(CANCER RESEARCH 52. 5604-5609, October 15, 1992]

Modulation of Protein Kinase C-e by Phorbol Esters in the Monoblastoid U937Cell1

D. Kirk Ways,2 Berniece R. Messer, Trudy O. Garris, Weixi Qin, Paul P. Cook, and Peter J. ParkerDepartments of Medicine and Microbiology/Immunology, East Carolina University, School of Medicine, Greenville, North Carolina 27858-4354 ¡D.K. W.,B. R. M., T. O. G„W. Q„P. P, C.J, and Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London,I'nited Kingdom HC2A 3PX [P. J. P.J

ABSTRACTExpression of protein kinase ( '-<was examined in the human mono-

blastoid U937 cell. This cell type contained the a, 0, and <isoforms ofprotein kinase C (PKC). While I'M '-<content was slightly higher in the

cytosolic than in the particulate fraction, the amount contained in theparticulate fraction was higher than the a and ßisoforms which werepredominantly localized to the cytosol. After an acute exposure to tet-radecanoyl-13-phorbol acetate (TPA), I'M -<translocated to the partic

ulate fraction. Acute or chronic exposure to ionomycin did not altercontent of the < isoform. Longer exposures to TPA decreased PKC-c inboth cellular fractions. I'M -< displayed a similar sensitivity to TPA-induced down-regulation as did PKC-0 while PKC-a was more resistantto this effect. After a 72-h exposure to 0.1 ini TPA, increases in the aand ßisoforms but not in I'M -< were observed. However, 1,25-dihy-

droxy vitamin I), and dibutyryl cyclic AMP which induce U937 differentiation enhanced I'M -<expression.

INTRODUCTIONPKC3 serves on a major signal transduction pathway utilized

by a variety of extracellular stimuli (1). PKC encompasses agene family consisting of at least nine members (2). The familycan be divided into two subgroups with the a, ß},ß2,and 7isoforms contained in one group and the 5, e, f, and j;/L isoformscomprising the second category (2, 3). Major differences between the groups reside in substrate specificity and calciumdependency (2-6). The o, «,»;/L,and f isoforms are calciumindependent and at least for eand Ãh́ave a more restricted rangeof substrate phosphorylations as compared to the a, ßt,ß2,and7 group (4-6). Such differences among individual isoforms maybe in part responsible for the diverse cell-specific responseselicited by agents activating the PKC pathway.

We examined the expression of PKC-«in the monoblastoidU937 cell. In response to phorbol ester induced activation ofPKC, the U937 cell ceases to proliferate and differentiates intoa monocyte/macrophage-like cell. In addition to PKC-e, thiscell type contained the a and ßisoforms. After exposure tophorbol esters, PKC-i translocated to the particulate fractionand was degraded in a time- and concentration-dependent fashion. Phorbol esters and other agents inducing U937 differentiation were found to selectively alter expression of individualisoforms.

MATERIALS AND METHODS

Reagents. I2sl-Protein A was purchased from New England Nuclear. The ECL Western blot detection system, goat anti-rabbit horse-

Received9/16/91; accepted8/10/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by NIH Grant CA43023, a Senior InternationalFogarty Fellowship, and American Cancer Society Junior Faculty AwardJFRA230.

2 To whom requests for reprints should be addressed.3 The abbreviations used are: PKC, protein kinase C; KLH, keyhole limpet

hemocyanin; PBS. phosphate-buffered saline; ECL, enhanced chemiluminescence;SDS. sodium dodecyl sulfate; TPA. tetradecanoyl-13-phorbol acetate; PDBu, phorbol 12.13-dibutyrate.

radish peroxidase antiserum, and molecular weight markers were obtained from Amersham. Reagents for antibody purification by proteinA chromatography were purchased from Pierce. KLH and /w-maleim-idobenzoyl-jV-hydroxysuccinimide ester were obtained from Calbio-chem. Penicillin, streptomycin, fetal calf serum RPMI-1640, and 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid were purchased fromGrand Island Biological Company, 1,25-dihydroxy vitamin D3 was generously provided by Dr. M. R. Uskovic (Hoffman-LaRoche, Nutley,NJ). All other reagents were purchased from Sigma.

