was not due to direct interference by calcium during the assay. Analysis of phosphatidylcholine metabolism of m e m b r a n e s isolated from cells grown in [14C]choline substantiated a role for calcium in activating P L A t .
TABLE I!
Metabolismof plasma membranephosphatidylcholine Cells were labeled with lJ4C]choline(1.4 p.Ci, 58.5 p.Ci/mmol) for 45 h, harvested and the plasma membranes isolated by aqueous two-phase partitioning. The isolated membranes (112 p.g protein) were treated for 10 rain at RT as described. The final concentrations of the reagents were: I mM CaCI2, 20 U/ml PLA 2, 5 mM EGTA, 0.2 mg/ml melinin ia a 120 p.I reaction volume. Lipids were extracted and aliquots of the organic and aqueous phase were ouantitated by scintillation counting. The remaining organic phase was analyzed by thin-layer chromntograpby and the [14CJPCand [14C]LPCquantified with a Bioscan 500 Imaging Scenner. Treatment
Ca2+ PLA2 + EGTA PLA 2 +Ca 2+ PLA2+Ca2++meliuin Melinin+Ca2+
LPC
dpm 357+ 23 1221 4-164 28214-107 25284- 14 13644- 21
PC
dpm 5250+336 4543 4- 86 14504- 21 244- 6 393+ 7
LPC/PC
0.06+ 0 . 0 1 0.194- 0.09 2.04- 0.l 90 4-36 12.84- 9.9
Organic
Aqueous
Organic
phase
phase
/aqeous" phase
dpm 6650+1300 8075 + 1700 59254-1050 39754- 650 30504- 513
dpm 47884-200 3700 4-169 53504- 15 57134-150 84254-100
1.64-0.2 2A + 0.5 IA4-0.4 0.84-0.3 0.54-0.2
a The ratios are the means of 4 to 8 values from two experiments. The dpm values are the average of two values from one representative experiment.
77 Adding PLA 2 in the presence of 1 mM CaCI 2 to the membranes resul(ed in a 2-fold increase in the amount of [14C]lysophosphatidylcholine (LPC) recovered compared to that recovered with PLA 2 in the absence of free calcium and an 8-fold increase compared to calcium alone (Table II). Treatment with calcium alone (1 mM or 4 mM for 10 rain) did not significantly increase in [14C]LPC but did result in a 15% and 50% loss, respectively, in [n4C]phosphatidylcholine (PC) compared to treatment with EGTA (data not shown). These data suggest that a calcium activated PLC or PLD was present, and they are consistent with the fact that exogenously added PLC, like calcium alone, decreased the release of PI kin~se activity. Melittin, a peptide present in bee venom, has been shown to activate endogenous phospholipases [22,23] and was used to increase the release of the opiate receptor [17] by enhancing membrane lipid hydrolysis. To determine whether further degradation of membrane lipids would enhance the release of the plasma membrane Pl kinase activity melittin was added in the presence and absence of PEA 2. Adding melittin (0.2 mg/ml) resulted in an almost total loss of [I4C]Fr~.~and a concomitant increase in water soluble products (Table II) suggesting the activation of PLC or PLD. However, at this concentration melittin decreased the release of the Pl kinase activity (Table 1) whether it was added in the presence or absence of PLA 2. The decrease in release of the Pl kinase activity when excess melittin was added may have been caused by the release of inhibitors as a result of increased membrane degradation. If less melittin (0.04 m g / m l ) was added there was an increase in the release of PI kinase activity (Table I). This was probably caused by the PLA 2 present in the melittin [23]. While the mechanism involved in the PLA2-induced release of Pl kinase activity is not known, one possibility was that calcium-dependent proteinases might be involved. However, there was no detectable proteinase activity in the PLA 2 preparations under these conditions (data not shown) and calcium alone resulted in a decrease in the recovery of soluble Pl kinase (Fig. 2). When BSA or several proteinase inhibitors were included in the PLA 2 treatment, there was less PLA2-released Pl kinase activity (Table 1). However, proteinase inhibitors have been shown to have an effect on PLAz activity [24]. Furthermore, when proteinase inhibitors were added to the membranes without PLA 2 there was less soluble Pl kinase recovered suggesting that the proteinase inhibitors may have a direct effect on the Pl kinase activity (Table !). Taken in toto, these data suggest that calcium-activated proteinases were not involved in the release of Pl kinase. in addition, we tested to determine whether or not phosphatases were involved in the PLA2-release of Pi kinase activity. The addition of alkaline phosphatase or
of 0.2 mM sodium molybdate (a phosphatase inhibitor) had little effect on the recovery of P! kinase activity in the PLA-fraction (Table I). To test for the involvemetlt of protein phosphorylation, we added [7-32p]ATP during the PLA 2 treatment. Although, the PLA-fraction exhibited three 32P-labeled bands at 34, 50 and 66 kDa on SDS gels, there was no difference in the PLA 2treated or control sample. Furthermore, addition of ATP during the PLA 2 treatment did not increase the activity in the PLA-fraction (Table !). Therefore, we have no evidence to suggest the release of Pl kin'ase from carrot cell plasma membranes involves the phosphorylation or dephe~phorylation of proteins. If the release of the Pl kinase by PLA 2 is specific, then one possible mechanism for release is via the cleavage of a fatty acid anchor from the protein. We labeled cells in vivo with [14C]myristate, [t4C]palmitate or [14C]stearate. While multiple bands were visible after SDS-PAGE and autoradiography of intracellular membranes, the incorporation of radiolabeled fatty acids into plasma membrane proteins was too low to be able to detect differences in the labelling pattern from PLA 2 treated and non-treated plasma membranes (data not shown). Activation o f released phosphatidylinositol kinase by a 70 kDa protein When plasma membranes were washed with 0.01% (v/v) Triton X-100 prior to the PLA 2 treatment, the recovery of P! kinase activity in the PLA-fraction was accompanied by a loss of activity in the plasma membranes with no significant change in total activity (106
TABLE ill The acti~,ator is trypsinsensitive The partially parified activator(0.78 mg wotein/ml) was incubated at RT in the presence and absence of 0.2 mg/ml trypsin(10000 units/mr) in 30 mM Tris-HCi buffer (pH 7.2) for 3 I1. Tr~in inhibitor (0.3 mg/ml) was added after 3 h or just ~ to the addition of tw0sin,and the activator(15.5/~g protein)was added to the PLA2-rel(.ased P! kinase and PI kinase activity was assayed as described in blaterials and Methods. PLA2-releused Pl kinase activi~ [32p]PIP Trypsininhibitorplus WJpsin Activator Activatorplus tvjpsin3 h; followedby trypsininhibitor Activator plustrypsininhibitor and tvjpsinat the sametime
pmol/min 40.8+5.1 84.05:6.8
activation"
2.2+0.2
36.0+4.4
none
63.6+i.8
2.0±0.5
a The pmol/minare averajevaluesfromone experimenLActivation was calculated re'l~ti~ to the control which was trypsin inhibitor plus trypsin added at the time of pimspho~atk)n. The values a r e the mean± S.D. of fourvaluesfromtwo experiments.
i~'°°°t
|,
"°°1/ I:'7
.
