Conditional Activation of cAMP Signal Transduction by Protein Kinase C

Vol. 269, No. 6, Issue of February 11, pp. 4065-4069, 1994 Printed in U.S.A.

THEJOWRNAJ. OF Blomlc/u. CHEMISTRY 0 1994 by The American Society for Biochemistryand Molecular Biology, Inc.

Conditional Activationof cAMP Signal Transduction by Protein Kinase C THE EFFECT OF PHORBOL ESTERS ON ADENYLYL CYCLASE IN PERMEABILIZED AND INTACT CELLS* (Received for publication, May 26, 1993, and in revised form, September 1, 1993)

Bruce H. MorimotoS and Daniel

E.Koshland, Jr.0

From the Wepartment of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393 and the §Department of Molecular a n d Cell Biology, University of California, Berkeley, California 94720

To understand the convergence ofCAMP and protein ing the effect of phorbol esters on forskolin-stimulated type I (3) or trankinase C signal transduction, adenylyl cyclase isozyme adenylyl cyclase activity in stably transfected cells identification and biochemical studies were performedsiently transfected cells(2) exemplifies the complexity of these on the HT4 neural cell line. In HT4 cells, basal C A M P potential regulatory interactions and indicate a need for furproduction by adenylyl cyclase typesI and VI were un- ther investigations. affected by phorbol esters, nor did phorbolesters have Neural cell lines provide a homogeneous population of cells any effecton forskolin-inducedCAMPproduction. How- in whichthe isozyme compositiona n d the functional regulation ever, phorbolesters synergistically increased CAMPpro- of adenylyl cyclase activity can be investigated. In one such cell duction when adrenaline receptors were simultaline, HT4, CAMP production is importantin the modulation of of neurotransmitter secretion(5,6).HT4 cells express both shortneously activated, indicating a conditional activation CAMPproduction by phorbol esters. and long-term potentiationof neurotransmitter secretion, and A permeabilized cell preparation was used to analyze the temporal elevationof CAMPcorrelates with the increase in the mechanism by which phorbol esters were affecting CAMPproduction. This preparation enables G-proteins cellular responsiveness (6). The persistent elevation in CAMP of CAMP and protein to be activated directly by GTP+ or bacterial toxins.In levels appears to involve co-activation the permeabilized cell preparation, phorbol esters en- kinase C signal transduction pathways' and may result in a hanced C A M P produced by GTPyS-activated G-protein. "molecular switch," changing the cell from short- to long-term A stimulatory G-protein pathway may be involvedsince information storage. Understanding the contribution of regulatory cross-talk in phorbol esters synergistically increasedCAMPproduca description no effect on that produced the formation of this molecular switch will require tion by cholera toxin, yet had of the signal transduction components involved. Focusing on by pertussis toxin. the regulation of CAMP production by phorbol esters in a clonal, In this cell culture model, phorbol esters stimulate cAMP production only whensomecomponent of the neural cell line may assist in unraveling the complex interaccAMP cascade is simultaneously activated. Moreover, tions between protein kinaseC and CAMPsignal transduction. the patternof modulation suggests that protein kinase C EXPERIMENTALPROCEDURES acts on an activated component of the second messenger or the coupling of the Gsystem, such as the G-protein Chemicals and Reagent~--'~~I-cAMP radioimmunoassay (RIA)2 was protein with adenylyl cyclase, and not on the resting obtained from Amersham. 