Cell Culture. U937 and HL-60 cells were maintained in RPMI 1640supplemented with 5% fetal calf serum, 15 HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, 100 /mniis/nil penicillin, and 100 ¿ig/mlstreptomycin as previously described (7).

Preparation of Cell Extracts. Cytosolic and solubilized particulatefractions were prepared by homogenizing cells in either Buffer A [20mm Tris/HCl (pH 7.4), 10 mm EDTA, 5 ITIMethyleneglycol bis(ßaminoethyl ether)-7VyvyVVV-tetraacetic acid, 5 HIM/3-mercaptoetha-nol, 10 HIMbenzamidine, 1 mg/ml leupeptin, 50 ng/m\ phenylmethyl-sulfonyl fluoride, 0.1 mg/ml ovalbumin, and 0.1 units/ml aprotinin] orBuffer B [identical to Buffer A except that the leupeptin concentrationwas reduced to 0.1 mg/ml, and ovalbumin and aprotinin were omitted].The cells were disrupted with 30 strokes in a type A Dounce homoge-nizer and centrifuged at 100,000 x g for 20 min in a Beckman TL-100ultracentrifuge. The cytosol was removed, and the pellet was washedand homogenized in the same buffer with 0.1% Triton X-100. After a20-min incubation at 4°C,the sample was centrifuged at 12,000 x g for

10 min yielding in the supernatant the solubilized particulate fraction.In certain experiments, a whole cell solubilized extract was prepared bydisrupting cells in the homogenization buffer which contained 0.1%Triton X-100. After a 30-min incubation at 4°C,the sample was cen

trifuged at 100,000 x g for 20 min yielding in the supernatant thecytosol and solubilized particulate fraction. Protein content was determined by the method of Bradford (8).

Antisera Preparation. Antisera were prepared using peptides corresponding to the variable regions of the deduced sequences of PKCisoforms [a residues 313-326 (9); 0 residues 313-329 (9); 7 residues306-318 (9); «residues 720-737 (4); and 6 residues 643-657 (10)]. Thea, ß,and 7 peptides were coupled to KLH via m-maleimidobenzoyl-A'-

hydroxysuccinimide ester (11). The «and 6 peptides were coupled toKLH using glutaraldehyde (4, 6). Rabbits were immunized with 1.7 mgof conjugate in complete Freund's adjuvant followed by booster injec

tions in complete adjuvant every 6 weeks. In some cases, the serum waspurified by protein A chromatography according to the instructions ofthe manufacturer (Pierce). The a, ß,and 7 antisera used in the studywere prepared in the laboratory of D. K. W. The 5 antiserum wasgenerated as described (6). The e antisera prepared in both laboratoriesgave identical results.

Western Blot Analysis. Equal concentrations of sample, either 100Mgof protein for cell fractions or 0.6 x IO6cells immediately denaturedby heating in Laemmli stop solution, were subjected to SDS-polyacry-lamide gel electrophoresis using a 10% running gel. Proteins weretransferred to nitrocellulose filters. Western blot analysis was performed as described elsewhere using I25l-protein A to detect antigen-

antibody complexes (12) or by the ECL detection system. For the latterprocess, nonspecific sites were blocked by incubation of the filters withPBS containing 5% low fat dry milk for l h at room temperature. Theappropriate antiserum was incubated with the filter for 12 h at 4°C.The

filters were washed for 3 x 10 min in PBS containing 0.05% Tween 20and incubated with a 1:4000 dilution of goat anti-rabbit horseradishperoxidase antiserum in the same solution except for the addition of 5%

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MODULATION OF PROTEIN KINASE C-< IN U937 CELLS

low fat dry milk, at room temperature for 1 h. Following this incubation, the filter was washed for 10-min periods in PBS and PBS with 0.5M NaCl and PBS. The filters were incubated for 1 min with the ECLdetection solution and the antigen-antibody complexes were detected by

fluorography.Identical results regarding isoform content were obtained using ei

ther the ECL or '"I-protein A detection modality. The antisera dilutions were as follows: a, 1:4000; 0, 1:4000; y, 1:1000; «,1:1000; and a,1:2500.