,o
.o
..o
l/PbesphnUdylinositol(mM)
AclivalorFrael|on(g¢ Protein) Fig. 3. Activation increases with increasing concentration of the activator fraction. The heat-treated, soluble fraction was added in increasing amountsto the PLA2-roleased Pl kinase (released from 27 p.g membraneprotein) and Pl kinase activity was assayed in a final reaction volume of 50 ,¢1. Values are the average of two numbers from one experiment. The error is indicated when it is larger than the symbols.The experiment has been repeated two timesand the trend~were similar. + 5%, mean + S.D. of four values). However, when the plasma membranes were not prewashed with 0.01% Triton X-100 prior to the PLA 2 treatment the total Pl kinase activity increased about 30% over the control (128 + 4%, mean + S.D. of four values). This suggested that a soluble factor entrapped in or loosely associated with the membrane vesicles could increase Pl kinase activity. Therefore, we collected the soluble fraction (e.g., the 45000 × g supernatant of a crude bomogenate), boiled it for 10 rain to inactivate the soluble Pl kinase present in this extract and added aliquots to isolated plasma membranes after PLA 2 treatment and to the PLA-fraction. The addition of the heattreated, soluble fraction to untreated plasma membranes or PLA2-treated plasma membranes increased the PI kinase activity only about 10% over the control (data not shown). However, the PLA 2-released Pl kinase activity increased from 2- to 8-fold after addition of the soluble fraction (Table III and Figs. 3 and 4).
tt
t
~
5~
i
J
....
o
" ;
" ,'o ,'~,'o2; FrscUon Number
~o°
Fig. 4. Gel filtration of crude activator protein on Sephaeryl-300. Peak fractions from O-Sepharose were pooled, concentrated and loaded onto a SephacrylS-300 column.The eluent was fractionated and an aliquot of each fraction tested for its ability to the activate PLA2-released PI kinase.
Fig. 5. Change of ["maxof solubilized Pl kinasc by the 70 kDa t'~rutein. PLA2-solubilized PI kinasewas assayedin the presenceand absence of partially purified 70 kD~.protein with increasingconcentration of Pl. The activation was eliminated by treating the heattreated, soluble fraction with trypsin (Table lll) indicating that the activator is a protein. Activation of the Pl kinase was concentration dependent. Using the heat-treated, soluble fraction as the source of activator, we found that adding greater than 1 # g of p r o t e i n / 5 0 ~.1 of reaction mixture was necessary for activation (Fig. 3) and 10 # g approached saturation. The activator preparation varied from day to day as indicated by the activation of the PLA2-released P1 kinase activity (compare Table III and Figs. 3 and 4). This may be be¢~.use of differences Jn the amount of activator or potential inhibitors present in the individual preparations. The activator was partially purified using the heattreated soluble fraction. The protein was filtered through a 0.2 /~m filter, chromatographed on a QSepharose column, concentrated by filtration (Centricon-30 filter) and further purified by Sephacryl S-300 column. The profile of activation versus absorbance at 280 nm is given in (Fig. 4). In the presence of this activator protein, the Pl kinase exhibits an increase in Vmx with no significant change in K m under the assay conditions used (Fig. 5). The estimated K m for Pl was approx. 1 mM. Discussion We have shown that Pl kinase activity is released from the plasma membranes of carrot cells by treating the iaembranes with PLA 2 in the presence of calcium. Rfiegg et al. [17] used PLA 2 to release the opiate receptor from rat brain membranes. With the opiate receptor, however, increased degradation of the membrane lipids by adding melittin enhanced the release suggesting that the release was the result of the loss of membrane integrity. This was not true for the release of carrot plasma membrane Pl kinase activity. The optimum release of the active enzyme occurred with a short term treatment with PLA 2. Treatment with PLD
did not result in the release of PI kinase activity and treatment with PLC inhibited the release of the active enzyme. Release of P! kinase activity by PLA_, required calcium; however, calcium had two effects on the release. Calcium (1 mM) enhanced the release of the active Pl kinase and enhanced PLA2 activity as evidence by the increase in LPC production. On the other hand, calcium alone or calcium greater than 1 mM in the presence of PLA z decreased the recovery of Pl kinase activity in the PLA-fraction. Since free calcium was removed by adding EGTA to the reaction mixture prior to assaying for Pl kinase activity, the decrease of PI kinase activity did not result from a direct effect of calcium on the kinase [25]. A calcium-dependent PLD has been reported to be associated with plant membranes [26]. Activation of endogenous PLD or PLC by calcium may explain why calcium alone or calcium at concentrations greater than ! m M in the presence of PLA 2 inhibited the release of P! kinase activity. Calcium-dependent proteinases appeared not to be involved in the PLA~:induced release of Pl kinase activity because there was no detectable proteinase activity in the PLA 2 preparations and because proteinase inhibitors had a greater effect on the control than the PLA2-released PI kinase activity. Phenylmethylsulfonyl fluoride has been shown to decrease PLA 2 activity [24], and this may have caused the decrease in the PLA2-induced release when proteinase inhibitors were added. Low [15] and Scailon et all [16] have shown that PLC and PLD, respectively, can release lipid anchored surface glycoproteins from the plasma membrane. Proteins also can be anchored by attachment to a single fatty acid which cannot be cleaved by PLC [27,28]. Although the treatment of various isolated acylated proteins with PLA 2 resulted in no release [29] or only limited release of fatty acids (4-5%) [30,31], a protein fatty acylesterase has been demonstrated in microsoreal membranes from mammalian cells [29]. It may he that PLA 2 plays a role in releasing fatty acid anchored proteins from the intracellular surface of the plasma membrane. We have not been able to recover enough fatty acid-labeled protein before or after PLA 2 treatment to determine whether or not this is true for the carrot plasma membranes. If only a small percentage of the fatty acylated proteins are affected, however, the differences may not be measurable ~ithout the specific antibodies to concentrate the proteins of interest. As a result of the PLA 2 treatment, only about 10% of the total plasma membrane Pl kinase activity was recovered in the PLA-fraction. The limited release of Pl kinase may be the result of the limited time of treatment or it may be that two populations of PI kinase are present in the plasma membranes and only one population can be released by PLA 2. Several dis-
tinct PI kinases have been isolated from mammals [32] and from yeast [33]. The estimated K m for PI was approx. I mM which is considerably higher than reported for most purified PI kinases [33-36] but is very similar to the partially purified enzyme from rat brain [371. The release and activation of the PI kinase were two different processes. While the release of P! kinasc activity was calcium dependent, activation was not. Activation was not caused by a factor in the P.LA 2 because, prewashing the membranes with Triton X-100 eliminated the activation even though the PI kinasc was released and PLA 2 was still present in the reaction mixture. Activation was recovered by adding back the Triton X- 100 wash. Cardiotoxin has been shown to activate PI kinasc [38]. Cardiotoxin, however, decreased the K m of the enzyme for P! and did not affect the P'm~. The opposite is true for the soluble protein activator of the PLA2-released PI kinasc, in addition, if cardiotoxin were presem in the PLA z preparations one would have expected to see activation using Triton-washed membranes and this was not found. Also, PLA 2 was used from several sources and all were effective in
