EXPERIMENTAL

CELL

RESEARCH

199.111-119

(19%)

Differential Expression of PKC lsoforms and PC1 2 Cell Differentiation MARIE W. WOOTEN Department

of Zoology and Wildlife

Science and Alabama Agricultural

Press,

Station, Auburn

University,

Alabama 36849

treatment of neuroblastoma cells with H7 alone induces morphological and functional differentiation [ll-131. In parallel, a recent study in PC12 cells shows that H7 is capable of blocking NGF-mediated PKC membrane association [14] as well as enhancing the effects of NGF [15]. Taken together, these studies suggest that inhibition of PKC enzymatic activity via the disappearance or a block in PKC translocation plays a direct role in modulating neurogenesis [ll-151. The genes that encode mammalian PKC have recently been cloned [ 161 and consist of a highly homologous gene family comprised of at least six members. Three of the PKC transcripts (Y, &ii, and y are expressed most abundantly [ 161. Each subspecies shows a slightly differential mode of activation and susceptibility to PMA-induced down-regulation, proteolytic fragment formation, cellular distribution, kinetic properties, and substrate specificities, suggesting differential roles within the cell [ 16, 171; however, the precise cellular function for the encoded transcripts is unknown. In addition, no study exists which has examined the role of pharmacological inhibitors, such as H7, in situ on the individual PKC protein isoforms expressed within a given population of cells. Two recent studies have examined both the translocation response of cells treated with H7 [14] and the cellular content of PKC within PC12 cells [ll]. However, since enzymatic activity constitutes the expression of several isoforms, the aforementioned papers have failed to address whether the individual isoforms responded to H7 in a similar or a distinct manner. Isoform heterogeneity could potentially account for the differences in the growth factor requirement for morphological differentiation documented in previous studies between the Neuro 2A and PC12 cells [ll-151. Here I report the effects of H7 on PC12 differentiation as assessed by functional and morphological differentiation. In addition, I have examined these cells with respect to PKC to determine the long-term effects of H7 on both PKC protein and mRNA for three of the encoded transcripts ((Y,@,and y). This study demonstrates that PKC isoforms are capable of selective isotype switching and their expression differs with respect to the inducer used to stimulate differentiation. Moreover, the degree of morphological and functional differentia-

Recent reports indicate that the protein kinase inhibitor H7 is capable of inducing both morphological and functional differentiation of a number of neural cell types. This investigation demonstrates that H7 potentiates the neurogenic properties of nerve growth factor (NGF) in PC12 cells with a concomitant change in the accumulation of the &-protein kinase C (&PKC) isoform protein without changes in either (Yor y. However, NGF alone stimulates a coordinate increase in all three isoforms. The assay of acetylcholine esterase as a functional marker of neuronal differentiation demonstrates that H7 alone is not capable of stimulating morphological or functional differentiation in PC12 cells. H7 synergizes with NGF through a PKC-dependent pathway and by differential expression of PKC subtypes. The expression of the PKC transcripts for (Y, &, and y all undergo simultaneous yet differential changes in their patterns of expression during treatment with H7 and/or NGF. These data suggest that isoform switching is regulated primarily at the protein level. Last, these findings suggest that expression of PKC isoforms is tightly coupled with neuronal differentiation and may play a role in the maintenance of the differentiated state. o 1992 Academic

Experiment

Inc.

INTRODUCTION Pheochromocytoma cells (PC12) have been widely used as a model system for studying the mechanisms associated with neural differentiation [l]. They are chromaffin-like cells which when treated with nerve growth factor (NGF) differentiate into sympathetic neurons [2]. The mechanism by which NGF mediates its cellular effects is unknown, but in part relies on coupling with protein kinase C (PKC) activation [3-51. A number of pharmacologic agents have been reported to inhibit PKC activity [6] and have thus been used to probe for the function of this kinase in receptormediated signal transduction pathways. The isoquinoline sulfonamide, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H7) has become popular in this regard [7,8]. More recently, several laboratories have reported that H7 is capable of mediating changes in cellular morphology [9, lo]. Several studies have demonstrated that 111

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Copyright 0 1992 rights of reproduction

0014-4827/92 $3.00 by Academic Press, Inc. in any form reserved.

