Regulation of protein kinase C by cyclic adenosine 3': 5 ...

Vol. 262, N o , 22, Issue of August 5, pp. 10497-10501 1987 Printed in d.S.A.

THEJOURNALOF BIOLOGICALCHEMISTRY 0 1987 by The American Society of Biological Chemista, Inc.

Regulation of Protein Kinase C by Cyclic Adenosine 3’:5’-Monophosphate and aTumor Promoter in Skeletal Myobl.asts* (Received for publication, December 4, 1986)

Suree Narindrasorasak,Ann Brickenden, EricBall, and BishnuD. Sanwal From the Department of Biochemistry, Faculties of Medicine and Dentistry, University of Western Ontario, London, Canhda N6A 5CI

The specific activity of protein kinaseC in rat skeletal myoblasts decreased when they were exposed for very short periods to isoproterenol, forskolin, dibutyryl cyclic AMP (BtacAMP),or the phorbolester, 12O-tetradecaaoylphorbol-13-acetate (TPA). In the presence of BtzcAMP or forskolin only the cytosolic but notthe membrane-boundkinase activity was found to decrease. Treatment with TPA, however, led to a decreaseinthe activity of theenzymebothinthe cytosolic as well as the membrane fractions. The effects extracts observed in vivo could be duplicated in crude of myoblasts incubatedwith cAMP analogues or TPA. In the presence of ATP, protein kinase C activity decreased considerably in crude cytosolic fractions treated with the cAMP analogues, but a requirement for ATP was not evident for the decrease in activity brought about by TPA. For the cAMP analogues the decrease in protein kinase C was also prevented by incubation of theextracts with an inhibitor of CAMPdependent protein kinase. The regulation of protein kinase C by BtzcAMP (but not by TPA)was altered in Rous sarcoma virus-transformed myoblasts. Itis considered likely that a componentaffected byCAMP (probablya substrate for CAMP-dependent protein kinase) participates in the regulation of protein kinaseC activity, and it is altered in unknown ways in transformed myoblasts.

Protein kinase C is an ubiquitous enzyme and is present invariably in all tissues and organs which havebeen examined so far (1). It is now amply clear that binding of certain hormones, neurotransmitters,and growth factors totheir receptors leads to the hydrolysis of inositol phospholipids, generating on the one hand inositol 1,4,5-triphosphate (2) which mediates calcium mobilization (3), and on the other, 1,2-diacylglycerolwhich activates protein kinase C. This activation results in the phosphorylation of several proteins which probably serve as mediators of cell proliferation, growth regulation, and several other cellular functions (4). Activation of protein kinase C, however, is not the only means by which some of the proteins are phosphorylated. Several substrates of protein kinase C also serve as substrates of the CAMPdependent proteinkinase (4, S), another enzyme whichenjoys as wide a distribution in various cells and tissues as protein kinase C (6). The CAMP-dependent protein kinase, however, constitutes apart of a completely different signaling pathway,

* This work wassupported by operational grants from the Medical Research Council of Canada (to E. B. and B. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertkepent” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact.

viz. that initiated by the activation of adenylate cyclase by the binding of certain hormones and neurotransmitters (7). Sharing of substrates may indicate a certain degree of interaction between the two signal-transducing systems. Indeed, the nature of interaction between the CAMP-generating and the phosphoinositide degradation pathways has been shown to depend upon the natureof the cellular system in which the two pathways operate (4).In monodirectional control systems, exemplified by some endocrine tissues, lymphoma (8) and pinealocytes (9) to name a few tissues among several (4), protein kinase C potentiates cAMP formation, which suggests some degree of synergism between the two signal-transducing pathways. In bidirectional control systems, on the other hand, the two signaling systems counteract each other (4). In certain tissues, such as ovarian granulosa cells (lo), glioma C6 cells ( l l ) , and several others (4, 12) protein kinase C inhibits the adenylate cyclase systems, while in others such as platelets, neutrophils (4), and lymphocytes (13) CAMP,the end product of the adenylate cyclase system, produces an inhibitory effect on phosphatidylinositol turnover. While several physiological studies on the interaction of the two signaling pathways in various tissues have been made, little information exists on the exact loci and the molecular mechanisms of the synergistic and antagonistic interactions between the two pathways. In an effort to understand the basis of the effect of cAMP on the differentiation of skeletal myoblasts (13, 14), we found that cAMP caused marked decreases in the activity of protein kinase C in vivo. This decrease resembled the “down regulation” brought about by the well known tumor promoter TPA.’ It was, thus, of interest to examine whether the mechanism of the down regulation was the same for cAMP and TPA. We show in the following account that TPA and cAMP regulate the activity of protein kinase C differently. EXPERIMENTALPROCEDURES