releasing the PI kinase. Activatio~ was ~aused by a heat-stable soluble component. The activator was trypsin sensitive indicating that it was a protein. Activation increased with increasing protein concentration. The relative molecular mass estimated by Sephacryl S-300 chromatography was 70 kDa. If one calculates the increase in P! kinase activity based on the total activity in the membrane and PLAfraction, the total increase was only about 30% when the activator was added. If however, one considers the amount of activation of only the solubilized enzyme in the PLA-fraction, adding activator increased the Pl kinase activity 2- to 8-fold. Regulation of Pi-4 kinase activity in animal cells was reviewed recently by Carpenter and Cantley [!1]. Phosphoproteins have been reported to regulate the Pl kinase in transformed fibroblast cultures [39] and the PiP kinase in rat brain [40]. However, there is no in vitro evidence that the Pl 4-kinase is phosphorylated or that purified Pl 4-kinase is regulated by a protein kinase [ I I]. Our data suggest that protein phosphorylation does not play a role in releasing the carrot plasma membrane Pl kinase. However, until we have'isolated the released PI kinase and the activator and determined whether or not they are phosphorylated, we cannot rule out a regulatory role for protein phosphorylation/dephospborylation in this system. The in vitro results presented in this paper lead one to question whether the release and activation of Pl kinase is physiologically relevant? in animals, as well as in plant cells, it is known that PLA 2 activation is part of signal transduction. Crouch and Lapetina [41] found
8O in platelets t h a t P L A 2 is p a r t o f a signal t r a n s d u c t i o n p a t h w a y t h a t is not directly c o r r e l a t e d with the phos* pholipase C / I P 3 pathway. Potential roles o f lysolipids a n d P L A 2 in h i g h e r p l a n t signal t r a n s d u c t i o n have b e e n reviewed [21,26,42]. In soybean, the levels o f lysolipids increase u p o n stimulation with auxin [43] indicating a n increase in p h o s p h o l i p a s e A activity. Previously, we have shown t h a t w h e n the specific activity o f the p l a s m a m e m b r a n e PI ~.nd P I P kina~;e increased, the specific activity o f the p l a s m a m e m b r a n e P L A 2 also incrcased [44]. Based o n these d a t a a n d the d a t a p r e s e n t e d in this p a p e r , we p r o p o s e as a w o r k i n g hypothesis the following signal t r a n s d u c t i o n c a s c a d e for the c a r r o t m e m b r a n e s . A stimulus activates the P L A 2 in the p l a s m a m e m b r a n e . T h e p l a s m a m e m b r a n e P L A 2 releases PI kinase which is activated by a 70 k D a protein. T h e activator-Pl kinase complex reassociates with the m e m b r a n e , ph0~phorylates PI a n d increases the level o f PIP in the m e m b r a n e . T h e inc r e a ~ in P I P stimulates the m e m b r a n e vanadate-sensitive A T P a s e [45], t h e r e b y causing a c h a n g e in cell physiology. Acknowledgements W e gratefully a c k n o w l e d g e the technical assistance o f I r e n a Brglez. This r e s e a r c h w a s s u p p o r t e d by g r a n t n u m b e r D C B - 8 8 1 2 5 8 0 - 0 3 f r o m the N a t i o n a l Science F o u n d a t i o n a n d in p a r t by the N o r t h C a r o l i n a Agricult u r a l R e s e a r c h Service. W e t h a n k A. G r a z i a n i a n d L.C. Cantley (Tufts University School of Medicine, Boston, M A ) for doing the headgroup analysis of PIP. References I Majerus, P.W., Connolly, T.M.. Deckmyn, H., Ross, T.S., Bross, T.E., Ishii, H., Bansal, V.S. and Wil~n, D.B. (1986) Science 234, 1519-1526. 2 Berridge, MJ. and Irvine, R.F. (1989) Nature 341,197-205. 3 Sch§fer, M., Behle, G., Varsanyi, M. and Heilmeyer, L.M.G. (1987) Biochem. J. 247. 579-587. 4 Chauhan, A., Chauhan, V.P.S., Deshmukh, D.S. and Brockerhoff, H. (1989) Biochemistry 28, 4952-4956. 5 Melin, P.M., Sommarih, M., Sandelius, A.S. and Jergil, B. (1987) FEBS Len. 223, 87-91. 6 Pfaffmann, H., Hartmann, E., Brishtman, A.O. and MorrO, DJ. (1987) Plant Physiol. 85,1151-1155. 7 McMurry, W.C and Iwine, R.F. (1988) Biochem. J. 249, 877-881. 8 Tare, B.F., Schaller, G.E., Sussm~n, M.R. and Crain, R.C. (1989) Plant Physiol. 91,1275-1279. 9 Merrin, J.E., Taylor, C.W., Rubin, R.P. and Putney, Jr., J.W. (1986) Biochem. J. 236, 337-343. 10 Fain, J.N. (1990) Biochem. Biophys. Acta 1053, 81-88. 11 Carpenter, C.L. and Cantley, L.C. (1990) Biochemistry 29, 11147-11156. 12 Memon, A.R. and Boss, W.F. (1990) J. Biol. Chem. 265, 1481714821.
13 Chen,Q. and Boss,W.F. (1990)Plant Physiol. 94. 1820-1829. 14 Lassiog,I. and Lindherg, U. (1990)FEBS LeU. 262, 231-233. 15 Low, M.G. (1989) FASEB J. 3, 1600-1608. 16 Scallon, BJ., Fuog, W-J.C., Tsaog, T.C., Li, S., Kado-Fong, H., Huaog, K-S. and Kochan, J.P. (1991) Science 252, 446-448. 17 Riiegg, U.T., Cuenoud, S., Fulpius, B.W. and Simon, E.J. (1982) Biochim. Biophys. Aeta 685, 241-248. 18 Wheeler, J.J. and Boss, W•F. (1987) Plant Physiol. 85, 389-392. 19 Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartnet, F.H., Provenzona, M.D., Fujimoto, E.K., Goeke, N.M., OIson, BJ. and Klenk, D.C. (1985) Anal. Biochem. 150, 76-85. 20 Blowers, D.P., Boss, W.F. and Trewavas, A.J. (1988) Plant Physiol. 86, 505-509. 21 Wheeler, J.J. and Boss, W.F. (1990) in Inositol Metabolism in Plants (Mort'~, D.J., Boss, W.F. and Loewus, F.A, ads.), pp. 163-172, Wiley-Liss, New York. 22 Fletcher, J.E., Jiang, ?,~.-S.and Gong, Q-H. (1991) Biochem. Cell Biol. 69, 274-281. 23 Fain, J.N. Kabnick, K.S. and Li, S.-Y. (1981) Biochim. Biophys. • Acta 677, 274-279• 24 Travers, J.B., Sprecher, H. and Fertel, R.H. (1990) Biochim. Biophys. Acta 1042, 193-197. 25 Carrasco, A., Magendzo, K., Jaimovich, E. and Hidalgo, C. (1988) Arch. Biochem. Biophys. 262, 360-366. 26 MorrO, D.J. (1990) in inositol Metabolism in Plants (Morr;', D.J., Boss, W.F. and Loewus, F.A., eds.), pp. 227-257, Wiley-Liss, New York. 27 Sefton, B.M. and Buss, J.E. (1987) J. Cell Biol. 104, 1449-1453. 28 Takeda, A. and Maizel, A.L. (1990) Science 250. 676-679. 29 Burger, M. and Schmidt, M.F.G. (1986) J. Biol. Chem. 261, 14912-14918. 30 Keenan, T.W. and Held, H.W. (1982) Cur. J. Cell Biol. 26, 270-276. 31 Bolanowski, M.A., Caries, B.J. and Lennarz, W.J. (1984) J. Biol. Chem. 259, 49."44-4940. 32 Carpenter, eL., Ducltwortb, B.C., Auger, K.R., Cohen, B., Schaflhausen, B.S. and Cantley, L.C (1990) J. Biol. Chem. 265, 19704-19711. 33 Belunis, CJ., Bae-Lee, M., Kelley, MJ. and Carman, G.M. (1988) J. Biol. Chem. 263,18897-18903. 34 Yamakawa, A. and Takenawa, T. (1988) J. Biol. Chem. 263, 17555-17560. 35 Porter, F.D., Li, Y.S. and Deuel, T.F. (1988) J. Biol. Chem. 263, 8989-8995. 36 Walker, D.H., Doogherty, N. and Pike, LJ. (1988) Biochemistry 27, 6505-6511. 37 Bostwick, J.R, and Eichber8, J. (1981) Neurochem. Res. 6,1053!065. 38 Walker, D.H. and Pike, 1.~. (1990) Biochim. Biophys. Acta 1055, 295-298. 39 Courlneidge, S.A., Kypla, R.M. and UIOg, E.T. (1988) Cold Spring Symp.Ouant. Biol. 53,153-160. 40 Van Doogen, CJ., Zwiers, H., De Graan, P.N.E. and Gispen, W.H. (1985)Biochem. Biophys.Res. Commun. 128,1219-1227. 41 Crouch,M.F. and Lapetioa,E.G. (1988) Biochem.Biophys. Res. Commun. 153,21-30. 42 Leshem, Y.Y. (1987) Physiol• Plantarum 69, 551-559. 43 Scherer, G.F.E. and Andre, B. (1989) Biochcm. Biophys. Res. Ccmmun. t63, I11-117. 44 Citeii, Q., Brglez, 1. and Boss, W.F. (1991) in Molecular Biology of Plant Development (Jenkins, G. and Schuch, W., ads.), pp. 159-175, Company of Biologist, Cambridge. 45 Maroon, A.R., Chen, Q. and Boss, W.F. (1989) Biochem. Biophys. Res. Commun. 162,1295-1301.