112

tion is tightly PKC isoform.

MARIE

coupled with the expression

MATERIALS

AND

W. WOOTEN

of the &-

METHODS

Cell culture and treatment of PC12 cells with H7. Pheochromocytoma cells were obtained from the American Type Culture Collection. They were cultured in RPM1 (Sigma) supplemented with 10% heat-inactivated horse serum (JC Biologics), 5% fetal calf serum, 50 pg/ml streptomycin, and 50 U/ml penicillin. The cells were incubated in a humidified atmosphere containing 8% CO, and 92% air. The cells were grown on cultureware coated with rat-tail collagen (Collaborative Research). The cells were maintained by passage every week at a ratio of 1:8. Cells used in this study were seeded at approximately 60% confluency and well attached. The B 2.5 NGF was obtained from BioProducts for Science and a stock was prepared by dilution in growth media at a concentration of 1 lg/ml for use in experiments. H7 was obtained from Seikagaku America and dissolved in DMSO at a stock concentration of 10 mM. Morphological assessment. At the end point day of treatment, the cells were scored by an individual blind to the experiment. Four random fields of cells were photographed (Olympus phase contrast microscope, 100X) and the percentage of neurite-bearing cells was determined. Typically 300 cells were scored for neurites equal to or greater in length than that of the cell body. A cell body was only scored once, although it may have had more than one process per cell. Acetylcholine esterase assay. To assess the functional differentiation of PC12 cells, acetylcholine esterase activity was assayed as a marker [18, 191. Cells were seeded at 2 X 10” cells onto loo-mm culture dishes in the absence or presence of test reagent. On the day of harvest, the cells were removed from the dish, washed twice with PBS, pH 7.4, and sonicated on ice in 1 ml of a 10 mh4 sodium phosphate buffer, pH 7.4, containing 0.5% Triton X-100. Samples of the homogenates were assayed for acetylcholine esterase (AChE) activity using the colorometric assay of Ellman et al. [18]. The change in absorbance at 420 nm was monitored using a Gilford spectrophotometer with a chart recorder. Protein was determined by using the Bio-Rad Coomassie protein assay kit with bovine serum albumin as the standard. AChE activity is expressed as nmol/min/mg protein. Protein kinase C activity assay. Cells were harvested from the plates and pelleted by centrifugation. The cell pellets were washed 2X in PBS, pH 7.4, and lysed and the total cellular PKC content was determined as previously described [20]. PKC activity (pmol/min/ mg) is expressed as histone kinase phosphotransferase activity obtained in the presence of activators Ca”/phosphatidylserine/diolein minus reactions containing Ca2+ only. All reactions were performed in triplicate. SDS-PAGE/Western blotting. Antibodies to PKC isoform proteins 01,&rr, and y were generously provided by Dr. T. Saitoh (Depart ment of Neurosciences, University of California, San Diego) [21]. Cells were harvested from the plates and pelleted by centrifugation. The cell pellets were washed 2X in PBS, pH 7.4, and lysed by sonication in a 20 mM Tris-HCI, pH 7.6, buffer containing 0.1 mM each of EDTA, NaF, aprotinin, leupeptin, PMSF, benzamidine, 20 mMparanitrophenyl phosphate, and 50 mM 2-mercaptoethanol. After sonication, NP-40 was added to a final concentration of 0.1% and 10 mM EGTA. The cell homogenate was rotated for 1 h at 4°C to release any membrane-associated PKC protein. The protein concentration in the samples was determined using the Bio-Rad kit. One hundred micrograms of protein in Laemmli sample buffer was electrophoresed on a 10% SDS-polyacrylamide gel, blotted to nitrocellulose, blocked with PBS containing 0.1% NP-40 plus 5% nonfat dry milk (NM-PBS) for 2 h at room temperature, and incubated with primary antibody at 1:1500 for 1 h at 4°C. Blots were then washed with NPBS, incubated with 0.5 &i/ml “‘I-protein A (NEN, DuPont) in NM-PBS, and then washed and autoradiographed on Fuji X-ray film at -70°C for 3 days (n and /3) to 1 week (7).