Cell Lines and Cell Culture-A highly myogenic clone of the rat myoblast line, L6 (15), and two of its transformed derivatives, JRu5 and LG(RSV) were used (14).The former was a spontaneously transformed line, and the latterwas transformed by Rous sarcoma virus. The cells were grown in a-modified minimal essential medium supplemented with 16 mM glucose, 10% horse serum, and 50 pg/ml gentamycin. Cells were usually plated at a density of 4 X lo6 cells/ 150-mmplate, and themedium waschanged every 2 days. To examine the effect of various drugs on protein kinase C activity, 2-3-day-old cells wereused. The growth mediumwas supplemented with the required drug for the statedlength of time.

* The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13acetate; AMP-PCP, adenyl-5’-yl (0.7-methy1ene)diphosphonatq AMP-PNP, adenyl-5”yl imidodiphosphate EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid MES, 2-(N-morpholino)ethanesulfonic acid; MIX, 3-isobutyl-l-methylxanthine; BkcAMP, dibutyryl cyclic AMP.

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Preparation of Cell Extract and Assay of Protein Kinase C-The cells were harvested at appropriate times by rinsing the monolayers twice with cold phosphate-buffered saline and scraping with a rubber policeman. The cells were collected in cold buffer A (10 mM 'l'risHCI, pH 7.5,5 mM EGTA, 0.25 M sucrose, 1mM phenylmethylsulfonyl fluoride, 2 pg/ml leupeptin, and 0.1% 2-mercaptoethanol) and disrupted hy 20-30 strokes of the pestle in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 40,000 X g for 30 min. The supernatant was desalted on a Bio-Gel P-6DG column and equilibrated with buffer B (10 mM MES, pH 6.7, 1 mM EGTA, 2 pg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 0.1% 2-mercaptoethanol). The desalted supernatant was applied to a DE52 cellulose column (1.5 X 5 cm) equilibrated with buffer B. After the addition of the sample, the column was washed with 30 ml of buffer B and then eluted with 100 mlof linear NaCl gradient (0-0.4 M ) in buffer B. Fractions of 1 ml were collected, and a 50-pl aliquot of each fraction was assayed for protein kinase C. The standard assay mixture (75 p l ) contained 20 mM Tris-HCI, pH 7.5, 25 pg of histone type I11 S, 20 p M [Y-~'P]ATP (specific activity, 200-300 cpm/pmol), 10 mM magnesium acetate, 0.8 mM CaCI2,9.75 pg of phosphatidylserine, and 0.3 pg of diolein. Nonspecific activity was measured in control aliquots without the addition of calcium and phospholipids. After 15 min of incubation a t 30 "C, a 50-pl aliquot was spotted on 2 X 2-cm phosphocellulose paper. The papers were washed four times with 5 min of stirring in 75 mM phosphoric acid. They were then rinsed in 95% ethanol and diethyl ether, respectively, and dried. Protein kinase C activity was determined by subtracting the activity measured in the absence of calcium and phospholipid from that measured in its presence. The activity has been expressed throughout as pmol of incorporated per min per fraction from 100 mg of protein (normalized value) loaded on the column. To determine the activity of the "membrane"-hound protein kinase C, the cells were homogenizedas described above, and the pellet after centrifugation at 40,000 X g was solubilized with 1%Triton X-100 by gentle mixing for 30 min at 4 "C. The preparation was centrifuged at 40,000 X g for 1 h, and the detergent-solubilized preparation was applied to a DE52 cellulose column and theenzyme eluted by applying a linear gradient of NaCL, as described above for the preparation of the cytosolic enzyme. Protein concentration in extracts was determined by the method ofLowry et al. (16). CAMP-dependent protein kinase activity was measured as described earlier (14). Materia/~-[r-~'P]ATPwas purchased from Du Pont-New England Nuclear. The isoquinolinesulfonamide protein kinase inhibitors H-8 and H-9 (17) were obtained from Seikagaku America,Inc., Florida. Tissue culture supplies werefromFlow Laboratories. All other chemicals were from various commercial sources.