RESULTS

Using a homogenization buffer containing conventionalquantities of protease inhibitors (0.1 mg/ml leupeptin, 50 jig/mlphenylmethylsulfonyl fluoride, 10 HIMbenzamidine), we wereunable to detect PKC-e in a whole cell solubili/al extract fromU937 cells using Western blot analysis (Fig. 1/4). Detection ofPKC-e in samples of U937 cells prepared by rapid denaturationin a SDS solution indicated that the isoform was present butunderwent degradation after cellular disruption (Fig. Iß).Inclusion of 0.1 mg/ml ovalbumin, 0.1 unit/ml aprotinin andincreasing the leupeptin concentration to 1.0 mg/ml enabled usto detect this isoform in samples that had undergone subcellularfractionation (Fig. 1, C and D). Unlike the a and ßisoforms thatare predominantly cytosolic (see below), PKC-e was evenly dis-

Antiserum:

WholeCellPreparation: Sokibiiized

Sample: Brain U937

Antiserum: i

RapidDenaturation

Preparation: Protocol

Sample: Brain U937

Antiserum:Homogenization Butter:

Preabsorption withImmunizing Peptide:

Cellular Fraction:

58kDa-48.5kDa —¿�

36.5KDa —¿�26.6kDa —¿�

B Antiserum:

Homogenization Butter:

Preabsorption withImmunizing Peptide:

Cellular Fraction:

1 16kDa —¿�84kDa —¿�58kDa —¿�

48.5kDa —¿�

36.5kDa26.6kDa

PCPC PC PC

58kDa-48 5kDa —¿�

36 5KDa -26 6kDa —¿�

58kDa —¿�

48.5kDa —¿�

36.5kDa —¿�

26 ekDa —¿�

Antiserum:

Cellular Fraction: Cytosoi

92kDa-

Antiserum:

Preparation: RDP wcs

84kDa —¿�

SSkDa —¿�

48.5kDa —¿�

36.5kDa —¿�

26.6kDa —¿�

Fig. 1. Detection of PKC-«in U937 cells. In A, 100 jig of whole cell solubilizcdfractions prepared in homogenization buffer B were used for Western blot analysis with PKC-* antiserum. In B, the LJ937 sample was prepared from 0.6 x IO6cells using the rapid denaturation protocol. The autoradiogram in B was exposedapproximately 3 times as long as was the autoradiogram in A. C, PKC-«contentin cytosolic and solubilized paniculate fractions (100 ^g) from U937 cells prepared by homogenization in Buffer A. D. PKC-< content from 11937 cells, compared between samples prepared by the rapid denaturation protocol and whole cellsolubilized fractions prepared by homogenization in Buffer A. Equal proteinconcentrations. 50 ni/lane, of these samples were loaded. Ordinales, molecularweight markers in thousands.

Fig. 2. o and fi isoform localization in the U937 cell. Western blot analysisusing .. and <isoform specific antiserum I I and B. respectively), coupled withpreabsorption of the antiserum with then respective immunizing peptide (seeabove the autoradiogram), was performed on cytosolic (C) and solubilizcd par-ticulate (/'! fractions obtained by homogenization in Buffer A or B. Ordinales,

molecular weight indicators in thousands. These results were duplicated in aseparate experiment.

tributed between cytosolic and paniculate fractions (Fig. 1C).Comparison between identical protein concentrations of samples prepared by the rapid denaturation protocol and homogenization in the buffer containing additional protease inhibitorsindicated that the recovery of PKC-e was greater in samplesprepared by the rapid denaturation protocol (Fig. ID). In someexperiments, a protein doublet with a molecular weight of approximately A/r 90,000 was detected by the PKC-e antiserum(Fig. 1, C and D). In the sample prepared by homogenization, aslightly slower molecular weight and less distinct band forPKC-t was observed (Fig. ID). These data suggest that limitedproteolysis or dephosphorylation may occur during homogenization. Preabsorption with the immunizing peptide of the antisera, prepared in either laboratory, abolished detection of theMr 90,000 protein (data not shown). In contrast to a previousreport (13), we did not detect a PKC-< species in the U937 cellwith a lower molecular weight than that observed using a ratbrain extract as a source for this isoform (Fig. \, B,C, and D).