Isolation, quantitation, and blotting of RNA. On the day of harvest the cells were removed from the plates in media, pelleted by centrifugation, and washed 2X with RNAse-free DEPC PBS, pH 7.4. RNA was isolated by using the RNAzol method [22]. The amount and purity of RNA was quantitated by the A,,,,, ratio. RNA with a purity of > 1.9 was used for all experiments. Typically 5-10 pg of RNA was used in all experiments. RNA was transferred to nitrocellulose membranes and subsequently annealled by baking under vacuum at 80°C for 2 h. The membranes were prehybridized at 62°C for 6-10 h in a solution containing: 5X Denhardt’s, 5X SSC, 7% SDS, 200 pg/ml tRNA in a 50 mM sodium phosphate buffer, pH 7.2 [23]. Membranes were hybridized in the same solution containing a “P-random-primed (Boehringer-Manneheim) cDNA probe specific for LY-,p-, or r-PKC (American Type Culture Collection 1241). The cDNA fragment was separated from the vector by a EcoRI digest of the three respective cDNA’s, electrophoresed on a 0.6% low-melt agarose gel, and recovered by GeneClean. Each probe was added at equivalent cpm/ng to the hybridization solution to obtain information regarding the abundance of the encoded transcripts amoung the samples. After 18 h of hybridization, the blots were washed with high stringency first using three washes in lx SSC, 0.1% SDS (55°C) for 1 h followed by a 20-min wash in 0.1x SSC, 0.2% SDS (62°C) and exposed to Fuji X-ray film at -70°C using intensifying screens. The blots were stripped by incubation in 0.1X SSC, 0.5% SDS at 68°C for 1 h followed by prehybridization/hybridization with a radiolabeled cDNA for P-actin. The resulting autoradiograms were scanned using a computer-interfaced scanning system. Representative experiments are presented as graphs reporting relative peak area (total optical density, corrected for background, of the treatment divided by the intensity of fl-actin as an internal control), in arbritrary units of PKC transcript for each sample. The reliability of these measurements was determined by first obtaining multiple autoradiographic exposures to ascertain the peak intensities were within the linear range of the Xray film. In addition, the reliability of this procedure was tested by producing standard curves using varying amounts of RNA from undifferentiated cells. Four curves were produced and analyzed separately, using the same set of standard samples. In all cases, the curves were linear over their entire range (correlation coefficient >0.99). This procedure provides a reliable estimate of relative PKC message expression within these cells.

RESULTS

The effects of H7 on PC12 cells were assessed by visual observation of morphological changes associated with acquisition of neurites. After 3 days treatment of PC12 cells with NGF alone (50 rig/ml), 80% of the cells possessed neurites with a length greater than or equal to the length of the cell body (Fig. 1, 11~). PC12 cells treated with lo-50 &f H7 alone and scored at this time displayed an increasing flattened morphology (Fig. 1, Ia-Ic). Cells that were treated with 50 PM H7 displayed a spiked appearance; however, there was no extension of neurites with increasing concentration of H7 or by changing the medium and adding H7 every 2 days for up to 14 days. In contrast, cells treated with H7 (10-50 PM) in the presence of a suboptimum differentiation concentration of NGF (10 rig/ml; Fig. 1, IIIa-IIIc) possessed well extended neurites, which were greater in lena-th than cells treated with NGF alone. In addition, it wai readily apparent that H7 directly synergized with NGF to induce neurite extension (Fig. 1,111~). As an additional measure of the neurogenic properties of H7, experiments were conducted to quantitate