5'-(N-Ethyl)carboxyamidoadenosine (NECA),isoproterenol (IPT), and Ro 20-1724 were obtained from Restate of the protein components. search Biochemicals (Natick, MA). Phorbol 12-myristate 13-acetate (PMA) and isobutylmethylxanthine (IBMX) were fromSigma. Cholera toxin A subunit (ctx), pertussis toxin A promoter (ptx), forskolin, and GTPyS wereobtained from Calbiochem. 2'-O-Anthraniloyl CAMP(antCross-talk between signal transduction pathways may play CAMP) wasobtained from Life Technologies Inc.The mouse neural cell an important role in the ability of single cells to integrate multiple signal inputs into a unitary ceIlular response. Protein line HT4 was obtained fromRonaldMcKay (7) and maintained on polyornithine-coated flasks and plates in Dulbecco'smodifiedEagle's kinase C-activating phorbol esters have been found to both medium supplemented with 10% fetal calf serum at 33 "C. Someof the in a number of cell lines, cell properties and characteristics of HT4 cells have been described previstimulate and inhibit cAMP levels types, and tissues (see Ref. 1). The diverse response of CAMP ously (8). Cyclic AMPAccumulation in Whole Cells"HT4 cells were grown to production to phorbolesters may be due in part the to multiple isozymes of both protein kinase C a n d the components of the confluence in 6-well plates. Tissue culture media were removed,and the cAMP cascade (heterotrimeric G-protein, adenylyl cyclase, andcells were washedwith buffered saline (125 m~ NaCl, 5 m~ KCl, 20 n" Hepes, pH 7.4, 1.2 m~ MgSO,, 1.2 n" K,HPO,, 2.5 m~ CaCl,, 10 m~ phosphodiesterase). glucose). The appropriate stimulus was presented in saline, and the Recently,phorbol esters havebeenshowntoselectively cells wereincubated for various lengths of time and then lysed with 0.4 stimulate particular isozymes of adenylyl cyclase( 2 4 ) . In cells N HClO,. Protein was pelleted by centrifugation at 2,000 x g for 3-4 transfected with the gene for adenylyl cyclase type 11, phorbol min. The protein-free acid extract was neutralized with l/6 volume of esters potentiatedbothbasal(unstimulated)andforskolinB. H. Morimoto and D. E. Koshland, Jr., unpublished observations. stimulated CAMPproduction ( 2 4 ) . Conflicting reports regardThe abbreviations used are: RIA, radioimmunoassay; GTPyS, guanosine 5'-043-thiotriphosphate);NECA, 5'-(N-ethyl)carboxyamidoad* This work was supported in part by Grant DK09765 from the Na- enosine; IPT, isoproterenol; PMA,phorbol 12-myristate 13-acetate; tional Institutes of Health (to D. E. K.) and by Grant IRG IN-17-33 from IBMX, isobutylmethylxanthine; KG, potassium glutamate buffer; the American Cancer Society (to B. H. M.). The costs of publication of PIPES, piperazine-N,l\r"bis(2-ethanesulfonic acid); MOPS,4-mOrphOthis article were defrayed in part by the payment of page charges. This linepropanesulfonic acid; ant-CAMP, 2'-O-anthraniloyl-cAMP; PCR, article must therefore be hereby marked "advertisement" in accordance polymerase chain reaction; X-Gal, 5-bromo-4-chloro-3-indolyl-~-~-galacwith 18 U.S.C. Section 1734 solely to indicate this fact. toside; IF'TG, isopropyl p-D-thiogalactopyranoside.