The «and ßisoforms were also detected in the U937 cell (Fig.2). In contrast to PKC-e which was more evenly distributedbetween these cellular fractions, the «and ßisoforms, whenextracted in the presence of chelating agents, were predominantly localized to the cytosolic fraction (Fig. 2). A similar

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distribution of PKC isoforms was observed in the promyelo-cytic HL-60 cell (data not shown). Unlike PKC-«,the a and 0isoforms were readily detected in cellular extracts prepared inthe absence of 0.1 units/ml aprotinin and 0.1 mg/ml ovalbumin,and with reduction of the leupeptin concentration to 0.1 mg/ml(Fig. 2). While cytosolic a and ßisoform content was similar insamples prepared in either buffer, homogenization in Buffer Aenhanced detection of these isoforms in the solubilized paniculate fractions. The 7 and 6 isoforms were not detected in theU937 cell (data not shown).

Given that differences in degradation of individual isoformsoccurred during preparation of cellular fractions in nonstimu-lated cells, the effect of differing methods of sample preparationwas examined in cells acutely exposed to TPA, an agent knownto enhance degradation of PKC (14). In samples prepared byrapid protein denaturation in an SDS solution, exposure to 600HMTPA for periods of up to 120 min only slightly reducedPKC-/3 content and had negligible effects on PKC-a (Fig. 3A).However, if cellular fractions were prepared using a conven

tional PKC homogenization buffer (Buffer B), the prior in vivoexposure to TPA caused a rapid and complete abolition ofPKC-/3 and a significant reduction in PKC-a content (Fig. 3Ä).

Thus, using certain methods of sample preparation, a portion ofTPA-induced down-regulation occurs not in the intact cell butduring cellular fractionation.

A 15-min exposure to 1 and 10 HMTPA decreased PKC-«inthe cytosol while concomitantly increasing the content of thisisoform in the paniculate fraction (Fig. 4A). Exposure to 100HMTPA for the same duration decreased both the cytosolic andpaniculate content of this isoform. During longer exposures to100 niviTPA, cytosolic PKC-t diminished to negligible levels(Fig. 4B). The paniculate content of this isoform remainedrelatively constant over a 7.5-h exposure to 100 nM TPA. Sincethe loss of cytosolic PKC-«appeared to reflect a dynamic trans-location with consequent degradation, the effect of treatmentwith 100 nM TPA was examined over a more extensive timecourse (Fig. 5). To minimize changes in PKC-e occurring aftercellular disruption, the samples were prepared by immediate

^ Antiserum:

Preparation:

Duration of Exposureto 600nM TPA (min): —¿�

Rapid Denaturation Protocol

15 30 60 90 120

BAntiserum: :

Preparation: Whole Cell Solubilized Fraction

Duration of Exposureto 600nM TPA (min): 15 30 45 60

Antiserum:Preparation:

Duration of Exposureto 600 TPA (min): —¿�

84kDa

58kDa

48.5kDa

Rapid Denaturation Protocol

15 30 60 90 120

—¿� 15 30 45 60

Antiserum:

Preparation: Rapid Denaturation Protocol

Duration of Exposureto 600nM TPA (min): '5 3° «s 60 90 120 -

Antiserum: PPreparation: WholeCell SolubilizedFraction

Duration of Exposureto 600nM TPA (min):

84kDa —¿�

58kDa —¿�

48.5kDa —¿�

Fig. 3. Effect of sample preparation on isoform content in TPA-treated cells. Cells, 5 x IO7, were treated with 600 HMTPA for varying periods of time shown abovethe autoradiograms. Samples for western blot analysis were prepared by rapid protein denaturation in a SDS-solution (A) or homogenization and extraction of a wholecell solubilized fraction in Buffer B (fi). Ordinales, molecular weight indicators in thousands. This experiment was repeated with similar results.

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MODULATION OF PROTEIN KINASE C-i IN U937 CELLS

Antiserum:

Cellular Preparation: CytosolSolubilizedPaniculate

Fraction

TPA Concentration (nM): o i 10 100 o 1 10 100

200kDa -

92kDa -

a more restricted TPA concentration range (0.01-1.0) alsofailed to increase the content of PKC-f (data not shown).