H7/NGF

AND

PC12 DIFFERENTIATION

FIG. 1. Phase contrast micrographs depicting the effect of H7 on PC12 cell morphology. PC12 cells were treated as indicated in the absence or presence of varying concentrations of NGF and/or H7. After 3 days treatment, the cells were photographed with an Olympus phase contrast microscope. Cells were treated as follows: I (H7 alone) at a (10 p&f), b (20 PM), c (50 PM); II (NGF alone) at a (10 rig/ml), b (30 rig/ml), c (50 rig/ml); and III (NGF 10 rig/ml) plus a (H7 10 PM), b (H7 20 PM), c (H7 50 pA4).

the effects this agent had on neurite extension. Treated cells were scored for the presence of neurites greater than or equal to the length of the cell body at Day 3. These data confirmed visual changes in morphology, documenting that in the presence of the two agents (H7 and NGF) the percentage of PC12 cells bearing neurites was approximately twofold greater than with just NGF alone (Fig. 2). To further examine the effects of H7 on the neurite enhancement properties of NGF, cells treated with H7 were assayed for the presence of a functional biochemi-

cal marker of differentiation, AChE [19]. The presence of H7 alone did not stimulate AChE activity as compared to control untreated cells (Fig. 3). In contrast, in the presence of suboptimal concentrations of NGF (10 rig/ml), H7 synergized with NGF to increase AChE activity over that of either agent alone, comparable to treatment with 50 rig/ml NGF. The increase in AChE activity was directly correlated with the degree of neurogenesis as assessed by the percentage of cells bearing neurites under these conditions (Fig. 1). Previous reports have documented changes in PKC

114

MARIE

W. WOOTEN

-/ Control

H7 lO/.iM

H7 2wM

NGFlO

NGFSO

NGFlO H7 1wM

NGF 10 H7 2OpM

NGF 10 H7 50pM

TREATMENT FIG. 2. Percentage of cells expressing neurites in H7- and NGF-treated PC12 cells. After 3 days, 300 random cells were scored for the presence of neurites by an individual blind to the experimental design. Only cells possessing neurites greater or equal in length to the diameter of the cell body were scored as positive. The data are expressed as X + SEM of three individual experiments in which the number of neurite positive cells were divided by the total (100).

during treatment with H7 [ll, 141. While these studies have demonstrated that H7 is capable of inhibiting the ability of PKC to respond to activation, as assessed by enzyme translocation from the cytosol to the plasma membrane, they have, however, failed to address whether H7 affects the individual isoforms in a differentiatial manner. In this study (data not shown) as well as others [25,26], changes in total PKC activity of PC12 cells treated with NGF over the time course of full morphological differentiation have been documented. The total activity of PKC rises approximately twofold during this period, concomitant with acquisition of the neuronal morphology (M. W. Wooten, unpublished results). Since NGF by itself and suboptimal concentrations of NGF combined with H7 are capable of inducing neurogenesis in PC12 cells, as well as documented changes in PKC activity, experiments were undertaken

to address whether changes in the individual isoforms of PKC could account for the observed differences in PKC activity. Whole cell lysates from treated PC12 cells were analyzed by Western blotting with PKC isoform-specific antibodies, 01,pi, &, and y (Fig. 4). In the case of the pi antibody, no immunoreactive PKC was detected in either unstimulated (control) cells or cells which had been induced with NGF, demonstrating the absence of this isoform in PC12 cells (data not shown). In the presence of NGF alone, all three PKC isoform proteins underwent coordinate dose-dependent changes in expression (Fig. 4, lanes l-4). However, increases in the & isoform protein were more pronounced relative to CYor y. Moreover, the increase in the expression of &-PKC was directly proportional to the concentration and/or length of exposure to NGF (Fig. 4, lanes l-4 compared to lanes