4065

Phorbol and Esters

4066

2.4 N =COB, and the potassium perchlorate was removed by centrifugation. Cyclic AMP accumulation was determined by competition binding with '261-cAMPusing the Amersham CAMP-RIAsystem. The rate of CAMP production was found to be linear for up to 20 min. All experiments were repeated at least 3 times, and CAMP production was determined in the time interval between 6 and 10 min. In any given experiment, the data presented were the mean 2 S.E.for n = 5. Protein concentrations were determined using Coomassie dyebinding with bovine serum albumin as standards (Pierce). Permeabilized CellPreparation"HT4 cells weregrown to confluence in 6-well plates. Tissue culture media were removed,and the cells were washed with buffered saline (125 m NaC1,5 n m KC1,20 m Hepes, pH 7.4, 1.2 m MgSO,, 1.2 m K2HP0,, 2.5 m CaC12,10 m glucose).The cells were then permeabilized in 1 ml of KG buffer (139m potassium glutamate, 20 m PIPES, pH 7,0.5 ~ M A T P1,m MgSO,, 5 m glucose, 20 p Ro 20-1724, and 50 p IBMX) containing 20 p digitonin for 15-20 min. The amount ofCAMP released into the KG buffer was measured as a function of time using the CAMP-RIA. The data presented represent the mean 2 S.E. for n = 5. PhosphodiesteraseAssay-Phosphodiesterase activity was measured by the decrease in fluorescence of 2'-O-anthraniloyl-CAMP(ant-4") as a function of time. A working solution of ant-cAMP (80 p)was prepared fresh by dilution into assay buffer (10 m MOPS, pH7,90 m KCl, 0.73m CaC12,5 m MgCI2). Fluorescence wasmeasured using a SLM fluorimeter with an excitation wavelength of 350 nmand an emission wavelength of 420 nm. Phosphodiesterase activity was measured in 2 ml of assay buffer containing 8 p an"t. Reactions were initiated by the addition of 10 pl of cell extract (0.64 mgof proteidml). The decrease in fluorescence was measured at 10-s intervals, and the initial rates were determined by comparison of the fluorescent intensity to ant-CAMP standards. Cholera and Pertussis Z'ozin T)-eatment--HT4 cells were permeabilized with 20 p digitonin in KG(-ATP) buffer (139 m potassium glutamate, 20 n m PIPES, pH 7, 1m MgSO,, 5 m glucose, 20 p Ro 20-1724, and 50 p IBMX) containing 0.2 m NAD and 0.4pglml pertussis toxinA oligomer or 5 pg/ml cholera toxinA subunit. The extent of ADP-ribosylation was monitored by the incorporation of [32PlADPribose into a 40-45 kDa protein. Permeabilization and ADP-ribosylation were allowed to proceed for 15-20 min, and the rate of CAMP production was initiated by presenting the permeabilized cells with the appropriate stimulus in an ATP-regeneratingbuffer (0.5 n m ATP, 10 nm MgCI2, 150 unitdml creatine kinase, 4 nm phosphocreatine, 33 unitdml myokinase, 5 unitdml adenosine deaminase). cAMP released into the media was measured by RIA over a time period of 20 min. MolecularDetermination ofAdenylyl Cyclase Isozymes-Poly(A) RNA was purified on oligo(dT)-cellulose from5 x lo6 cells (MicroFast Track, InVitrogen). First strand cDNA was random-primed and synthesized using murine Moloney leukemia virus reverse transcriptase in the presence of methyl mercuric hydroxide (cDNA cycle, InWtrogen). Adenylyl cyclase genes were amplified from first strand cDNAbyPCR using degenerate oligonucleotides of the following sequence: AC1 = 5'-AGC ATC CTG TlT GCA GAY ATY GTG and AC2 = 5'-RGA CCA YAC ATC ATACTGCCA. As a control to avoid amplification of genomic DNA sequences, mRNA not treated with reverse transcriptase was alsoused as template for amplification. Only the first strand cDNA template resulted in the expected 354-base pair PCR fragment which was subsequently purified on a 1.7% agarose gel using Qiaex resin (Qiagen)and cloned into SmaI-digested pBluescript (Stratagene) d e r the ends of the PCR fragment were repaired with Qenow fragment (Stratagene). Recombinant plasmids were transformed by eledroporation into XL1 blue cells (Stratagene) andscreened by inactivation of lacZ cx-complementation on X-GaVisopropyl /3-D-thiogalactopyranosideindicator plates. Recombinant clonesweresequenced using Tog polymerase(Cycle sequencing, Life Technologies Inc.). RESULTS

To understand the role of protein kinase C in the regulation presented with increasing of CAMP production, HT4 cells were concentrations of the P-adrenergic receptor agonist isoproterenol (IPT), which resulted in an increase in CAMPproduction (Fig. lA). In theabsence of receptor stimulation, phorbol esters had no effect on CAMP production. However, when increasing concentrations of phorbol esters were included simultaneously with p-adrenergic receptor stimulation, a synergistic increase in CAMPproduction resulted (Fig. lA). The increase in cAMP PMA. production by phorbol esters saturates at about 0.1

CAMPProduction

o . b Y t : ~ " ~ : - - " ; ~ " - : - - - ~ - " ~ - " - ~ 0

50

100

150

200

250

300

350

IPT (nM)

0

0.03

0.06

0.09

0.12

0.15

Vls (pmolslmin/mg/nM)

FIG.1. Effect of phorbol esters on fl-adrenergic receptopinduced CAMPproduction. A, HT4 cells were presented with various concentrations of the /3-adrenergicreceptor agonist, IPT, along with 0, 10,100, or 1000I ~ PMA. M The incubations were terminated with HClO,, CAMPlevels were measured on the neutralized extract, and the initial rates were plotted as a function of IPT concentration. The data presented represent the mean 2 s.E. for n = 6.B , the data presented in A were replotted on a reciprocal plot to determine the K,,,and Vmm.

