Different isozymes of protein kinase C mediate feedback inhibition of ...

Communication

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 4, Issue of February 5, pp. 2280-2283,1993 Printed in U.S.A.

Different Isozymes of Protein Kinase C Mediate Feedback Inhibition of PhospholipaseC and Stimulatory Signals for Exocytosis in Rat RBL-2H3 Cells* (Received for publication, August 27, 1992)

Koichiro OzawaS, Koji YamadaSS, Marcelo G. Kazanietzq, Peter M. Blumbergq, and Michael A. BeavenS(1 From the $Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, and the 7Laboratot-y of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutesof Health, Bethesda, Maryland 20892

Previous studies indicated that rat basophilic RBL2H3 cells contained the Ca2+-dependenta and ,tl and the Ca2+-independent6, t, and 5 isoforms of protein kinase C (PKC); of these, PKC@ and6 were the most potent transducers of signals for exocytosis in antigenstimulated permeabilized cells. Exocytosis, nevertheless, wasstill dependent onan elevatedfree Ca2+. (Ozawa, K., Szallasi, Z., Kazanietz, M. G., Blumberg, P. M., Mischak, H., Mushinski, J. F., and Beaven, M. A. (1993) J. Biol. Chem. 268, 1749-1756). We now demonstrate that PKCa and t, exclusively, inhibit antigen-induced hydrolysis of inositol phospholipids in the same permeabilized RBL-2H3 cells. Unlike secretion, the inhibitory actions occurred at a basal concentration (0.1 PM) of free Ca2+.The inhibitory actions of the two isozymes were potentiated by 20 nM phorbol 12-myristate 13-acetate.As indicated by the effects of the phorbol ester, the probable mechanismwas reduced tyrosine phosphorylation of phospholipase C y l . The negative regulation of phospholipase C was apparent in intact cells, because the PKC inhibitor Ro31-7549 or down-regulation of PKC with phorbol esterenhanced antigen-induced hydrolysis of inositol phospholipids. The concentrations of the various isozymes of PKC in RBL-2H3 cells, as estimated by immunoblotting studies, were sufficient for promotion ofexocytosis (i.e. /3 and 6) and inhibition of phospholipid hydrolysis (i.e. a and 6 ) .

Although cells generally contain more than one isozyme of protein kinase C (PKC),’ little is known about the distinct * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. 5 Present address: Dept. of Biochemistry, Tsukuba Research Laboratories, Eisai Co., Ltd., 1-3Tokodai 5-chome, Tsukuba-shi, Ibaraki 300-26, Japan. I( To whom all correspondence should be addressed Rm. 8N114, Bldg. 10, National Institutes of Health, Bethesda, MD 20892. ‘The abbreviations used are:PKC,proteinkinase C; [CaZ+li, concentration of free cytosolic calcium;DNP-BSA, antigen consisting of 24 molecules of dinitrophenol conjugated with 1 molecule of bovine serumalbumin;DNP,0-dinitrophenol; IgE, immunoglobulin E; PIPES, 1,4-piperazinediethanesulfonicacid.