H7/NGF

AND

115

PC12 DIFFERENTIATION

r

H7 l@iM

H72wM

H7 5@M

NGF 10

NGF 30

NGF 50

NGF 10 H7 1QiM

NGF 10 ti72Q1M

NGF 10 H7 5@M

TREATMENT FIG. 3. Acetylcholine esterase assay of PC12 cells treated with H7 and NGF. For measurement of AChE activity, cells were seeded at 2 X 10s onto loo-mm collagen-coated cultureware for 3 days in the presence or absence of test reagent as indicated. The results are represented as X + SEM in which triplicate samples were assayed and are representative of three independent experiments.

13-18). In comparison, treatment with H7 induced little or no change in either (Yor y isoforms (Fig. 4, lanes 5-8). On the other hand, treatment of PC12 cells with H7 for 3 days induced a selective increase in the &-PKC isoform protein (Fig. 4, lanes 5-8), which correlated with the dose of H7 treatment. PC12 cells treated with H7 50 +‘U for 3 days (Fig. 4, lane 8) possessed levels of &-PKC which were similiar to those found in cells treated with NGF (50 rig/ml) for 3 days (Fig. 4, lane 4). In the presence of a suboptimum concentration of NGF (10 rig/ml) and H7 lo-50 pM, an increase was noted in &-PKC expression over that observed with either treatment alone (Fig. 4, lanes 10-12). To quantitate changes in the expression of the PKC isoforms, the autoradiograms (Fig. 4) were scanned with a computer-interfaced densitometer. These data (Table 1) confirm visual observation of the blots in Fig. 4, demonstrating that: (1) in control untreated cells all three PKC isoform proteins are expressed; (2) treatment of PC12 cells with increasing concentrations of NGF stimulated a dose-depen-

dant accumulation of the three isoform proteins; (3) treatment of PC12 cells with H7 alone resulted in a selective increase in the Pi,-PKC isoform; (4) NGF treatment of PC12 cells over the time course of differentiation resulted in coordinate increases in LYand & isoform proteins, with ,& displaying the greatest change in expression; and (5) suboptimal concentrations of NGF (10 rig/ml) plus H7 produced increases in &-PKC over treatment of PC12 cells with either agent alone. H7 was capable of stimulating differential accumulation of PKC isoform proteins and synergizing with NGF to induce morphological differentiation. I hypothesized that the change in isoform proteins was perhaps due to transcriptional induction of the respective isoform gene. Therefore, studies were undertaken to examine the expression of the individual transcripts for (Y, /Iii, and y isoforms in PC12 cells treated with H7 and/or NGF. First, in undifferentiated cells the relative abundance of the individual PKC transcripts was y > (Y > p (Fig. 5). This finding did not parallel the Western blot-

116

W. WOOTEN

MARIE 1234

5

G

7

city between isoform-specific antibodies. Upon treatment of PC12 cells with NGF, the amount of the individual transcript declines. A parallel decline was also noted for H7 treatment (Fig. 5). In contrast, the expression patterns during treatment with NGF/H7 increased for the fl-PKC transcript, while only modest changes were noted for LYand y.

8 9 10 11 12 13 14 15 16 17 16

DISCUSSION

1234567

8 9 10 11 12 13 14 15 16 17 18

-97 466

In this study, I have extended previous observations on the effects of H7 on PKC and neurogenesis in PC12 cells. These results demonstrate that H7 is capable of regulating the amount of PKC transcripts expressed within the cell and this in turn accounts for the parallel increase in both activity and isoform protein levels. Acute, short-term treatment of PC12 cells with H7 is capable of blocking NGF-induced translocation of PKC to the plasma membrane [ 141; however, it is also evident that H7 has an unreported effect via its ability to differentially modify the amount of PKC transcripts present within the cell during long-term exposure. It is possible, since H7 is capable of blocking receptor-mediated translocation of PKC to the plasma membrane [ 141 for degradation by Ca2+-dependent proteases, that this also may account for the accumulation of the PKC protein.