C

-A "

A.

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FIG. 1. The effect of isoproterenol, forskolin, and BtacAMP on protein kinase C activity in L6 myoblasts. For each drug treatment 20 plates (150 cm') of 4-day-old myoblasts were utilized. The duration of exposure and concentrations of the drugs were: 10 p M isoproterenol, 45 min; 0.7 mM BkcAMP, 45 min; 10 pM forskolin, 60 min. In addition, each plate also received 0.1 mM MIX. At the end of the incubation period, the cells were harvested, and protein kinase C in cytosol preparations was measured as described in the text. Panel A , control; Panel B, isoproterenol; Panel C, BtSAMP; Panel D, forskolin. The symbols represent: A"-A, NaCl concentration; O"-O, activity in the presence of calcium and phospholipid X- - -X, activity in the absence of calcium and phospholipid.

RESULTS

Decrease in Protein Kinase C Activity in Vivo-We noted that protein kinase C activity of L6 myoblasts decreased in cytosol fractions after treatment of the cells for less than an hour by Bt,cAMP or compounds which are capable of generating CAMP in vivo in myoblasts, uiz. isoproterenol and forskolin. This is shown in Fig. 1. Protein kinase C is eluted as a single peak between 75 and 80 mM NaCl under our experiFraction numbe! mental conditions. The activity of the enzyme which in unFIG. 2. Protein kinase C activity inthe particulate fractions tre.lt,ed myoblasts is 105 units/mg of protein isreduced to 67, of myoblasts treated vivo with forskolin. Cells were treated 48, and 50 units/mginthepresence of isoproterenol, with or without 10 p~ in forskolin and 0.1 mM MIX for 60 min. Cell BhcAMP, and forskolin, respectively. Prolonged treatment homogenates and particulate fractions were prepared as described in with these reagents (4-40 h) reduces the protein kinase C the text. Panel A , control; Panel B, forskolin-treated cells. The symbols have the same meaning as in Fig. 1. activity up to 70-80%. In several systems it has been demonstrated (18-20) that a decrease in cytosolic protein kinase C levels in thepresence of hormones (18, 19) or tumor the diminution of enzyme activity in cytosolic fractions. As reported in some other type of cells (20-22), treatment promoter,TPA (20-22), isdueto a translocation of the enzyme from the cytoplasmic to the membrane compartment. of the myoblasts with TPA decreases the cytosolic protein This is distinctly not so in myoblasts treated with forskolin kinase C activity (Fig. 3). This decrease isconcentrationor Bt2cAMP, as shown in Fig.2. In response t o forskolin dependent. At a concentration of 0.02 PM TPA more than 65% of the activity is lost (Fig. 3). In the presenceof 1.0 p M treatment, the enzyme activity present in particulate fractions TPA for 30 min, no activity at all was discernible in the remainsunchangedas compared tothecontrols.Similar results are obtained (data not shown) when cells are treated cytosol. At a very low concentration of TPA (0.01 FM) inwith Bt2cAMP or isoproterenol. Translocation to the mem- creased activity of the enzyme was found in the particulate higher concentrations the particulate activity brane component, thus, does not appear to be the reason for fractions, but at