roles of the individual isozymes (1)except that they collectively transduce stimulatory signalsfor cell activation and, in many types of cells, inhibitory signalsfor negative regulation of effector systems (2). The isozymes of PKC are activated by binding of diacylglycerol to putative binding sites on the isozymes. PKCa, @,and y have, in addition, a Ca2+-binding domain that allows Ca2+ anddiacylglycerol to act synergistically on these isozymes (3). Themore recently described 6, c, 5; and 7 (3-7) isozymes of PKC lack this Ca2+-binding site, and, in addition, PKCr lacks one of two cysteine-rich Zn2+binding domains, which together are the probable binding sites for diglyceride and exogenous activators suchas phorbol 12,13-dibutyrate (8,9). When cells are activatedby physiological stimulants or by phorbol ester that actvia PKC, thea , p, y (3), and the6 (10-12), t (10-13), and possibly { (Ref. 13 but see Ref. 14) isozymes of PKC become tightly associated with the membrane fraction. Prolonged exposure to phorbol ester results in degradation of at least someof the isozymes of PKC in many typesof cells (1, 15, 16), a process otherwise referred to as down-regulation (2). We have used the permeabilized rat RBL-2H3 cell (17) to study the role of the individual isozymes as transducers of signalsforantigen-induced exocytosis (11). The necessary stimulatory events in antigen-stimulated RBL-2H3 cells include an increase in [Ca2+Ii and the activation of PKC (11, 18)preceded by the tyrosine phosphorylation of various proteins (19)including phospholipase C y l (20). Thelatter phosphorylation is thought to be essential for the activation of phospholipase C (20, 21). All of the isozymes of PKC that are normally present in these cells ( a , p, 6, t, and {) can be eliminated by washing permeabilized cells, and in so doing the secretoryresponse to antigen islost. The response can be restored by addition of nanomolar amounts of PKCp and 6, but only partially restored by addition of >lo0 nM amounts of PKC a and t. Additional studies in which PKCa and p could be selectively down-regulated in intactcells by phorbol ester indicate a probable role for both PKCp and6 in exocytosis (11). Here we have identified,using permeabilized cells, the individual isozymes of PKC that mediatefeedback inhibition of phospholipase C in antigen-stimulated cells. Although activators of PKC, such as phorbol12-myristate 13-acetate and oleoyl acetyl glycerol, markedly suppressedantigen-stimulated production of inositol phosphates and mobilization of Ca2+, the extent to which these responses were negatively modulated in antigen-stimulatedcells was uncertain (22, 23). An additional objective was to assess the physiological relevance of our observations by comparing the stimulatory and inhibitory potencies of the isozymes of PKC with the actual concentrations of the individual isozymes of PKC in RBL2H3 cells as determinedby immunoblotting techniques. EXPERIMENTALPROCEDURES

The reagents for the immunoblotting experiments with phospholipase C y l were obtained from previous sources (21). Isozymes of PKC were prepared using the baculovirus expression system from Invitrogen (SanDiego, CA) and purified as previously described (11). Monoclonal antibodies against PKCa and (3 were from Upstate Biotechnology, Inc., Lake Placid,NY; polyclonal antibodies against other isozymes of PKC were from GIBCO/BRL. The antigen, DNP-BSA, and DNP-specificmonoclonal IgE were kindly supplied by Dr. Henry Metzger (NIAMSD, National Institutes of Health). Other reagents

2280

Inhibitory Protein KinaseC Isozymes i n RBL-2H3 Cells RBL-2H3 PURIFIED EXTRACT

RECOMBINANT ISOZYME

-

"

10

5

" - 0

TABLE I Isozymes of PKC i n RRL-2H3 cells: estimated concentrations and activities PKC y and 7 were not detectable. PKC {was not studied. Intracellular concentration"

50 75 100 1 -/j

--a

20 10

uu 10 5

50 75 loo

30 20

f

?.&.d

50 75 100

fmol protein FIG. 1. Quantitation of the isozymes of PKC in RBL-2H3 cells. The indicated amounts of soluble extracts of RBL-2H3 cells ,ug

(pg of protein) and of purified recombinant isozymes were subjected t o electrophoresis for immunoblotting with isozyme-specific antibodies. The densitiesof the bandswere determined with a scanning laser densitometer; standard curves (density uersus amount) were prepared for each isozyme. The concentration of each isozyme was based on a cell volume of 1.1p1/10' cells as determined in a Coulter counter. The data are summarized in Table I.

Particulate

ECwb

-PMA

+PMA

~~

d

- : ,v

ICW' Total

z

Activity

Isozyme

50 75 100

L -

2281

nM

Stimulatory

P 8 Inhibitory

35 60

-10 -35

5 45

>200 >200

-75 -200

3 -20 175 0.75 85 3 -7.5 >250 1.5 12.5 Estimated concentration in whole cells extracts and particulate fraction 1 min after stimulation with 10 ng/ml DNP-BSA. bConcentration of isozyme required to support 50% of maximal secretory response to antigen (data from Ref. 11). 'Concentration of isozyme required to attain 50% of maximal inhibitory response to antigen in the absence (Fig. 2 A ) and presence (Fig. 2B) of phorbol ester. (Y