73
© 1992ElsevierScience PublishersB.V. All rights reserved0167-4889/92./$05.00
BBAMCR 13107
Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A 2 and activation by a 70 kDa protein W o l f g a n g G r o s s t, W a n n i a n Y a n g a n d W e n d y F. B o s s Botany Department, North Carolina State Unh'ersity, Raleigh, NC (U.S.A.)
(R~eeived 16July 1991) (Revisedmanuscriptreceived 15 October 1991)
Key words: Phosphatidylinositolkinase; PhospholipaseA..; Phosphatidylinositol: Phosphatidylinosito]monophosphate
Plasma membranes were isolated from carrot (Daucus carota L) cells grown in suspension culture and treated with phospholipase A 2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinasc or phosphatidylim~itol monophesphate kinase activity released from the membranes after the phospholipase A 2 treatment. Treating the plasma membranes with phospholipaso C or D did not release PI kinase activity. The phespholipase A -released Pl kinasc was activated over 2-fold by a beat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the V,,~, but does not affect the K , of the phospholipase A2-released Pl kinase.
Introduction Whether phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP 2) are precursors of the second messengers dia~lglycerol and inositol 1,4,5-trisphosphate (IP 3) [1,2], or directly affect membrane enzymes [3,4], regulation of the biosynthesis of these lipids is critical. Although PIP- and PIP2-specifie phospholipase C have been well characterized with regard to the activation by Ca 2+ in higher plants [5-8] and by Ca 2+ and GTP in animals [9,10], very little is known about the regulation of the phosphatidylinositol 4-kinase [11]. Rapid changes in
t Present address: Instimt far Pflanzenphysiolollieund Mikrobiolo8ie. Fmie Universit'ttBedin, 1000Berlin 33, Germany. Abbreviations: lP3, inceitol 1,4,.%.trisplmsphate; LPI, lysophos* phatidylia~itol; PC, phesphalidykholine;Pl, plmsphaticb/linositol; PLA2, phmpbolipase A2; PLC, phospholipase C; PLD, phospholipa~ D; PIP, phosphatidylino~itol 4-monophosphate; PIP2, phmphatidylinesitol4,5.bisplmsphate;SDS, sodium dode~l sulfate; PAGE, polyacrylamide gel electmphoresis. Co~nce: W.F. Boss, Botany Department, North Carolina State University,Ra'eigh, NC 27695,U.S.A.
the activity of P! kinasc and PIP kinase occur in response to external stimuli in higher plants [12,13]. Recently, Lassing and Lindherg [14] reported that in" stimulated platelets, the levels of PIP and PIP2 increased prior to activation of PIP2-phospholipase C. These data suggest an important role for the in~itol phospholipid kinases in signal transduction. Our focus was to characterize the plasma membrane P! kinase from carrot cells grown in suspension culture. It is well accepted that some membra'~e surface glycoproteins are anchored to the membrane with lipid anchors which can he released by phospholipase C (PLC) [15] and D (PLD) [16]. We reasoned that intracellular facing proteins on the plasma membrane also might be released from the membrane by phospholipases. We report here that phospholipase A 2 (PLA 2) released Pl kinase activity from isolated plasma membranes but PLC and PLD did not. The release of PI kinase by P L ~ z appeared not to result from nonspecific loss of the membrane lipids as was reported for the opiate receptor [17]. In addition, we observed that a heat stable, soluble 70 kDa protein activated the PLA2-released P! kinase. The release and activation of the plasma membrane PI kinase are two separate phenomena which may together or separately play a role in regulating the P! kinase activity in vivo.
74 Materials and Methods
Materials Wild carrot (Daucus carota L.) cells were maintained in suspension culture according to Chen and Boss [13]. Ceils were transferred serially every 7 days and Used for experiments at day 4. PLA 2 from Crotalus darissus terrificus venom, PLA 2 from Naja mocambique mocambique, PLA 2 from Apis mellifera venom, PLC from CIostridium perfringens Type XIV, PLD from peanut Type Ill, melittin, trypsin I, trypsin inhibitor type IV-O (chicken egg white), lysophosphatidylcholinc (LPC) and phosphatidylcholine (PC) were purchased from Sigma. [3,-32p]ATP (7000 Ci/mmol) was purchased from ICN and [mC]eboline (58.5 /.tCi/mmol) was purchased from New England Nuclear.
Plasma membrane isolation Plasma membranes were isolated by aqueous twophase partitioning as previously described [18], except that the isolated membranes were washed once with 20 mM Tris-HCI (pH 7.2), 1 mM CaCI 2, 0.01% (v/v) Triton X-100 before they were used for treatments unless indicated otherwise.
PLA z treatment of plasma membranes The isolated plasma membranes were resuspended in buffer A (20 mM Tris-HCI (pH 7.2), 1 mM CaCI 2, 0.01% (v/v)Triton X-100) and phospholipase in buffer A or buffer A alone was added to give a final concentration of 30/~g membrane protein/20 p.I buffer. The samples were incubated for 10 rain at room temperature under constant shaking (200 rpm). At the end of incubation time, EGTA was added to give a final concentration of 1.5 raM. The samples were centrifuged for 30 min at 45000 × g and the supernatant and the membrane pellet were collected. The resuspended pellet and the supernatant were assayed immediately for phosph,~lipid kinase activity.
Enzyme assays PI kinase activity was assayed using the following procedure: 20 p.I enzyme preparation (released from up to 30 p.g of membrane protein) was added to 30 p.I reaction mixture to give a final concentration of 50 mM Tris-HCl (pH 7.0), 20 mM MgCI 2, 0.6 mM PI (prepared as mixed micelles using 3% (v/v) Triton X-100), 0.2 mM sodium molybdate, 0.25% (v/v) Triton X-100 and 0.6 mM AT, ~ containing [3,-32p]ATP (0.1 tzCi/nmol). For studies of the trypsin sensitivity of the 70 kDa activator and of the effect of increasing activator concentration, the pbosphorylation mixture contained 30 mM Tris-HCI (pH 7.2), 7.5 mM MgCI2, 0.6 mM el, 1 mM sodium molybdate, 0.3% (v/v) Triton X-100 and 0.9 mM ATe. To measure PIP and diacyl-
glycerol kinase activity, either PIP or dipalmitoylglycerol was added instead of el. The reaction mixture was incubated for 10 min with constant shaking. The reaction was stopped by the addition of 1.5 ml of ice-cold CHCI3/MeOH (1:2, v/v). HC[ (0.5 ml of 2.4 M) was added and the lipids were extracted and chromatographed on thin-layer plates as previously described [13] except that TLC plates were developed in C H C I 3 / M e O H / 1 5 M N H 4 O H / H 2 0 (86:76:6:18, v/v). Radioactivity was quantified with a Bioscan System 500 Imaging Scanner. Protein was determined using the bicinchoninic acid method as described by Smith et al. [19].