Regulation Kinase of Protein

C

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membrane-bound activity. These findings would support the contention that protein kinaseC is degraded in the presence of TPA, although productionof the inhibitor cannotbe ruled out. As with TPA, more than 10 h was required in treated cells for theenzyme to return to control levels after Bt,cAMP treatment. Effect of TPA and CAMP Analogues on the Decrease of Enzyme Activity in Vitro-In search for a mechanism for the effect of TPA and cAMP analogues on protein kinase C, we investigated whether the events we had found in vivo could be reproduced in vitro. When cytosol preparations from myoblasts were incubated separatelyfor 10 min at 30 "C with 0.2 ~ L MTPA or 0.7 mM Bt,cAMP, almost 50% of thekinase activity was lost in TPA-treated extracts. Bt,cAMP by it,self showed no effect unless ATP was also added to the cytosol TPA(pM1 the ofATP, Bt2cAMP, FIG. 3. Protein kinase C activity in the soluble (cytosol) and preparations along with it. In presence particulate fractions of myoblasts after exposure to TPA. The and an inhibitor of cAMP phosphodiesterase (MIX), a 50cell monolayers were exposed to the various indicated concentrations 60% reduction occurred in the activity of protein kinase C in of TPA in vivo for 30 min. The cells were treated as described in the 10 min (Table I). ATP could not be substituted by GTP or text. The symbols represent: W,enzyme activity in thesoluble the nonhydrolyzable analogues, AMP-PNP or AMP-PCP, for fraction; A-A, enzyme activity in particulate fractions. the inhibition of protein kinase C to occur in vitro (Table I). declined in parallel with the enzyme activity in the cytosol The effect of TPA was neither potentiated nor antagonized (Fig. 3). At a concentration of 1 ~ L MTPA, only 20% of the by ATP or Bt,cAMP and ATP together. Forskolin did not control level of enzyme remained in the particulate fractions. cause any diminution of the kinase activity in cell-free exThe effect of T P A was thus in contrast with that obtained intracts. Addition of protease inhibitors, leupeptin (100 pg/ml) the presence of BhcAMP, where only the cytosolic form of and phenylmethylsulfonyl fluoride (50 pg/ml), either singly protein kinase was affected, but not that bound to the mem-or in combination tocytosol preparations failed to relieve the brane compartment (Fig. 2).The tumor promoter, Bt2cAMP, reduction of protein kinaseC activity obtainedin the presence of Bt,cAMP and ATP. forskolin, andisoproterenolhadno effectdirectly on the Reversal of Bt,cAMP Effect by Protein Kinase Inhibitorsenzymewhen assayedunderourexperimentalconditions. It is the general consensus that all cAMP effects in various Both in the presence of BtzcAMP and TPA the decrease in the Ca2+/phospholipid-activatableprotein kinase activitywas biological systems are mediatedby the CAMP-dependent pronot accompanied by a n increase of the phospholipid-inacti- tein kinases. CAMP-dependent protein kinases I and I1 have myoblasts vatable form of the enzyme (Fig. 1). Myoblast extracts show been shownto occur in a soluble form in rat skeletal (14). If these kinases were involved in the decrease of protein the presence of two small peaks of protein kinase activities, kinase C activity