c

tions of PKCP andc were sufficient for physiological activity. Although permeabilization and washing of RBL-2H3 reand materials were from the sources listed previously (11) sulted in virtually complete loss of all isozymes of PKC and RBL-2H3 cultures were incubated overnight withDNP-specific IgE and, as required, ['H]inositol in complete growth medium in 96- the secretory response to antigen, stimulated hydrolysis of well plates (4X lo4cells/80 pl of medium/well), or for immunoblotting inositol phospholipids was enhanced about 2-fold when comprocedures in 150-cm2 tissue culture dishes (30 X 10' cells/20 ml of pared to theresponse in intact cells (11).The possibility that medium/dish) (11).Experiments were performed with the following loss of PKC eliminated feedback inhibition of phospholipase buffers:aglucose-saline, PIPES-buffered medium that contained either 1 mM Ca2' (Ca2+-containing medium)or 0.1 mM EGTA instead C by certain isozymes of PKC was tested by addition of PKC of Ca2+ (Ca2+-free medium)for experiments with intact cells, or for isozymes individually to washed, permeabilized cells 15 min 100ng/ml);only studies with permeabilized cells a potassium glutamate, PIPES-buff- before addition of antigen(DNP-BSA, ered medium thatcontained 5 mM ATP, 1 mM EGTA,andthe PKCa and t caused partial (about 40%) suppression of anticalculated concentration of Caz+to give the desired concentration of gen-inducedhydrolysis of inositolphospholipids (Fig. 2 A ) , free Ca2' (11).Cells were permeabilized with streptolysin 0 (24, 25), and this suppressionwas markedly enhanced in the presence but as noted previously (ll), the critical maneuver was that the phorbol 12-myristate 13-acetate (Fig. 2B): Addipermeabilized cells were washed twice (each wash was left for 60 s) of20nM tional experiments revealed (data not shown) that the extent before addition of the same buffer. Release of hexosaminidase (11) of inhibition was exactly the same whether[Ca2++Ii was buffand ['Hlinositol phosphates (26) were measured as described previously. ered a t 0.1 p M (as in Fig. 2B) or 1 PM (as in Fig. 2 A ) . This Immunoblotting of the isozymes of PKC was performedwith minor was in contrast to exocytosis which required the presence of modifications of previously described techniques (11).Specifically, PKC and elevated [Ca2+Ii(11).The data, nevertheless, were cells (30 X lO'/dish) were homogenized with a Dounce homogenizer of PKCa and (100 strokes) in 200 pl of an extraction buffer (50 mM Tris-CI, pH consistent with the fact that redistribution 7.4; 2 mM EGTA; 2mM dithiothreitol; 0.5 mM phenylmethanesulfonyl PKCP from cytosolic to membrane fractionsoccurred a t low a t 0.1 pM [Ca2']i, fluoride; 0.5 mM leupeptin; 1% TritonX-100)andcentrifuged a t [Ca2+Ii, withsignificantredistribution 105,000 X g for 15 min to remove insoluble material. Samples of this whereas exocytosis occurred only when [Ca2+Ii was between extract (5-30 pg of protein) and known amounts (50-100 fmol) of 0.16 and 0.4 P M . ~Of note, the antigen-inducedresponse was purified recombinant isozymes of PKC were separated by electropho- suppressed to the level of response that was observed in intact resis on 8% sodium dodecyl sulfate-polyacrylamide gels. Detection cells, either without (Fig. 2.4, solid triangle) or withprior and quantitationof immunoreactive isozymes were performed exactly as described (11)as was the immunoblotting of tyrosine phosphoryl- exposure to phorbol ester(Fig. 2B, solid triangle). PKCP and 6 possessed slight inhibitory activity a t high concentrations ated phospholipase C y l (21). (30-200 nM) in the presence of the phorbol ester (Fig. 2B) but had no inhibitory activity when cells were stimulated with RESULTS AND DISCUSSION antigen alone (Fig. 2 A ) . Treatment with the phorbol ester As in previous studies (11), immunoblotting revealed the caused much more extensive translocationof the isozymes of presence of the a,p, 6, t, and { isozymes of PKC in extracts PKC to the cell membrane fraction thandid antigen (ll),and of whole cell? PKCr and 9 were again not detectable. The presumably the inhibitory potencies of these isozymes were concentration of the various isozymes, except PKC{, was dependent on the extent of this translocation.Collectively the determined by comparison of immunoblotspreparedfrom data suggested that PKCa andt suppressed antigen-induced known concentrationsof the purified baculovirus-derived iso- activation of phospholipase C by 40-50% in intact cells but zymes of PKC (Fig. 1and TableI). The predominantisozymes that additional recruitment of PKCa andt and possibly PKCp of PKC were the Ca2+-dependent3a and Ca2+-independent6 isozymes, although, as will be discussed later, the concentraAntigen-stimulation of permeabilized cells elicited a prompt and The significance of the two bands isdiscussed elsewhere (11). 'The terms "Ca2+-dependent" and "Ca2+-independent" were used to designate the two groups of isozymes instead of the designations "conventional" and "novel."

sustained increase in levels of membrane diglycerides through the activation of phospholipase D and, toa lesser extent, of phospholipase C (H. M. S. Gonzaga and M. A. Beaven, unpublished observations). K. Ozawa, K. Yamada, M. G. Kazanietz, P. M. Blumberg, and M. A. Beaven, unpublished data.