Phorbol esters may increase CAMPproduction by phosphorylation of the P-adrenergic receptor, thereby increasing the receptor's affinity for ligand. To evaluate this possibility, the data obtained in Fig. lA were subjected to a modified Eadie-Hofstee analysis in which the velocity of CAMP production was plotted as a function of the velocity divided by the concentration of IPT (Fig. 1B). The negative slope of the curve is equal to the concentration of IPT required for half-maximal CAMP production and is an indicator of receptor afinity. Such a transformation revealed that only the maximal activity of adenylyl cyclase (the y-intercept) was affected by phorbol esters. The concentration of IPT required for half-maximal activation was essentially identical either in the presence or absence of phorbol esters. This suggests that the mechanism by which phorbol esters enhance adenylyl cyclase activity was not mediated by alterations in receptor sensitivity for ligand. To determine the effect of phorbol esters on adenylyl cyclase activity, CAMP levels were elevated by the diterpene forskolin. Forskolin binds to, and directly activates, adenylyl cyclase,and increasing concentrations of forskolin resulted in an increase in the rate ofCAMP production (Fig. 2). However, forskolin-induced CAMP production was unaffected by the inclusion of phorbol esters. This indicates that phorbol esters were not elevating CAMP production by inhibiting the degradation of CAMP, since inhibition of phosphodiesterase activity would result in the accumulation of CAMP. To verify this conclusion directly, the effect of phorbol esters on phosphodiesterase activity was measured using a fluorescent analog of CAMP. Hydrolysis of ant-CAMP results in a decrease in the fluorescence intensity at 420 nm. The basal rate of CAMP hydrolysis was 36.3 2 1.3 pmol/min/mg. The inclusion

4067

Phorbol Esters and CAMP Production 5

1

-2

0

2

4

6

8

1 0 1 2

Forskolin (pM) Forskolin (pM)

FIG.2. Effect of phorbol esters on forskolin-induced C A M P production in whole cells. HT4 cells were presented with increasing concentrations of forskolin in Hepes-buffered saline, in the presence (open circles) or absence (closed circles) of 1 p~ PMA. Cyclic AMP production was measured on the neutralized acid extract, and the initial rates were plotted as a function of forskolin concentration. The data presented represent the mean = S. E. for n = 6.

of 1p~ PMA did not change the rate of hydrolysis, remaining at 35.2 2 2.1 pmol/min/mg. The inclusion of phosphodiesterase inhibitors, 20 p~ Ro 20-1724 and 50 p~ IBMX, completely abolished ant-CAMP hydrolysis. It appeared that phorbol esters were not modulating CAMP production by affecting either receptor activation or phosphodiesterase activity; thus, to further dissect the interaction between phorbol esters andCAMP production, direct activation of the heterotrimericG-protein was necessary. Unfortunately, reagents commonly used to activate G-proteins are essentially impermeable to the plasma membrane, so an alternative experimental preparation to whole cells needed to be developed. The cholesterol-binding detergent digitonin had been used to permeabilize the plasma membrane of PC12 and C6-2B cells to small molecules (9, lo), so this protocol was adapted to the HT4 cell culture system. The release of CAMP from permeabilized cells was measured as a function of time. In the absence of digitonin, the basal releaseof cAMP into themedia was 25 2 3 fmol/min/mg. The inclusion of 20 V M digitonin resulted in a 3-fold increase of CAMPrelease to 74 2 5 fmol/midmg. CAMP production wasthen stimulatedwith 1p~ NECA, a n adenosine receptor agonist, resulting in very little CAMP released in the absence of detergent, 33 & 5 fmol/min/mg; however, the inclusion of digitonin resulted in a 7-fold increase in the rate of cAMP release to 210 f 20 fmol/min/mg. The effect of phorbol esters on forskolin-stimulated CAMP production was verified inthe digitonin-permeabilized cell preparation. Inpermeabilized HT4 cells, an increase in forskolin concentration resulted in anincrease in CAMP release into the media (Fig. 3). As seen with whole cells, the inclusion of 1 p~ PMA had littleeffect of CAMPproduction. The concentration of forskolin required for half-maximal CAMP production was 5 p~ in thepermeabilized cell preparation, and approximately 8 p~ in whole cells (Fig. 21, confirming that in many ways the permeabilized cell preparation functioned similarly to whole cells with respect tocAMP metabolism. Using digitonin-permeabilized HT4 cells, G-proteins could then be activated with increasing concentrations of the GTP analog, GTPyS. In the absence of phorbol esters, increasing concentrations of GTPyS resulted in a slight increase inCAMP production (Fig.4). The inclusion of phorbol esters dramatically increased GTPyS-induced CAMP production. This indicates that the site of phorbol ester and CAMP convergence was distal to receptor activation and may involve G-protein activation or coupling between the G-protein and adenylyl cyclase. It is unlikely that phorbol esters were altering G-protein inactivation