Since exposure to TPA induces U937 differentiation whichcould independently alter the levels of this isoform, the effectsof differentiation alone and other agents inducing U937 maturation on PKC-i were examined. A 72-h exposure to 10 nM1,25-dihydroxy vitamin D, or 0.1 m\i dibutyryl cyclic AMPincreased PKC-f content (Fig. 8). Interferon y or tumor necrosis factor a negligibly altered the content of this isoform, while

69kDa -

B Antiserum: C

Cellular Preparation: Cytosol

Duration of Exposureto 100nM TPA (hr): o i 2 3.5 55 7.50 1 2 3.55.57.5

SolubilizedPaniculate

Fraction

Antiserum:

Cellular Preparation:

Duration of Exposureto 100nM TPA (Hr): 0

200kDa-

RapidDenaturatoci

Protocol

8 24 72

69kDa-92kDa

69kDa-

48kDa-

Fig. 4. Effect of acute exposures of TPA on PKC-< localization. U937 cells, 5x IO7, were exposed to varying concentrations of TPA for 15 min (A) or to 100n.viTPA for varying times (B). Cytosolic and solubilized paniculate fractions wereprepared for Western blot analysis with PKC-i antiserum using homogenizationBuffer A. Ordinales, molecular weight indicators in thousands. Similar resultswere obtained in a second experiment.

placement in preheated SDS stop solution. The total cellularcontent of PKC-f declined slightly over a 6-h exposure to TPAand diminished to negligible levels between an 8- and 24-hincubation with the phorbol ester (Fig. 5).

The effects of ionomycin on PKC-f content were analyzed. Aprevious report indicated that chronic exposure to this calciumionophore decreased PKC-f content (13). Treatment with ionomycin (0.6 MM)for 1 or 24 h did not decrease PKC-f content(Fig. 6). Content of the a and ßisoforms was also unchanged(data not shown). Thus, we were unable to confirm this observation regarding down-regulation of PKC-e by ionomycin.

PKC content was analyzed after a 72-h exposure to varyingTPA concentrations (Fig. 7). Since, in cells treated with vehicleor 0.1 nM TPA, viability of nonadherent cells exceeded 90%,total cells (adherent and nonadherent) were used for Westernblot analysis. Treatment with higher TPA concentrations (>1.0nivi)not only stimulates a percentage of U937 cells to differentiate and adhere to plastic but also induces cell death in asignificant portion of the nonadherent cell population. Underthese circumstances, a substantial portion (40%) of nonadherent cells are nonviable, as assessed by trypan blue exclusion.Thus, only adherent, viable cells were used in samples exposedto > 1.0 HMTPA. Equal numbers of cells (0.6 x IO6) from eachtreatment were loaded per lane. In a concentration-dependentfashion, a 72-h exposure to TPA down-regulated the a, ß,and«isoforms (Fig. 7). The t and ßisoforms were equally sensitiveto this effect, while PKC-a was slightly more resistant to TPA-induced down-regulation. Unlike the a and ßisoforms whichincreased after exposure to 0.1 n%i,PKC-f did not increase inresponse to TPA. A 72-h treatment with 0.001-0.01 nMTPA or

Fig. 5. Effect of chronic exposures of TPA on PKC-< content. U937 cells, 1 x10', were exposed to 100 nMTPA for varying durations. Samples for Western blotanalysis using the <antiserum were prepared by placing the cells in heated SDS-stop solution to rapidly denature proteins. Ordinate, molecular weight indicatorsin thousands. This experiment »asrepeated with similar results.

Sample:

Duration of Exposureto lonomycin (hr):

116kDa-

84kDa-

58kDa-48.5kDa-

36.5kDa-26.6kDa-

U937 Brain

24

Fig. 6. Effect of ionomycin on PKC-< content. Cells, 30 x IO6, were exposed to0.6 (jMionomycin. After 1 or 24 h. whole cell solubilized samples were preparedby homogenization in Buffer A. Equal protein concentrations. 100 tintane, wereloaded and Western blot analysis was done using PKC-< antiserum. Ordinate,molecular weight indicators in thousands. This experiment was repeated on threeoccasions with similar results.

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Antiserum: a

TRA Concentration (nM): o 0.1 1.0 10 100 eoo

116kDa-

84kDa- «.