Metabolism of phosphatidylcholine in isolated plasma membranes Cells were labeled with [Inc]choline (1.4 p.Ci/0.3 to 0.4 g fresh wt.) for 45 h and harvested by filtration on Whatman No. 1 filter paper. Plasma membranes were isolated by aqueous two-phase partitioning [18], resuspended in Tris-HCI buffer (pH 7.2) and treated for 10 min with PLA 2, calcium, EGTA and melittin as indicated. After treatment, the reaction was stopped by adding 5 mM EGTA. The membrane lipids were extracted in C H C I 3 / M e O H / 2 . 4 M HCI (1:2:1, v/v) as previously described [13]. The 14C recovered in both the organic and aqueous phases was quantified by scintillation counting in a Beckman LS 7000 scintillation counter using ScintiVerse (Fisher Scientific). LPC and PC in the organic phase were separated by thinlayer chromatography using LK-5 plates (Whatman) and C H C i 3 / M e O H / N H 4 O H (65:25:5, v / v ) as a solvent. [mC]PC and [14C]LPC were identified by comigration with standards and quantified with a Bioscan System 500 Imaging Scanner.
Partial purification of the 70 kDa protein Cells were homogenized as described above and the homogenate was centrifuged for 60 rain at 45 000 × g. The supernatant was collected, boiled for I0 min and filtered (0.2 p.m). The filtered solution was then loaded onto a Q-Sepharose column (1.5 × 20 cm) equilibrated with buffer B (10 mM Tris-HCl (pH 7.5), 2 mM KCI, 0.2 mM EDTA, 0.2 mM MgCl2). The column was washed with 0.25 M KCI in butter I~, |oiJowed by a salt gradient from 0.25 to 0.8 M KCI. Aliquots of the fractions eluted from the column were tested for activation. For this assay, 10 /~l of PLA2-released PI kinase was incubated for 2 min with 2 to I0/zl of the column fractions and EGTA (5 mM final concentration) was added to inhibit phospholipasc A 2 activity. The PLA2-released PI kinase was incubated for 2 min with 2-10 pA of the. c~lumn fraction and the reaction was started by the addition of reaction mixture (see above). Fractions that activated PI kinase were pooled, concentrated using Centricon-30 (Amicon), and loaded
75 o n t o a Sephacryl S-300 c o l u m n (I × 55 cm). Fractions w e r e eluted with b u f f e r B a n d tested for activation. P e a k fractions w e r e p o o l e d a n d used for f u r t h e r studies.
Determination of Mr T h e native M r o f the activator w a s estimated by gel filtration o n Sephacryl S-300 (1 × 80 cm) using the following Me s t a n d a r d s : 7 - g l u t a m y l t r a n s p e p t i d a s e (80000), bovine s e r u m albumin (66000), peroxidase (40000) a n d c h y m o t r y p s i n (25 000).
F o r protein p h o s p h o r y l a t i o n 4 /.LCi of [ y - 3 2 p ] A T P (3.5 / . t C i / n m o l ) was a d d e d to 80 p l m e m b r a n e s in b u f f e r A. A f t e r 10 rain at r o o m t e m p e r a t u r e , the s a m p l e w a s c e n t r i f u g e d 90 min at 4 5 0 0 0 × g a n d 80 #1 o f S D S - P A G E (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) s a m p l e b u f f e r (62.5 m M Tris-HCI ( p H 6.8), 10% glycerol, 2 % SDS, 5 % /3-mercaptoe t h a n o l ) w a s a d d e d to the s u p e r n a t a n t . S D S - P A G E a n d a u t o r a d i o g r a p h y w e r e d o n e as d e s c r i b e d by Blowers et al. [20]. Results
Release of phosphatidylinositol kinase activity by phospholipase A z treatment W o r k i n g with c a r r o t suspension cell cultures, we f o c u s e d o n the c h a r a c t e r i z a t i o n o f a p l a s m a m e m b r a n e - a s s o c i a t e d Pl kinase. W h e n w a s h e d p l a s m a m e m b r a n e s f r o m c a r r o t cells w e r e i n c u b a t e d in 0.01% T r i t o n X-100 a n d 1 m M CaCI 2 f o r 10 m i n at r o o m t e m p e r a t u r e , a b o u t 1% o f the total P l ~ n a s e activity w a s r e c o v e r e d in the soluble fraction. A d d i t i o n o f P L A 2 to t h e i n c u b a t i o n m e d i u m g a v e a 5- t o 1 0 - f o l d i n c r e a s e o f Pl kinase activity in t h e soluble fraction ( P L A - f r a c t i o n ) (Table I), w h i c h w a s a c c o m p a n i e d by a loss o f activity in t h e t r e a t e d p l a s m a m e m b r a n e s . T h e P L A 2 itself c o n t a i n e d n o kinase activity a n d only back-
All t~ealmcnt~",~'.src~,nth~ prese~e of ! mM CaCI: and the calcium was removed by adding EGTA prior to phospho~lation as described in Materials and Methods. Data are the meaas of four values from two experiments. The [32p]PlP recovered in the control was 3150 dpm. The enzyme activity is e:~prasscdper ml PLA-fraction and not per mg protein, because the addition of the PLA 2 preparation made it difficult to extrapolate the amount of released iffotein in the fraction. However. for each treatment the same amount of starting material (plasma membrane protein)was used. PLA2-released PI kinase activity [32P]Plp
none PLC 20 U/ml PLD 20 U/ml Pt.A 2 a 20 U / m l PLA2" (0~ C) PI-A2~* + 5 mg BSA/ml PLA2 h PLA2b +0.2 mg melittin/ml Melittin 0.2 m~/ml Melittin 0.(Mmg/ml Proteinase inhibitors ~ PLA2 a + proteina~ inhibitors c PLA2;' +20 ttM ATP PLA2 ~ +alkaline p;,osphatase d PLA2 a + 0.2 mM Na2Mo 4
~'u ~
'
.,'~
' ,,'o ' A~(,mi~=.~)
pmol/min /ml
% of control
21.7 14.5 28.6 270.2 Q.8 171.9 127.8 69.2 6.2 48.4 7.4 212.7 256.1 261.5 284.3
100:[: 2 67+ 14 132+ 8 1245 -+I I 45± 7 792=1:12 589+ 14 319± 9 27-1:6 223 d:22 34:t: I I 980_+13 I 180+ 12 1205+ 15 1310± 4
a !,L~ 2 was from Crotalus dun~sus teniflcus. PLA 2 was used at 20 U/ml in all instances. b PLA2 was from Apis mellifera venom. I mM phenylmethylsolfowJl fluoride, 1O0 Fg pepstatin A, 2 /~g leupeptin, 20 p.g trypsin inhibiter from chicken egg white. d Alkaline phosphatase attached to agarose beads was used and removed by centrifugation after incubation.
g r o u n d levels o f PI kinase activity w e r e d e t e c t e d in the P L A - f r a c t i o n w h e n t h e P L A 2 t r e a t m e n t was c a r r i e d o u t o n ice (Table !). T r e a t m e n t o f m e m b r a n e s with
_*"I B < ,i
ii:i[ ,
Effect of furious phospholipa~s and compounds on the release of P! kinase from washedplasma membranes
Additions
Protein phosp.:~orytation
o;
TABLE I
---.........
go
3o "lime (mla)
Fig. I. Concentration-dependent release of PI kinase activity by PLA 2. (A) Isolated plasma membranes were incubated with increasing concentrations of PLA 2 fm 1Orain at rc~m t¢inl~aZurc. Th~ o.mpl~ wcrc centrifu~:cd and the ngmbrane-free supernatant was assayed for Pl kinase activity. (13)Isolated plasma ,membranes were treated with 20 U/ml PLA2 and the released P! kinase activity was measured over time. Values are the average of two numhets from one experiment. The effor is indicated when it is larger than the symbols. The experiment has been repeated two times and the trends were similar.