FIG.3. Effect of phorbol esters on forskolin-induced cAMP production in permeabilized cells. HT4 cells were permeabilizedin KG buffer containing 20 p~ digitonin and various concentrations of forskolin. PMA was included at 1 p~ (open circles) or carrier dimethyl sulfoxide (closed circles). The amount of CAMP released into 1 mlofKG buffer was determined as a function of time by RIA. The data presented represent the mean * S.E. for n = 5.

1

-0.1 -10

0

10

20

30

40

50

60

G T W (PM)

FIG.4. Effect of phorbol esters on GTP+-stimulated C A M P production in permeabilized cells. HT4 cells were permeabilizedin 1ml of KG buffer containing 20 p~ digitonin and various concentrations of GTPyS, in the presence (open circles) or absence (closed circles) of 1 p~ PMA. The amount of CAMP released into the media was determined as a function of time by RIA. The data presented represent the mean * S. E. for n = 5.

by reducing the intrinsic GTPase activity, since GTPyS is not hydrolyzed to any appreciable extent. Two G-protein pathways are capable of affecting CAMPproduction. The stimulatory a subunit of the heterotrimeric Gprotein (Gas) directly interacts and activatesadenylyl cyclase, whereas the inhibitory a subunit (Gai) inhibits adenylyl cyclase by an unknown mechanism (11).Thus, if the increase in CAMP production upon phorbol ester presentation is the result of G-protein activation or coupling between the G-protein and adenylyl cyclase, either stimulation of a Gas or inhibition of a Gai pathway canbe the mechanism. To assess which G-protein pathway wasinvolved in phorbol ester-induced synergism,bacterial toxins which covalently modify either of the G-proteins wereused.Cholera toxin ADP-ribosylates the Gas subunit, resulting in theinhibition of the intrinsicGTPase activity and stabilization of the activatedconformation (121, while pertussis toxin inactivates the Gai subunitprobably by uncoupling this G-protein from its receptors (13). To circumvent the uncertainty of toxin transport into the cell, digitonin-permeabilized HT4 cells were once again used, and the ADP-ribosyltransferase subunit of either cholera toxin (ctx) or pertussis toxin (ptx) was presented. As expected, both of these treatments resulted in an increase in CAMPproduction (Fig. 5). The inclusion of phorbol esters had no effect on pertussis toxin-induced CAMPproduction, but phorbol esters synergistically increased choleratoxin-mediated CAMP generation. This suggests that phorbol esters andprotein kinase C are probably modulating CAMP production via a Gas mechanism.

4068

Phorbol Esters and CAMP Production 0.3

'

Bovine type I HT4 type I

HT4 type V I

Mouse type V I

Bovine type I HT4 type I HT4 type V I

FIG.5. Effect of phorbol esters on bacterial toxin-induced C A M P production in permeabilized cells. HT4 cells were permeabilized in 1 ml of KG(-ATP) buffer containing 20 p digitonin, 0.2 m NAD, and either 0.4 p g / d pertussis toxin A oligomer or 5 p g h l cholera toxin A subunit for 20 min at 33 "C. Cyclic AMP production was initiated by the addition of an ATP-regenerating bufferin the presence or absence of 1 p PMA. The amount of CAMP released into the media was monitored as a function of time over 20 min. The data presented represent the mean f s. E. for n = 5.