TABLE

1

Effect of H7 and NGF on the Expression of Individual

PKC

Isoform

Proteins

'97

Protein

kinase C isoforms

-66

Treatment

FIG. 4. Expression of PKC isoform proteins from PC12 whole cell lysates in cells treated with H7 or NGF. Cell lysates (100 pg) were probed independently for the expression of the individual isoforms proteins 01,&ii, or y. Note no immunoreactivity toward @,was present in treated or control lysates. PC12 cells were treated for 3 days unless otherwise noted and prepared for SDS-PAGE/Western blotting. Separate replica blots were probed with polyclonal PKC isoform-specific antibodies. Shown are representative autoradiograms of the Western blots for four independent experiments conducted under identical conditions. Concentrations of NGF (N) are in rig/ml, while that of H7 (H) is in ).&4. Lane 1, control; lane 2, NlO, lane 3, N30; lane 4, N50; lane 5, H5; lane 6, Hl0; lane 7, H20; lane 8, H50; lane 9, N10; lane 10, NlO + Hl0; lane 11, NlO + H20; lane 12, N10 + H50; lane 13, N502da; lane 14, N50-4da; lane 15, N50-6da; lane 16, N50-8da; lane 17, N50-10da; lane 18, N50-14da.

ting data where the expression of isoform proteins was found to be @> a! > y. However, it should be noted that this might be due to differences in the immunospecifi-

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Control N IO-3 da N 30-3 da N 50-3 da H7-5 PM-3 da H7-10 PM-3 da H7-20 pM-3 da H7-50 pM-3 da N IO-3 da N 10 + H7 10-3 da N 10 + H7 20-3 da N 10 + H7 50-3 da N 50-2 da N 50-4 da N 50-6 da N 50-8 da N 50-10 da N 50-14 da

(Y

011

Y

23 36 52 49 23 23 23 23 23 23 20 43 32 a2 130 *

37 131 221 239 163 81 144 188 139 121 133 218 140 236 242 *

40 59 61 66 40 40 40 40 40 40 40 40 40 20 23 *

120 106

204 162

37

Note. Replica Western blots probed with isoform-specific antibodies to 01,&,, and y. PKC (Fig. 4) were scanned with a computer-interfaced densitometer. Arbritrary units for the individual treatments are reported. N, concentration of NGF in rig/ml; da, length of exposure to agent in days. * This sample was not scanned due to protein underload in this respective sample.

H7/NGF

AND

117

PC12 DIFFERENTIATION

Alpha

Beta

H72i&M

H750yrM

NGFlO

I

NGF30

NGFlO H72QA

NGFlO H75Q.1t.4

FIG. 5. Expression of PKC isoform-specific transcripts in PC12 cells treated with various agents. Cells were exposed to the indicated reagents for 3 days prior to isolation of mRNA, followed by analysis of the levels of expression for the three PKC isoform transcripts. The resulting autoradiograms were quantitatively analyzed using a computer-interfaced scanning system to determine the effect which various agents have upon the expression of individual PKC mRNA’s. The data are plotted as relative expression of each individual PKC transcript, corrected for the expression of @-actin as an internal control. The results are representative of five independent experiments conducted under identical conditions.

A similar accumulation of PKC isoform protein was noted during treatment with NGF alone and therefore accumulation of PKC may represent a coupling to the process of differentiation as well as being regulated by protein turnover mechanisms. This study differs with respect to those conducted in neuroblastoma cells [ 11-131. First, in these cells a rela-

tively high concentration of H7 was used to stimulate differentiation ~200 pM. Second, in that system H7 alone is capable of inducing neurogenesis. However, it should also be noted that in Neuro-2A cells, NGF is not required for morphological or functional differentiation. In this study, no direct effect of H7 treatment alone is evident for morphological differentiation. Al-