76
PLA 2 from either Apis mellifera, Naja mocambique mocambique (data not shown) or Crotalus durissus terrificus resulted in release of PI kinase activity. O f the lipid kinases tested, only PI kinase was released from the plasma membrane vesicles. No PIP kinase or diacylglycerol kinase activity was detected (data not shown). Phospholipase C or phospholipase D did not release PI kinase activity from plasma m e m b r a n e s (Table I). In fact, PLC treatment resulted in a decrease in the recovered PI kinase activity compared to the control value. As shown in Fig. IA, PI kinase activity was released from the membrane by PLA 2 in a concentration-dependent manner. Incubation with about 20 U PLA 2 per ml for 10 rain at R T gave maximum recovery of PI kinase activity in the 45 000 × g supernatant. A t higher PLA 2 concentrations the kinase activity decreased slightly. The release of PI kinase activity in the presence of 20 U P L A 2 / m l increased gradually over the 10 rain incubation time (Fig. 1B). Longer incubation periods, however, did not result in an increase in the PI kinase activity released. The decrease in PI kinase activity observed with higher P L A 2 concentrations or with increasing treatment times may have been caused by an increase in free fatty acids or lysolipids which have been shown to inhibit PI kinase [21] or by some other factor released during the treatment. In any event, these data indicate that continued treatment with PLA 2 did not facilitate the release of active PI kinase. Therefore, the routine t r e a t m e n t of plasma ;;.~tni~.,iles was with 20 U / m l P L A 2 for 10 rain. U n d e r these conditions 10 to 20% of the total PI kinase was released from the plasma membranes. It should be noted that u n d e r the assay conditions used only P1-4 kinase activity was measured. H P L C analysis of the deacylated reaction products verified that more than 99% PI-4-P and less than 1% PI-3-P was formed
plus Phosphollpese A=
~|,on
mM Calcium chloride Fig. 2. Effect of calcium on the release of PI kinase by PLA 2. Plasma membranes were treated as described, except that increasing concentrations of CaCI2 were used. Following the incubation. EGTA was added to give an excess of 0.5 mM relative to the Ca2+ concentration. The samples were centrifuged and the membrane-free supernatant was assayed for PI kinase activity. Values are the average of two numbers from one experiment. The error is indicated when it is larger than the symbols. The experiment has been repeated two times and the trends were similar.
by the PI kinase (A. Graziani and L.C. Cantley, unpublished data). Calcium was required for the PLA2-induced release of PI kinase activity (Fig. 2). The optimum calcium concentration for PLA2-induced release was between 0.5 to 1 m M CaCI 2. Increasing the calcium concentration to 2 mM inhibited the release of the active kinase in the presence of P L A 2 while calcium alone decreased the recovery of soluble PI kinase activity at all concentrations tested (Fig. 2). Note that E G T A was added at the end of the 10 rain incubation period to chelate the free calcium; therefore, the observed decrease in activity was not due to direct interference by calcium during the assay. Analysis of phosphatidylcholine metabolism of m e m b r a n e s isolated from cells grown in [14C]choline substantiated a role for calcium in activating P L A t .
TABLE I!
Metabolismof plasma membranephosphatidylcholine Cells were labeled with lJ4C]choline(1.4 p.Ci, 58.5 p.Ci/mmol) for 45 h, harvested and the plasma membranes isolated by aqueous two-phase partitioning. The isolated membranes (112 p.g protein) were treated for 10 rain at RT as described. The final concentrations of the reagents were: I mM CaCI2, 20 U/ml PLA 2, 5 mM EGTA, 0.2 mg/ml melinin ia a 120 p.I reaction volume. Lipids were extracted and aliquots of the organic and aqueous phase were ouantitated by scintillation counting. The remaining organic phase was analyzed by thin-layer chromntograpby and the [14CJPCand [14C]LPCquantified with a Bioscan 500 Imaging Scenner. Treatment
Ca2+ PLA2 + EGTA PLA 2 +Ca 2+ PLA2+Ca2++meliuin Melinin+Ca2+
LPC
dpm 357+ 23 1221 4-164 28214-107 25284- 14 13644- 21
PC
dpm 5250+336 4543 4- 86 14504- 21 244- 6 393+ 7
LPC/PC
0.06+ 0 . 0 1 0.194- 0.09 2.04- 0.l 90 4-36 12.84- 9.9
Organic
Aqueous
Organic
phase
phase
/aqeous" phase
dpm 6650+1300 8075 + 1700 59254-1050 39754- 650 30504- 513
dpm 47884-200 3700 4-169 53504- 15 57134-150 84254-100
1.64-0.2 2A + 0.5 IA4-0.4 0.84-0.3 0.54-0.2
a The ratios are the means of 4 to 8 values from two experiments. The dpm values are the average of two values from one representative experiment.