58kDa-

48.5kDa -

Antiserum: ß

TRA Concentration (nM): o o.t 1.0 10 100 eoo

116kDa -

84kDa-

SBkOa -

48.5kDa -

Antiserum: e

TPA Concentration (nM): o 0.1 1.0 10 100 eoo

116kDa -

84kDa -

SSkDa -

48.5kDa -

Fig. 7. Effect of varying TPA concentrations on PKC isoform content after a72-h exposure. Cells, 1 x IO7, were exposed to varying concentrations of TPA orvehicle for 72 h. In controls and cells exposed to 0.1 nM TPA. nonadhcrent andadherent cells were combined for use in Western blot analysis. In treatmentsutilizing TPA concentrations >1.0 nM. the adherent cells were scraped from theflask and used for analysis. At TPA concentrations > 1.0 nw. a significant decreasein viability occurred in the nonadherent cells. Thus, only adherent cells were usedunder these conditions. The samples, 0.6 X IO6cells/tone, were prepared using therapid denaturation protocol for Western blot analysis using the a (lop), ß(middle)and < (bottom) antisera. The lower molecular weight protein (~M, 58,000) detected by the ft antiserum was nonspecific. The experiment was repeated withsimilar findings. Ordinales, molecular weight in thousands.

1.0 HMTPA abolished its detection. To selectively examine theeffects of differentiation on this isoform, cells were stimulatedto differentiate by exposure to PDBu, a less lipophilic phorbolester. After a 48-h treatment with 100 HMPDBu, the differentiated, adherent cells were extensively washed and placed inPDBu-free medium for 72 h. In U937 cells differentiated in thismanner, PKC-e content was similar to that observed in non-

treated cells (data not shown).

DISCUSSION

Unlike the a and ßisoforms, PKC-e could only be detected ifU937 cells underwent rapid denaturation following cellular disruption, or if the leupeptin concentration was increased to 1mg/ml and aprotinin and ovalbumin were added to the homog-enization buffer. The ability to detect PKC-e in cytosol preparedfrom brain in the absence of increased leupeptin concentrationsand inclusion of aprotinin and ovalbumin may reflect either therelative initial content of the isoform or differences in the quantity of factors responsible for degradation of PKC-e.

The individual isoform content, after an acute exposure toTPA, was influenced by the method of sample preparation.Using a conventional homogenization buffer, content of the aand ßisoforms markedly decreased after an acute exposure toTPA. However, if samples from identically treated cells wereprepared using the rapid denaturation protocol, a and ßisoformcontent was less significantly affected. Thus, in extracts prepared using conventional homogenization buffers, TPA-in-duced degradation occurred not only in the intact cell but alsoafter cellular disruption and during sample preparation. TPA-

induced increases in PKC degradation occurring during samplepreparation have been noted previously in other systems (16).Given these findings, caution must be exercised in ascribingchanges in isoform content as to be solely due to in vivo manipulations.

The a, ß,and e isoforms were down-regulated in a concentration-dependent manner after exposure to TPA. lonomycinexposure did not alter the content of PKC-e. The ßand e isoforms were equally sensitive to TPA-induced down-regulation.The a isoform was more resistant but nevertheless decreased toundetectable levels after exposure to 100 nw TPA for 72 h.These findings differ from a report which demonstrated that thea and e isoforms were resistant to TPA-induced down-regulation in the U937 cell (13). We also found that PKC-a containedin U937 cells was susceptible to TPA-induced down-regulation.Using a second antiserum against a different epitope in PKC-a(17), we also found that PKC-a was depleted to negligible levelsby TPA treatment (data not shown). Except for possible differences in subclones of the U937 cell used in these studies, we areunable to explain the basis for these discrepancies. In the previous report (13), the PKC-e antiserum detected a U937 proteinwith a significantly lower molecular weight than was observedin samples derived from a brain extract. Our antisera, whichwere generated independently in two separate laboratories, specifically detected a poorly resolved protein doublet that essentially comigrated with PKC-t found in brain extracts. The molecular weight of the doublet detected by the PKC-«antiserumin our study is in agreement with the deduced molecular weight

<o

U937 cells contain the a, ß,and e isoforms of PKC. We havealso described the existence of PKC f in the U937 cell (15).