in vitro, it should be possible to prevent the both of which are independentof Ca2+and phospholipids(Fig. 1) in treated as well as untreatedcells. These peaks are CAMP-decrease by treating the extracts with inhibitors of CAMPdependent kinases. An inhibitor well suited for this purpose dependent protein kinases but appear not proteolytically to be (17). produced catalytic fragmentsof protein kinase C, such as the is N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide This inhibitor (H8) has been reported to have Ki values of 1 one reported by Kishimoto et al. (23).In any event, thelevels and 15 p~ for the CAMP-dependent and protein kinase C, of these peaks do not increase with a decrease in the level of of these enprotein kinase C under different treatmentregimens (Fig. 1). respectively. We ascertained that the activity In fetal rat skin keratinocytes (22)it has been shown that zymes in cytosolic preparations of myoblasts was inhibited to the loss of protein kinase C on TPA treatment is prevented the extent of about 80% for the CAMP-dependent protein by exposure of the cells to the proteinase inhibitor, leupeptin.kinasesbut only about 10% for proteinkinase C inthe We made several attempts to prevent the decrease in enzyme presence of 5 p~ kinase inhibitor. From Table I1 it will be activity in myoblasts in the presence of Bt,cAMP and TPA noted that incubationof cytosol preparation with 5 p~ inhibwith 0.1 mg/ml leupeptin but did not succeed in doing so. TABLEI Leupeptin by itself caused a small decrease in the activity of Requirement of both BtZcAMP and ATP for inhibition of protein protein kinase C in vivo. C activity in crude cytosol of the myoblasts ___ ..kinase Recovery of Protein Kinase C Activity after Treatment-In Additions" Specific activityb view of the results obtained above, it seemed possible that pmol/rnin/mg protein treatment of myoblasts with BhcAMP or TPA either leads addition Control, 99 to a degradation of protein kinase C or a masking of the no andATP, 43 activity of the enzyme by, for example, the production of an MIXBbcAMP, Bt2cAMP andMIX 88 in vivo inhibitor in the presenceof the drugs. We, therefore, ATP 95 sought to find the time required for the treatedcells to recover AMP-PNP, Bt2cAMP, and MIX 94 completely their normal contentof protein kinase C activity. AMP-PCP, BbcAMP, and MIX 100 ATP, Bt2cGMP, and MIX 101 For t,his purpose cells were treated with 0.2 p~ TPA for 10 BtZcAMP, and GTP, min when thecytosolic protein kinase C activity was reduced118 MIX .." . . from the initial value of 132 to 33 units/mg and the memEach cytosol preparation containing 2.9 mg of prot.ein WHS incubrane-bound enzyme from 36 to 30 units/mg. The drug was bated with the chemicalslisted. At the end of the incubation period. removed after 10 min by changing the medium. At various the mixture was desalted, and the activityWRS measured after chromatography as described in the text. The concentrat.ion o f the reactime intervals thereafter (1, 5, 10, and 24 h) enzyme activity tants were: ATP,AMP-PCPandGTP, 0.1 mM; Ht.,cAMP and was measured. Almost complete return to control levels was BtZcGMP, 0.7 mM; MIX, 0.1 mM. obtainedafter 10 h of culture for both the cytosolic and *Average values from three different experiments. ~~~~