77 Adding PLA 2 in the presence of 1 mM CaCI 2 to the membranes resul(ed in a 2-fold increase in the amount of [14C]lysophosphatidylcholine (LPC) recovered compared to that recovered with PLA 2 in the absence of free calcium and an 8-fold increase compared to calcium alone (Table II). Treatment with calcium alone (1 mM or 4 mM for 10 rain) did not significantly increase in [14C]LPC but did result in a 15% and 50% loss, respectively, in [n4C]phosphatidylcholine (PC) compared to treatment with EGTA (data not shown). These data suggest that a calcium activated PLC or PLD was present, and they are consistent with the fact that exogenously added PLC, like calcium alone, decreased the release of PI kin~se activity. Melittin, a peptide present in bee venom, has been shown to activate endogenous phospholipases [22,23] and was used to increase the release of the opiate receptor [17] by enhancing membrane lipid hydrolysis. To determine whether further degradation of membrane lipids would enhance the release of the plasma membrane Pl kinase activity melittin was added in the presence and absence of PEA 2. Adding melittin (0.2 mg/ml) resulted in an almost total loss of [I4C]Fr~.~and a concomitant increase in water soluble products (Table II) suggesting the activation of PLC or PLD. However, at this concentration melittin decreased the release of the Pl kinase activity (Table 1) whether it was added in the presence or absence of PLA 2. The decrease in release of the Pl kinase activity when excess melittin was added may have been caused by the release of inhibitors as a result of increased membrane degradation. If less melittin (0.04 m g / m l ) was added there was an increase in the release of PI kinase activity (Table I). This was probably caused by the PLA 2 present in the melittin [23]. While the mechanism involved in the PLA2-induced release of Pl kinase activity is not known, one possibility was that calcium-dependent proteinases might be involved. However, there was no detectable proteinase activity in the PLA 2 preparations under these conditions (data not shown) and calcium alone resulted in a decrease in the recovery of soluble Pl kinase (Fig. 2). When BSA or several proteinase inhibitors were included in the PLA 2 treatment, there was less PLA2-released Pl kinase activity (Table 1). However, proteinase inhibitors have been shown to have an effect on PLAz activity [24]. Furthermore, when proteinase inhibitors were added to the membranes without PLA 2 there was less soluble Pl kinase recovered suggesting that the proteinase inhibitors may have a direct effect on the Pl kinase activity (Table !). Taken in toto, these data suggest that calcium-activated proteinases were not involved in the release of Pl kinase. in addition, we tested to determine whether or not phosphatases were involved in the PLA2-release of Pi kinase activity. The addition of alkaline phosphatase or
of 0.2 mM sodium molybdate (a phosphatase inhibitor) had little effect on the recovery of P! kinase activity in the PLA-fraction (Table I). To test for the involvemetlt of protein phosphorylation, we added [7-32p]ATP during the PLA 2 treatment. Although, the PLA-fraction exhibited three 32P-labeled bands at 34, 50 and 66 kDa on SDS gels, there was no difference in the PLA 2treated or control sample. Furthermore, addition of ATP during the PLA 2 treatment did not increase the activity in the PLA-fraction (Table !). Therefore, we have no evidence to suggest the release of Pl kin'ase from carrot cell plasma membranes involves the phosphorylation or dephe~phorylation of proteins. If the release of the Pl kinase by PLA 2 is specific, then one possible mechanism for release is via the cleavage of a fatty acid anchor from the protein. We labeled cells in vivo with [14C]myristate, [t4C]palmitate or [14C]stearate. While multiple bands were visible after SDS-PAGE and autoradiography of intracellular membranes, the incorporation of radiolabeled fatty acids into plasma membrane proteins was too low to be able to detect differences in the labelling pattern from PLA 2 treated and non-treated plasma membranes (data not shown). Activation o f released phosphatidylinositol kinase by a 70 kDa protein When plasma membranes were washed with 0.01% (v/v) Triton X-100 prior to the PLA 2 treatment, the recovery of P! kinase activity in the PLA-fraction was accompanied by a loss of activity in the plasma membranes with no significant change in total activity (106
TABLE ill The acti~,ator is trypsinsensitive The partially parified activator(0.78 mg wotein/ml) was incubated at RT in the presence and absence of 0.2 mg/ml trypsin(10000 units/mr) in 30 mM Tris-HCi buffer (pH 7.2) for 3 I1. Tr~in inhibitor (0.3 mg/ml) was added after 3 h or just ~ to the addition of tw0sin,and the activator(15.5/~g protein)was added to the PLA2-rel(.ased P! kinase and PI kinase activity was assayed as described in blaterials and Methods. PLA2-releused Pl kinase activi~ [32p]PIP Trypsininhibitorplus WJpsin Activator Activatorplus tvjpsin3 h; followedby trypsininhibitor Activator plustrypsininhibitor and tvjpsinat the sametime
pmol/min 40.8+5.1 84.05:6.8
activation"
2.2+0.2
36.0+4.4
none
63.6+i.8
2.0±0.5
a The pmol/minare averajevaluesfromone experimenLActivation was calculated re'l~ti~ to the control which was trypsin inhibitor plus trypsin added at the time of pimspho~atk)n. The values a r e the mean± S.D. of fourvaluesfromtwo experiments.
i~'°°°t
|,
"°°1/ I:'7
.
,o
.o
..o
l/PbesphnUdylinositol(mM)
AclivalorFrael|on(g¢ Protein) Fig. 3. Activation increases with increasing concentration of the activator fraction. The heat-treated, soluble fraction was added in increasing amountsto the PLA2-roleased Pl kinase (released from 27 p.g membraneprotein) and Pl kinase activity was assayed in a final reaction volume of 50 ,¢1. Values are the average of two numbers from one experiment. The error is indicated when it is larger than the symbols.The experiment has been repeated two timesand the trend~were similar. + 5%, mean + S.D. of four values). However, when the plasma membranes were not prewashed with 0.01% Triton X-100 prior to the PLA 2 treatment the total Pl kinase activity increased about 30% over the control (128 + 4%, mean + S.D. of four values). This suggested that a soluble factor entrapped in or loosely associated with the membrane vesicles could increase Pl kinase activity. Therefore, we collected the soluble fraction (e.g., the 45000 × g supernatant of a crude bomogenate), boiled it for 10 rain to inactivate the soluble Pl kinase present in this extract and added aliquots to isolated plasma membranes after PLA 2 treatment and to the PLA-fraction. The addition of the heattreated, soluble fraction to untreated plasma membranes or PLA2-treated plasma membranes increased the PI kinase activity only about 10% over the control (data not shown). However, the PLA 2-released Pl kinase activity increased from 2- to 8-fold after addition of the soluble fraction (Table III and Figs. 3 and 4).
tt
t
~
5~
i
J
....
o
" ;
" ,'o ,'~,'o2; FrscUon Number
~o°
Fig. 4. Gel filtration of crude activator protein on Sephaeryl-300. Peak fractions from O-Sepharose were pooled, concentrated and loaded onto a SephacrylS-300 column.The eluent was fractionated and an aliquot of each fraction tested for its ability to the activate PLA2-released PI kinase.
Fig. 5. Change of ["maxof solubilized Pl kinasc by the 70 kDa t'~rutein. PLA2-solubilized PI kinasewas assayedin the presenceand absence of partially purified 70 kD~.protein with increasingconcentration of Pl. The activation was eliminated by treating the heattreated, soluble fraction with trypsin (Table lll) indicating that the activator is a protein. Activation of the Pl kinase was concentration dependent. Using the heat-treated, soluble fraction as the source of activator, we found that adding greater than 1 # g of p r o t e i n / 5 0 ~.1 of reaction mixture was necessary for activation (Fig. 3) and 10 # g approached saturation. The activator preparation varied from day to day as indicated by the activation of the PLA2-released P1 kinase activity (compare Table III and Figs. 3 and 4). This may be be¢~.use of differences Jn the amount of activator or potential inhibitors present in the individual preparations. The activator was partially purified using the heattreated soluble fraction. The protein was filtered through a 0.2 /~m filter, chromatographed on a QSepharose column, concentrated by filtration (Centricon-30 filter) and further purified by Sephacryl S-300 column. The profile of activation versus absorbance at 280 nm is given in (Fig. 4). In the presence of this activator protein, the Pl kinase exhibits an increase in Vmx with no significant change in K m under the assay conditions used (Fig. 5). The estimated K m for Pl was approx. 1 mM. Discussion We have shown that Pl kinase activity is released from the plasma membranes of carrot cells by treating the iaembranes with PLA 2 in the presence of calcium. Rfiegg et al. [17] used PLA 2 to release the opiate receptor from rat brain membranes. With the opiate receptor, however, increased degradation of the membrane lipids by adding melittin enhanced the release suggesting that the release was the result of the loss of membrane integrity. This was not true for the release of carrot plasma membrane Pl kinase activity. The optimum release of the active enzyme occurred with a short term treatment with PLA 2. Treatment with PLD