Interpreting the mechanism for cross-talk between protein kinase C and CAMPsignal transduction is complicated by the uncertainty of the isozymes of adenylyl cyclase expressed. Recently, several groups have found that various isozymes of adenylyl cyclase are differentially sensitive to phorbol ester-induced synergism (2-4). Inordertounderstandthe role of multiple isozymes in the convergence of protein kinase C and CAMPsignal transduction, the identityof the adenylyl cyclase isozymes expressed in the HT4 cell line was determined. Degenerate oligonucleotide primers were designed to a region of homology between adenylylcyclase types I (14) and I11 (15)and used to PCR-amplify the adenylyl cyclase genes expressed in HT4 cells. Fifty recombinant clones were isolated, and restriction mapping indicatedtwo distinct classes of genes. Representatives of each class were sequenced, and the predicted amino acid sequence was compared to the bovine type I and mouse type VI (Fig. 6).The nucleotide sequence of first class of clones from HT4 cells was identical with the mouse adenylyl cyclase type VI (16). The predicted amino acid sequence of the other class of clones was identical with the bovine type I adenylyl cyclase (14). Two nucleotide differences betweenthe HT4 type VI and mouse type VI occur in the region defined by the oligonucleotide amplificationprimers andprobably do not reflect a n actual difference in the genesequence.

AC 1

I

SILFADIVGFTGLASQCTAQELVXLLNELFGKFDELATE 111111111111l111111IIIIIIIIIIIIIlllllll SILFADIVGFTGLASQCTAQELVKLLN'ELFGKFDELILTE IIllllIIIII 11111111111 IIIII I II I I SILFADIVGFTSLASQCTAQELVMTLN'ELFARFDKLAAE IIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII SILPADIeGFTSLASQCTAQELVMTLNELFARPDKLAAE

NHCRRIKILGDCYYCVSGLTQPKTDHAHCCVEMGLDMID IIIIIIIIIIIIlIIIIIl1111l111111111111III NHCRRIKILGDCYYCVSGLTQPTKDHAHCCVEMGLDMID IIIIIIIIII Ill I I IIIIIIIIIIIIIII NHCLRIKILGDCYYCVSGLPEARADHAHCCVEMGVDMIE IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

MOUEe type VI

NHCLRIKILGDCYYCVSGLPEARADHAHCCWGVDMIE

Bovine type I

TITSVAEATEVDLNMRVOLHTGRVLCGVLGLRXWQYDWS IIIIIIIIIIIIIIIIIIIIllllllllllllIlTlIIII TITSVAEATEMLNMRVGLHTGRVLCGVLGLRXWQYDWS I I I I I IIIII I Ill IIIIIIIIIIIIIII

HT4 type I

AISLVREVTGVNVNMRVOIHSGRVHCGVLGLRKWQYDVWS

HT4 type V I

Mouae type

VI

1 1 t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 IIII AISLVREVTGVNVNMRVGIHSGRVHCGVLGLRKWQfDVWS c "

AC2

FIG.6. Predicted amino acid sequence of the two isozymes of adenylyl cyclase genes expressed HT4 in cells. First strand cDNA from HT4 cells was used as template forPCR amplification of the adenylyl cyclase isozymes. In the bovine adenylyl cyclase type I sequence (14), the AC1 primer would begin at nucleotide position 1015, and AC2 primer would end at nucleotide position 1368.