Inhibitory Protein Kinase C Isozymes in RBL-2H3 Cells

2282 12Oc A.

DNP-BSA 11Wng/rnl): No P M A ICa”1, l.O(pM)

1

1201 B.

DNP-BSA Ila)ng/rnl); PMA 120nM): 1Ca“l. O.l(pM1

v)

z

I* PMAl

PROTEIN KINASE C (nM)

FIG. 2. Suppression of inositol phospholipid hydrolysis in antigen-stimulated permeabilized cells by different isozymes of P K C and phorbol 12-myristate 13-acetate. RBL-2H3 cellswerepermeabilized and washedtwicebefore addition of the indicated concentrations of isozymes. Antigen (DNP-BSA) was added 15 min later (panel A ) . Alternatively, phorbol 12-myristate 13-acetate ( P M A ) was added 10 min later and antigen5 min thereafter (panel B ) . Release of [‘H]inositol phosphates was determined 15min after the addition of antigen. Release of inositol phosphates in unstimulated (8 f 0.6%) and stimulated (61 f 1.5%) cells was unaffected by change in [Ca”]i i.e. from 0.1 to 1 PM (see also Ref. 25) or the presence of phorbol ester. Values are mean f S.E. of 3 cultures. The effectof 20 nM phorbol 12myristate 13-acetate on tyrosine phosphorylation of phospholipase C y l in intact cells was also determined by immunoprecipitation with antibody tophospholipase C y l and immunoblotting with anti-phosphotyrosine antibody (21)(panel C ) . As indicated, cells were unstimulated or stimulated with DNP-BSA (20 ng/ml for intact cells and 100 ng/ml for permeabilized cells) for 10 min in the absence or presence of phorbol ester.

and 6 byt.he phorbol ester resulted ineven more marked suppression (about 80%). The probable mechanism for the above inhibitory actions of PKCa and c was a reduction in antigen-induced tyrosine phosphorylation of phospholipase C y l , which was apparent after treatment of intact cells with phorbol ester (Fig. 2C). A similar attenuation of tyrosine phosphorylation of phospholipase C y l by phorbol ester has been observed in T cells stimulated via the antigen receptor, probably as a consequence of the phosphorylationof a serine residue in the phospholipase C yl by PKC (28, 29). Identification of the sites of phosphorylation by peptide mapping has, however, been unsuccessful because of the small amounts of phospholipase C yl in RBL-2H3 cells? Furthermore,althoughtyrosinephosphorylation is one mechanism of activation of phospholipase C yl inpermeabilized RBL-2H3 cells, the involvement of other isozymes cannot be excluded in the production of inositol phosphates (21). HOURS If PKCa and c normally suppressed activation of phosphoFIG. 3. Enhancement of antigen-induced hydrolysis of inolipase C in intact antigen-stimulated cells, then inhibition by sitol phospholipids in intactcells by the PKC inhibitor Ro31these isozymes of PKC should enhance the tyrosine phos7549 alone or in combination with phorbol 12-myristate 13phorylation of phospholipase C y l and hydrolysis of inositol acetate. Unexposed RBL-2H3 cells or cells that had been exposed phospholipids. Re-examination of earlier datarevealed a mod- to 20 nM phorbol 12-myristate 13-acetate ( P M A ) for the indicated est increase in extent (about 50%) of tyrosine phosphorylation times to deplete cells of the Ca2+-dependent isozymes of PKC (see of phospholipase C y l (e.g. see Fig. 5 , lower panel, in Ref. 21) “Results andDiscussion”), were stimulated with antigen (DNP-BSA, 20 ng/ml) for 15 min for measurement of release (mean k S.E. from when RBL-2H3 cells were treated with 10 PM of the PKC 3 cultures) of inositol phosphates. Ro31-7549 wasadded, a t the inhibitor, Ro31-7549 (30). In all but oneof our earlier studies, indicated concentrations,5 minbefore addition of antigen. Thepanels treatment with Ro31-7549 also caused a modest enhancement indicatethephosphoinositide response toantigen withdifferent (50-100% increase) of inositol phospholipidhydrolysis in concentrations of Ro31-7549 (panel A ) and with different times of a similar exposure to the phorbol ester in the absence and presence of Ro31antigen-stimulated cells. Inthepresentstudies, enhancement was noted infour of five experiments (increases 7549 (panel B ) . For comparison, the responses of normal intact and permeabilized cells are shown in panel B. of the response ranged from about 50 to 150%). Enhancement also occurred after prolonged treatment of cells with 20 nM phorbol 12-myristate 13-acetate, and theeffects of treatment ester was attributed to activation and subsequentdisappearwith both phorbol ester and Ro31-7549 were additive (Fig. ance of the Ca”-dependent isozymes of PKC (23) and,from 3 A ) . As previously observed (23), treatment with the phorbol the hindsight of the present data, can now be attributed to ester resulted initially in marked suppression (by -80%) of the activationof PKCa andc and thedisappearance of PKCa (data not shown but similar to data inFig. 4B of Ref. 16). In the phosphoinositide response to antigen, which then ultimately rebounded to give a response about 1.5 times that in combination with Ro31-7549, the initial suppressive action untreated cells (Fig. 3B). This biphasic response to phorbol of phorbol ester was blocked and the phosphoinositide remanse to antigen increased 2-fold over the course of 5-10 h S. G . Rhee, personal communication. tb achieve lev& of responses comparable those to obtained