5608

Exposure to:ll6kDa-

84kDa-58kDa-

48.5kDa_

36.5kDa-26.6kDa-

Fig. 8. Effects of differentiation-inducing agents on PKC-t. U937 cells, 1 XIO7, were exposed for 72 h to 10 units/ml ^-interferon (y-INF) 1 ¿ig/mltumornecrosis factor «(TNF-a), 10 nM 1,25-dihydroxy vitamin D3 (D3), 0.1 HIMdibu-tyryl cyclic AMP, or 1 nMTPA. Samples for Western blot analysis were preparedby the rapid denaturation protocol. This experiment was repeated with similarresults.

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MODULATION OF PROTEIN KINASE C-< IN U937 CELLS

of PKC-f (4). We did not detect a lower molecular weight species of PKC-i found in the earlier report (13). Thus, the differences between our observations and those previously published(13) reside in the proteins detected by our respective antisera.

The susceptibility of PKC-«to TPA-induced down-regulationin the U937 cell does not imply that this isoform is equallysusceptible to degradation in other cell types. For instance,PKC-i content is not diminished in murine myeloid cells chronically exposed to 32 ni\t TPA (18). Thus, phorbol ester-induceddown-regulation of PKC isoforms is a function not only of theisoform examined, but also of the cell type and the method usedfor sample preparation.

Despite being activated, as evidenced by translocation to thepaniculate fraction, an 8- to 24-h exposure to TPA was required to observe total down-regulation of PKC-e. While theseresults do not disprove the in vitro observation that activationrenders PKC more susceptible to proteolytic degradation (14),they suggest that in vivo activation is not sufficient to immediately enhance degradation and other processes must occur priorto degradation. For instance, translocation of PKC-f to a specific fraction within the membrane which facilitates interactionbetween the kinase and protease may have to occur prior toPKC degradation. Clearly, further studies will be required toelucidate the in vivo processes mediating PKC down-regulation.

Factors controlling the intracellular distribution of PKC isoforms are not fully understood. Activation is the predominantfactor known to enhance the paniculate association of PKC(19). The finding of a predominantly cytosolic localization ofPKC-i in the GH4 cell (20), while a more even distributionbetween the cytosolic and paniculate fractions in the U937 cell,suggests that factors specific to the nonstimulated U937 cellelicit the membrane association. Furthermore, the selective increase in the paniculate content of PKC-f over the a and ßisoforms in the U937 cell indicate that the factors controllingmembrane association are specific to individual isoforms. Onepossibility is that in the U937 cell, PKC-f undergoes selective

activation by factors present in fetal calf serum or produced inan autocrine fashion which stimulate translocation of the iso-form to the paniculate fraction. Another possibility is thatPKC-e has a longer half-life in the membrane-associated statethan the a and ßisoforms, although the relative rates of down-regulation might suggest otherwise.

The vitamin D and dibutyryl cyclic AMP-induced increase inPKC-f content could be due to mechanisms independent of thedifferentiation process since TPA, which potently induces differentiation, did not increase the content of this isoform. However, TPA-induced acceleration of PKC-e degradation couldobscure observation of an increase in synthesis of this isoform.In an attempt to directly examine the effects of differentiationon PKC-f, cells were induced to differentiate by exposure toPDBu followed by extensive washing and three days maintenance in phorbol ester-free medium. These treatment conditions were chosen to minimize continued exposure of U937cells to the phorbol esters. Under these circumstances, PKC-fcontent was unchanged relative to the undifferentiated state.The similar content of PKC-e under these circumstances suggests that differentiation is not a sufficient stimulus to increaseexpression of this isoform. However, the possibility exists thatthe phorbol ester increased PKC-f expression but the increasewas masked by the presence of PDBu which still contaminatedthe cells despite the extensive washings.

The ability of TPA, at low concentrations, to increase thecontent of the a and ßisoforms, but not PKC-e, indicates that

expression of individual isoforms can be selectively altered.This selective alteration of isoforms provides another potentialmechanism by which PKC-dependent signal transduction may

be regulated.

ACKNOWLEDGMENTS

The authors are indebted to Amanda Wilkinson and Nancy Hammfor their expert editorial assistance in the preparation of this article.

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1992;52:5604-5609. Cancer Res   D. Kirk Ways, Berniece R. Messer, Trudy O. Garris, et al.   Monoblastoid U937 Cell

by Phorbol Esters in theεModulation of Protein Kinase C-

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