cance of such diversity is unclear, but itis reasonable tobelieve that a particular subset of the G-protein and adenylyl cyclase isozymes are expressed in a single cell type, and this results in a specific organization and regulation of subsequent signal transduction. The genetic and functional diversity of the various signal transduction components is illustrated by the multiple adenylyl cyclase isozymes. The regulation of the variousadenylyl cyclase isozymes varies widely. For example, types I and I11 adenylyl cyclase are activated by calciudcalmodulin (27, 28), whereas typeVI cyclase is inhibited by submicromolar concentrations of calcium (16). The other isozymes appear to be unaffected by calcium. Additionally, the ability of other signal transduction components to interact with adenylyl cyclase is also isozyme-dependent. Increasing concentrations of the Gprotein /3y subunits inhibit typeI cyclase, but activate typeI1 (29-31). For the regulationof cAMP production by protein kinase C, various isozymes of protein kinase C have also been DISCUSSION postulated to havea modulatory role, in which the (x isozyme of The finding that phorbol ester enhancement of CAMP pro- protein kinase C may inhibit forskolin-mediated CAMPproducduction in thewhole and permeabilized cells adds a new facet tion, whereas the y isozyme may be facilitory (32). to the integration of protein kinase C and cAMP signal transThe possibility also exists thatadenylyl cyclase forms heterduction. The specific interaction between protein kinase C and ologous multimers. For example, a cell which expresses both CAMPsignal transduction hasalso been observed in platelets, types I and VI cyclase may be subjected to regulation by phorhepatocytes,adipocytes, and lymphocytes (17-20). Although bo1 esters, whereas theexpression of either individual isozyme the precise mechanism by which protein kinase C regulates alone is not responsive to phorbol esters. Thiswould provide a n CAMPproduction in thesecell types remainsinconclusive, sev- additional level of potential regulation for a particular cell to eral proposed mechanisms for protein kinase C and CAMP integrate signal transductionpathways. The lack of a n effect of phorbol esters on basal or forskolincross-talk include the phosphorylation of Gai, which decreases stimulated adenylyl cyclase activity in HT4 cells is consistent its ability to inhibit adenylyl cyclase (17, 18, 21, 22) or the direct phosphorylation of adenylyl cyclase (23, 24). Although with that observed in transfected kidney 293 cells with either phosphorylation of these individual components has been ob- type I or VI adenylyl cyclase (2). Although phorbol esters have served, the direct biochemical consequence of phosphorylation no effect on basal or forskolin-stimulated type I or VI cyclase has yet tobe determined, and theisozymes of adenylyl cyclase activity in HT4 cells, phorbol esters synergistically increase receptor-, cholera toxin-, or GTPyS-stimulated CAMP producaffected in these preparations is unknown. One explanation for the mechanistic ambiguity of protein tion from these two adenylyl cyclase isozymes. This indicates kinase C and CAMPcross-talk may be related to the numerous that cross-talkbetween CAMP and protein kinase C signal transduction pathwaysnot only requires theexpression of speisozymes of both the heterotrimeric G-protein subunits and adenylyl cyclase (see Refs. 25 and 26). The functional signifi- cific isozymes of adenylyl cyclase, but it also requires co-acti-

Phorbol and Esters vation of the two pathways. One possible mechanism may involve the phosphorylation of theheterotrimeric G-protein of the and/or adenylylcyclase which results in an enhancement interaction between the two, having no effect on the unstimulated activity of each component. This form of synergism may allow the cell to integrate two signal inputs, one activating CAMPproduction and one activating protein kinase C. Acknowledgments-We thank Dr. Dermot Cooper for providing the clone for mouse adenylyl cyclase type V I , Ann Fischer for assistance with cell culture, and Dr. Phil McFadden and Diana Chu for suggesting the use of digitonin permeabilizedcells.

REFERENCES 1. Houslay, M. D.(1991)Eur: J. Biochem. 195, %24 2. Yoshimura, M., and Cooper, D.M. F. (1993)J. Biol. Chem. 268,46044607 3. Choi, E . J . , Wong, S. T., Dittman, A. H., and Storm, D.R. (1993)Biochemistry 32. 1891-1894 4. Jacobowitz, O., Chen, J., Premont,R. T., and Iyengar, R.(1993)J. Biol. C h m . 268.38293832 5. Morimoto, B. H., and Koshland, D. E., Jr. (1990)Neuron 5,875-880 6. Morimoto, B. H., and Koshland, D.E., Jr. (1991)Proc. Natl. Acad. Sei. U. S . A. 86, 10835-10839 7. Whittemore, S . R., Holets, V.R., Keane, R. W., Levy, D. J., and McKay, R. D. G. (1991)J. Neurosci. Res. 28, 156-170 8. Morimoto, B. H., and Koshland, D.E., Jr. (1990)Proc. Natl. Acad. Sei. U. S . A. 87,3518-3521 9. Peppers, S . C., and Holz, R. W. (1986)J. Biol. Chem. 261, 1466514669 10. Brooker, G., and Pedone, C. (1986)J. Cyclic Nucleotide Protein Phosphoryla. tion Res. 11, 113-121 11. Levitzki, A. (1990)G-Proteins as Mediators of Cellular Signalling Processes,

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