Inhibitory Protein KinaseC Isozymes in RBL-2H3 Cells

2283

in washed permeabilized cells (Fig.3B). Our interpretationof mediated by one Ca2+-dependent isozyme and one Ca2'-inthese results was that Ro31-7549 blocked feedback inhibition dependent isozyme. of phospholipase C y l by phorbol ester, initially via PKCa REFERENCES and t and subsequently via PKCc after down-regulation of 1. Jaken, S. (1990) Curr. Opin. Cell Biol. 2 , 192-197 2. Ogita, K., Koide,H., Kikkawa, U., Kishimoto, A,, and Nishizuka, Y. (1990) PKCa. A paradox was the failure of Ro31-7549 by itself to Adu. Second Messenger Phos hoprotein Res. 2 4 , 218-223 enhance the phosphoinositideresponse to that obtained with 3. Nishizuka, Y. (1989) Cancer 63q 1892-1903 4. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. the combination of reagents in intact cells (Fig. 3A) or that (1988) J. Biol. Chem. 263,6927-6932 5. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. in washed, permeabilized cells (Fig. 3B). Preliminary studies (1989) Proc. Natl. Acad. Sci. U. S. A. 8 6 , 3099-3103 have indicated that Ro31-7549 was a less effective inhibitor 6. Osada, S., Mizuno, K., Takaomi, T. C., Akita, Y., Suzuki, K., Kuroki, T., and Ohno, S. (1990) J. Biol. Chem. 2 6 6 , 22434-22440 of PKCa than PKCt when concentrations of isozymes were 7. Schaa D and Parker, P. J. (1990) J. E d . Chem. 2 6 5 , 7301-7307 matched for those within the cell (see below). Irrespective of 8. Ono f:, Fujii, T., Igarashi, K., Kuno, T., Tanaka, C., Kikkawa, U., and Nishizuka, Y. (1989) Proc. Natl. Acad. Scz. U. S. A. 86,4868-4871 whether Ro31-7549 was effective against all isozymes, the 9. Burns, D. J., and Bell, R. M. (1991) J. Bzol. Chem. 266,18330-18338 effects of the pharmacological agentsadequatelydemon10. Kiley, S. C., Parker, P. P., Fabhro, D., and Jaken, S. (1991) J. Bzol. Chem. 2 6 6 , 23761-23768 strated that inhibitory signals were generated via PKC in 11. Ozawa K.Szallasi 2. Kazanietz M. G., Blumberg, P. M., Mischak,H., Muihiniki, J. F.,'and Beaven, M . A. (1993) J. Biol. Chem. 2 6 8 , 1749intact cells and probably of sufficient intensity to reduce the 17!x phosphoinositide response to antigenby half. 12. Ryves, W. J., Evans, A. T., Olivier, A. R., Parker, P. J., and Evans, F. J. (1991) FEBS Lett. 2 8 8 , 5-9 The permeabilized cells provided an alternative model to 13. Baldassare, J. J., Henderson, P. A,, Burns, D., Loomis, C., and Fisher, G. the overexpression of PKC isozymes in cells (for example as J. (1992) J . Biol. Chem. 2 6 7 , 15585-15590 14. Ways D. K., Cook P. P., Webster, C., and Parker, P. J. (1992) J. Biol. in Ref. 31) for studies of isozyme function and, in addition, C d m . 267,4796-4805 allowed direct comparison of the properties of each isozyme 15. Ase, K., Kikkawa, N., Kishimoto, A,, and Nishizuka, Y. (1988) FEBS Lett. 236,396-400 within the samecell culture. Were, however, these properties 16. Huan , F. L., Yoshida, Y., Cunha-Melo, J. R., Beaven, M. A,, and Huang, K.