~~

~~~

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C

TABLEI1 Effect of protein kinase inhibitor, Ha, on the reversal of the inhibition of protein kinase C activity in vitro The cytosol preparation from myoblasts was incubated for 10 min at 20 "cwith 0.7 m M Bt2cAMP, 0.1 mM MIX, and 20 PM ATP. The concentration of H8 was 5 PM when used. Protein kinase C activity was measured after desalting and columnchromatography as described in the text. Additions Protein

kinase C activity

pmol/min/mg protein

Control, no additions BtsAMP, and ATP, MIX H8 BtsAMP, ATP, MIX, and H8

69.2 37.0 63.7 62.5

itor for 10 min at30 "C reduced the specific activity of protein kinase C from 69.2 in the absence of the inhibitor to 63.7 in its presence. Also, in conformation of previous results, incubation of the extracts with BtzcAMP and ATP reduced the kinase activityt o 37. However, simultaneous incubation with the kinase inhibitorreversed this decrease almost completely. It would thus appear that the effect of BtzcAMP on protein kinase C activity isprobably mediated by the CAMP-dependent protein kinase. In view of the in vitro reversal of the inhibition of protein kinase C broughtabout by thekinaseinhibitor,H8, we attempted to reverse the in vivo inhibitioncausedinthe presence of BtzcAMP. Inclusion of 50 PM H8 in the culture medium along with BtzcAMP, however, failed to reverse the decrease in the level of protein kinase C. It is quite possible that myoblasts are not permeable to H8, although the isoquinolinesulfonamides have been shown to be permeable in some cell lines (24).

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FIG. 4. The effect of Bt2cAMP,isoproterenol, and forskolin on protein kinase C activity in Rous sarcoma virus-transformed myoblasts. The concentrations of the drugs, time of exposure of the cells, and preparation of soluble enzyme fractions was exactly the same as described inthe legend to Fig. 1. Panel A , control; Panel B, Bt,cAMP; Panel C, forskolin; Panel D,isoproterenol.

ment is presented in Fig. 4. It may be recalled that exposure of wild-type myoblasts (Fig. 1) to these drugs causes a 40Absence of Direct Effect of Catalytic Subunit in Protein 50% reduction of protein kinase C activity. Results similar to Kinase C-The simplest explanation to account for the results those presented in Fig. 4 were also obtained with JRu5, a described above would be to assume that theCAMP-depend- spontaneously transformedmyoblast line. As expected, treatent protein kinases directly phosphorylate protein kinase C ment of cytosol preparations from the transformed cell lines and alter its catalytic activity. Autophosphorylation of protein with BtzcAMP and ATP also did not result in any loss of kinase C in the presenceof Ca2+ andphospholipids has been protein kinase C activity. demonstrated to occur (4)without significant alterations of its activity. To examine if CAMP-dependent protein kinase DISCUSSION has any effect on protein kinase C, we purified this enzyme We have demonstrated that both TPA and cAMP analogues from myoblasts' to a specific activity of 9000 units/mg of (or compounds which cangeneratecAMP in vivo by the protein.Thepreparation was free of anycontaminating stimulation of adenylate cyclase in myoblasts) decrease the CAMP-dependent protein kinases. When this preparationwas incubated with variousconcentrations of the catalytic subunit activity of the soluble protein kinase C within a few minutes of beef heart protein kinasepurified according to the method of the exposure of myoblasts to thedrugs. In othercell systems cells (27), keratinocytes (22), of Beavo et al. (25) in the presence of ATP, no significant such as Friend erythroleukemia Swiss 3T3 cells (28), and HL-60 human promyelocytic leueffect on the activity of protein kinaseC was discernible after C in the 10 and 30 min of incubation. We did not attempt to ascertain kemiacells (29), the decrease inproteinkinase presence of the tumor promoter is due to a down regulation whether protein kinaseC was a t all phosphorylated under our of the enzyme, which serves as the receptor for TPA. This experimental conditions. Effect of C A M P and TPA on Protein Kinase C of Trans- down regulation has variously been considered to be due to thetranslocation of the enzymefrom the soluble tothe formedMyoblasts-In work from our laboratory published earlier (26),we had shown that regulation of cAMP phospho- membrane compartment of the cell (18, 22) and the proteolytic degradation of the enzyme (22). It is possible that degdiesterase by cAMP or its analogues in wild-type myoblasts was altered in transformed myoblasts. To examine if this is a radation of protein kinase C also occurs in myoblasts. What general phenomenon exhibited by CAMP-regulated systems, distinguishes myoblasts from the other cell types so far studdown regulation of protein kinaseC is the we tested theeffect of cAMP and TPA on levels the of protein ied in regard to the speed with which it occurs in these cells. Within 10 min of kinase C in transformed myoblasts. TPA caused a decrease in enzyme activity both i n vivo and in vitro exactly like the treatment in vivo almost 80% of the kinase activity is down regulated. If down regulation is indeed due toproteolysis, this wild-type cells. In contrast, exposure of the Rous sarcoma virus-transformed myoblasts to BtzcAMP, isoproterenol, or observation may point to the very active proteolytic system forskolin for various lengths of time did not cause any dimi- that probably exists in myoblasts. The down regulation of view of the nution of protein kinase C in vivo. A representative experi- protein kinase C to TPA is not surprising in results obtained in other systems but what is indeed a novel * S. Narindrasorasak andB. D. Sanwal, unpublishedobservations. observation in myoblasts is that protein kinase C activity is ~"