did not result in the release of PI kinase activity and treatment with PLC inhibited the release of the active enzyme. Release of P! kinase activity by PLA_, required calcium; however, calcium had two effects on the release. Calcium (1 mM) enhanced the release of the active Pl kinase and enhanced PLA2 activity as evidence by the increase in LPC production. On the other hand, calcium alone or calcium greater than 1 mM in the presence of PLA z decreased the recovery of Pl kinase activity in the PLA-fraction. Since free calcium was removed by adding EGTA to the reaction mixture prior to assaying for Pl kinase activity, the decrease of PI kinase activity did not result from a direct effect of calcium on the kinase [25]. A calcium-dependent PLD has been reported to be associated with plant membranes [26]. Activation of endogenous PLD or PLC by calcium may explain why calcium alone or calcium at concentrations greater than ! m M in the presence of PLA 2 inhibited the release of P! kinase activity. Calcium-dependent proteinases appeared not to be involved in the PLA~:induced release of Pl kinase activity because there was no detectable proteinase activity in the PLA 2 preparations and because proteinase inhibitors had a greater effect on the control than the PLA2-released PI kinase activity. Phenylmethylsulfonyl fluoride has been shown to decrease PLA 2 activity [24], and this may have caused the decrease in the PLA2-induced release when proteinase inhibitors were added. Low [15] and Scailon et all [16] have shown that PLC and PLD, respectively, can release lipid anchored surface glycoproteins from the plasma membrane. Proteins also can be anchored by attachment to a single fatty acid which cannot be cleaved by PLC [27,28]. Although the treatment of various isolated acylated proteins with PLA 2 resulted in no release [29] or only limited release of fatty acids (4-5%) [30,31], a protein fatty acylesterase has been demonstrated in microsoreal membranes from mammalian cells [29]. It may he that PLA 2 plays a role in releasing fatty acid anchored proteins from the intracellular surface of the plasma membrane. We have not been able to recover enough fatty acid-labeled protein before or after PLA 2 treatment to determine whether or not this is true for the carrot plasma membranes. If only a small percentage of the fatty acylated proteins are affected, however, the differences may not be measurable ~ithout the specific antibodies to concentrate the proteins of interest. As a result of the PLA 2 treatment, only about 10% of the total plasma membrane Pl kinase activity was recovered in the PLA-fraction. The limited release of Pl kinase may be the result of the limited time of treatment or it may be that two populations of PI kinase are present in the plasma membranes and only one population can be released by PLA 2. Several dis-
tinct PI kinases have been isolated from mammals [32] and from yeast [33]. The estimated K m for PI was approx. I mM which is considerably higher than reported for most purified PI kinases [33-36] but is very similar to the partially purified enzyme from rat brain [371. The release and activation of the PI kinase were two different processes. While the release of P! kinasc activity was calcium dependent, activation was not. Activation was not caused by a factor in the P.LA 2 because, prewashing the membranes with Triton X-100 eliminated the activation even though the PI kinasc was released and PLA 2 was still present in the reaction mixture. Activation was recovered by adding back the Triton X- 100 wash. Cardiotoxin has been shown to activate PI kinasc [38]. Cardiotoxin, however, decreased the K m of the enzyme for P! and did not affect the P'm~. The opposite is true for the soluble protein activator of the PLA2-released PI kinasc, in addition, if cardiotoxin were presem in the PLA z preparations one would have expected to see activation using Triton-washed membranes and this was not found. Also, PLA 2 was used from several sources and all were effective in
releasing the PI kinase. Activatio~ was ~aused by a heat-stable soluble component. The activator was trypsin sensitive indicating that it was a protein. Activation increased with increasing protein concentration. The relative molecular mass estimated by Sephacryl S-300 chromatography was 70 kDa. If one calculates the increase in P! kinase activity based on the total activity in the membrane and PLAfraction, the total increase was only about 30% when the activator was added. If however, one considers the amount of activation of only the solubilized enzyme in the PLA-fraction, adding activator increased the Pl kinase activity 2- to 8-fold. Regulation of Pi-4 kinase activity in animal cells was reviewed recently by Carpenter and Cantley [!1]. Phosphoproteins have been reported to regulate the Pl kinase in transformed fibroblast cultures [39] and the PiP kinase in rat brain [40]. However, there is no in vitro evidence that the Pl 4-kinase is phosphorylated or that purified Pl 4-kinase is regulated by a protein kinase [ I I]. Our data suggest that protein phosphorylation does not play a role in releasing the carrot plasma membrane Pl kinase. However, until we have'isolated the released PI kinase and the activator and determined whether or not they are phosphorylated, we cannot rule out a regulatory role for protein phosphorylation/dephospborylation in this system. The in vitro results presented in this paper lead one to question whether the release and activation of Pl kinase is physiologically relevant? in animals, as well as in plant cells, it is known that PLA 2 activation is part of signal transduction. Crouch and Lapetina [41] found
8O in platelets t h a t P L A 2 is p a r t o f a signal t r a n s d u c t i o n p a t h w a y t h a t is not directly c o r r e l a t e d with the phos* pholipase C / I P 3 pathway. Potential roles o f lysolipids a n d P L A 2 in h i g h e r p l a n t signal t r a n s d u c t i o n have b e e n reviewed [21,26,42]. In soybean, the levels o f lysolipids increase u p o n stimulation with auxin [43] indicating a n increase in p h o s p h o l i p a s e A activity. Previously, we have shown t h a t w h e n the specific activity o f the p l a s m a m e m b r a n e PI ~.nd P I P kina~;e increased, the specific activity o f the p l a s m a m e m b r a n e P L A 2 also incrcased [44]. Based o n these d a t a a n d the d a t a p r e s e n t e d in this p a p e r , we p r o p o s e as a w o r k i n g hypothesis the following signal t r a n s d u c t i o n c a s c a d e for the c a r r o t m e m b r a n e s . A stimulus activates the P L A 2 in the p l a s m a m e m b r a n e . T h e p l a s m a m e m b r a n e P L A 2 releases PI kinase which is activated by a 70 k D a protein. T h e activator-Pl kinase complex reassociates with the m e m b r a n e , ph0~phorylates PI a n d increases the level o f PIP in the m e m b r a n e . T h e inc r e a ~ in P I P stimulates the m e m b r a n e vanadate-sensitive A T P a s e [45], t h e r e b y causing a c h a n g e in cell physiology. Acknowledgements W e gratefully a c k n o w l e d g e the technical assistance o f I r e n a Brglez. This r e s e a r c h w a s s u p p o r t e d by g r a n t n u m b e r D C B - 8 8 1 2 5 8 0 - 0 3 f r o m the N a t i o n a l Science F o u n d a t i o n a n d in p a r t by the N o r t h C a r o l i n a Agricult u r a l R e s e a r c h Service. W e t h a n k A. G r a z i a n i a n d L.C. Cantley (Tufts University School of Medicine, Boston, M A ) for doing the headgroup analysis of PIP. References I Majerus, P.W., Connolly, T.M.. Deckmyn, H., Ross, T.S., Bross, T.E., Ishii, H., Bansal, V.S. and Wil~n, D.B. (1986) Science 234, 1519-1526. 2 Berridge, MJ. and Irvine, R.F. (1989) Nature 341,197-205. 3 Sch§fer, M., Behle, G., Varsanyi, M. and Heilmeyer, L.M.G. (1987) Biochem. J. 247. 579-587. 4 Chauhan, A., Chauhan, V.P.S., Deshmukh, D.S. and Brockerhoff, H. (1989) Biochemistry 28, 4952-4956. 5 Melin, P.M., Sommarih, M., Sandelius, A.S. and Jergil, B. (1987) FEBS Len. 223, 87-91. 6 Pfaffmann, H., Hartmann, E., Brishtman, A.O. and MorrO, DJ. (1987) Plant Physiol. 85,1151-1155. 7 McMurry, W.C and Iwine, R.F. (1988) Biochem. J. 249, 877-881. 8 Tare, B.F., Schaller, G.E., Sussm~n, M.R. and Crain, R.C. (1989) Plant Physiol. 91,1275-1279. 9 Merrin, J.E., Taylor, C.W., Rubin, R.P. and Putney, Jr., J.W. (1986) Biochem. J. 236, 337-343. 10 Fain, J.N. (1990) Biochem. Biophys. Acta 1053, 81-88. 11 Carpenter, C.L. and Cantley, L.C. (1990) Biochemistry 29, 11147-11156. 12 Memon, A.R. and Boss, W.F. (1990) J. Biol. Chem. 265, 1481714821.
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