-f'. (1989) J. Biol. Chem. 264,4238-4243 physiologically relevant? The studies demonstrated, at least, 17. Metzger,H.,Alcaraz, G., Hohman, R., Kinet, J. P.,Pribluda, V., and that the isozymes had distinct and diverse activities. Other Quarto, R. (1986) Annu. Rev. Immunol. 4,419-470 Beaven, M. A,, and Cunha-Melo,J. R. (1988) Progr. Allergy 4 2 , 123-184 studies (data not shown) further support this conclusion. For 18. 19. Benhamou, M., Stephan, V., Rohbins, K. C., and Siraganian, R. P. (1992) example, coupling of the IgE receptor to phospholipase A2 J. Bid. Chem. 2 6 7 , 7310-7314 20. Park, D. J., Min,H. K., and Rhee,S. G. (1991) J. Biol. Chem. 266,24237was totally blocked by PKCa specifically. In addition, the 24240 estimated concentrations of the various isozymes of PKC in 21. Yamada, K., Jelsema, C. L., and Beaven, M. A. (1992) J. Immunol. 1 4 9 , 1031-1037 cytosol and membrane fractionsof antigen-stimulated RBL- 22. Beaven, M. A., Guthrie, D. F., Moore, J. P., Smith, G . A., Hesketh, T. R., and Metcalfe, J. C. (1987) J. Cell Bzol. 1 0 6 , 1129-1136 2H3 cells andthepotency of theindividual isozymes in 23. Cunha-Melo, J. R., Gonzaga, H. M. S., Ali, H., Huang, F. L., Huang, K.promoting exocytosis andinhibitinginositolphospholipid P., and Beaven, M. A. (1989) J. Immunol. 143,2617-2625 H., Cunha-Melo, J. R., and Beaven, M. A. (1989) Biochim. Biophys. hydrolysis in permeabilized cells are summarized in Table I. 24. Ali,Acta 1010,88-99 The data indicated that PKCp 6 and were present insufficient 25. Ali, H., Collado-Escobar, D. M., and Beaven, M. A. (1989) J. Immunol. 143.,"" 2628-2633 ~~~quantities, either in the whole cell or associated with the 26. Maejama, K., Hohman, R. J., Metzger, H., and Beaven, M. A. (1986) J. Biol. Chem. 261,2583-2592 membrane fraction, to promote secretion and PKCa and t 27. Tanaka, Y., Miyake, R., Kikkawa, U., and Nishizuka,Y. (1986) J. Biochem. were present in sufficient quantities to inhibit hydrolysis of (Tokyo) 99,257-261 D. J., Min, H. K., and Rhee, S. G. (1992) J. Biol. Chem. 2 6 7 , 1496inositol phospholipids. On the basis of these datawe conclude 28. Park, 1 Knl that in RBL-2H3 cells, positive stimulatory signals for secre- 29. Rtl:'S. G . , and Choi, K. D. (1992) J. B i d . Chem. 267,12393-12396 L. H., Wilkinson, S. E., Sedgwick, A. D., Hill, C. H., Lawton, G., tion were mediated primarily by PKCp and 6 and inhibitory 30. Elliott, Davis, P. D., and Nixon, J. S. (1990) Biochem. Bzophys. Res. Commun. 171,148-154 signals for phospholipase C were mediated by PKCa and t . 31. Pachter, J. A,, Pai, J.-K., Ma er Ezell, R., Petrin, J. M., Dobek, E., and Thus, and of possible significance, each set of signals was Bishop, W. R. (1992) J. Bzor &em. 267,9826-9830 _ . Y V