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Regulation of Protein Kinase C also considerably reduced i n vivo when the cells are exposed to cAMP analogue^.^ This reduction in the enzyme obviously cannot be due to down regulation, because CAMP orCAMPrelated enzyme systems are not known to bind to protein kinase C. The rapid loss of activity after treatment with CAMP-elevating drugs is similar to the TPA-induced down regulation in some ways. In both cases, the decrease in activity in cells is rapid and can only be reversed after a long period (10 h) in culture, suggesting degradation rather than inhibition of protein kinase C as the mechanism. The reduction due to cAMP is more selective than that induced by TPA, however, in thatonly soluble protein kinase C activity is affected. We could find no evidence that cAMP affects the membrane association of protein kinase C. From these observations it would appear that TPA and cAMP cause a reduction in the levels of protein kinase C through different pathways. Our finding that theCAMP-mediatedloss of protein kinase C is also observable in high speed supernatants allowed us to begin characterizing the mechanism. The results are consistent with an action via the CAMP-dependent protein kinase. ATP is required, and the effect is inhibited by inhibitors of the kinase. Since we have shown that protein kinase C activity is notaltered when it is incubated with purified CAMPdependent protein kinase: it is likely that kinase-sensitive components (presumably substrates) are involved in the decrease of protein kinase C observed i n vitro. The components affected by cAMP and TPA,however, must be different from each other since in TPA-treated cells membrane-bound protein kinase C remains unaffected and in transformed myoblasts only TPA (but not CAMP) is able to reduce protein kinase C levels, indicating a loss or abnormality inthe CAMPregulated system in these cells. Interestingly, we have earlier demonstrated (26) that CAMP-related proteolytic conversion of cAMP phosphodiesterases in L6 myoblasts is not demonstrable in Rous sarcoma virus-transformed L6 derivatives. The situation we had described with regard to thephosphodiesterases is similar to the observation made in this work with regard to the regulation of protein kinase C except that we have not been able to show a direct involvement of proteases in this process. It is too early to say whether other CAMP-regulated systems will be found to be similarly affected in transformed cells, but as we had suggested before (26), an alteration in a proteolytic system is one possible mechanism for the effects. More than themechanism of partial inactivationof protein kinase C, the present studies are important in regard to the mechanisms of interaction between the two signal-transducing pathways, one initiated by the transformation of phosphoinositides and another by activation of adenylate cyclase (4). Both these pathways lead to theactivation of the protein kinases specific to theindividual pathways, uiz. protein kinase C for the phosphoinositide pathway and CAMP-dependent We (G. A. Cates, D. Nandan, A. Brickenden, S. Narindrasorasak, E. H. Ball, and B. D. Sanwal, unpublished observations) have recently found that phosphorylation of an in vivo substrate of protein kinase C in myoblasts, a 46-kDa glycoprotein, is reduced to an extent of about 30-40% when the cells are exposed to Bt2cAMP. This observation is in accord with the findings in this paper that the in vivo levels of protein kinase C are regulated by CAMP. Purified rat brain protein kinase C incubated in the presence of [T-~'P]ATPwith or without the catalytic subunit of CAMP-dependent protem kinase is found to be phosphorylated to the same extent, which suggests that the CAMP-dependent kinase probably is unable to phosphorylate the autophosphorylated protein kinase C further (D. W. Litchfield and E. H. Ball, unpublished observations).

protein kinases for the adenylate cyclase pathway. These kinases are known to share some of the same substrates, whose seryl/threonyl residues they phosphorylate (4).However, while phosphoinositide breakdown is connected with growth proliferation (4), cAMP causes growth retardation in myoblasts. It is possible that one of the reasons cAMP can do so is by inhibiting protein kinase C, i.e. by antagonizing the positive proliferation signal. Clearly, the phenomenon we have described in the myoblasts should be studied in other cellular systems whichshow retardation of growth in the presence of CAMPor of compounds which can generate cAMP

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