118

MARIE

W

though at concentrations between 20 and 50 pM the cells had a spiked appearance, however, extended neurites were never present (up to 14 days). In addition, AChE activity was similar to control values in H7treated cells alone. The role of PKC in differentiation has not been as well studied as its role in proliferation. In cell lines such as HL60, phorbol 12-myristate 13-acetate (PMA) can induce down-regulation of PKC activity with concomitant morphological differentiation [27]. However, other compounds which are capable of inducing differentiation in this system do not down-regulate PKC, but rather stimulate an accumulation in PKC activity [28]. Thus, while PMA may be a useful tool in modulating PKC activities and in mediating cell growth, the role of PKC in differentiation may be somewhat more complex. This hypothesis is supported by observations in HL60 cells where PKC activity increases during differentiation are attributed to changes in specific PKC isoforms [29]. Likewise, reports in the literature are present which document that NGF itself is capable of eliciting increases in PKC activities during the course of neurogenesis in PC12 cells [25,26] (M. W. Wooten, unpublished results). Studies by Hashimoto et al. [30] have clearly restricted the expression of the r-PKC transcript to neural tissue. Thus, it is not surprising that this isoform is expressed in PC12 cells which are of neural crest origin. The data herein demonstrate a correlation between the differentiated phenotype and the accumulation of the Pi,-isoform protein. This study parallels a recent finding of Abraham et al. [31] documenting increases in /3-PKC during differentiation of neuronal NT2/Dl cells by retinoic acid. Likewise, they noted a simultaneous increase in a-PKC [31]. Therefore, it appears that acquisition of neurites is tightly coupled to the expression and accumulation of these two isoforms. It is worth noting that a recent study in MEL cells has demonstrated that direct introduction of the &PKC isoform is capable of stimulating enhanced differentiation of these cells as well as acquisition of differentiated markers [32]. Similarly, it has been noted that increases in P-PKC in MEL cells lead to faster rates of HMBAinduced differentiation [ 331. It will be of future interest to examine the localization of P-PKC protein with respect to its temporal distribution pattern during neurite extension in PC12 cells. Several proteins which participate in neurofilament reorganization during differentiation of PC12 cells are themselves substrates for PKC [34, 351. Thus, it is possible that both the &PKC isoform and particular substrates colocalize during neurite extension and that proximal distribution of the kinase and substrate plays a critical role in the acquisition of neuronal morphology. Recent studies with both the CAMP- and cGMP-dependent protein kinase support the concept that the spatial dis-

WOOTEN

tribution of kinase and substrate plays a key role in the cellular function of a respective protein kinase [36, 371. This is an attractive model worth consideration in regard to the function of the fi-PKC isoform protein and its accumulation during neuronal differentiation. The expression of both a and y proteins was unchanged during treatment with H7. These results parallel other observations demonstrating that specific inducers are capable of eliciting differential changes in the individual isoforms [29, 31-331. Moreover, the results from this study demonstrate that PKC isoform protein levels cannot be predicted from their respective mRNA levels. Similar discordance between PKC protein and mRNA levels has been demonstrated previously in two different systems expressing two or more PKC isoforms [29, 311. In addition, this study further strengthens the hypothesis that PKC activity may be regulated primarily at the protein level rather than the transcriptional level. These results clearly demonstrate modulation of PKC isoform proteins and mRNA levels via specific induction agents and support the notion that each of these isotypes plays a discriminate role in the cell. Moreover, this study demonstrates that each isoform is capable of both selective and differential modulation in response to differentiation induction. It will be of interest for future studies to ascertain if situations exist under which the a and y transcripts and isoform proteins may be modulated and their role in relationship to differentiation and neurogenesis determined. I am endebted to M. L. Seibenhener for technical assistance, to K. R. White for the initial observations which led to this study, and to the members of my laboratory for helpful discussions regarding this work. This work was supported in part by funds from the AAES (Project 740, Journal 15-913081) and PHS Grants CA49647 and AA08753.

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Received August 14, 1991 Revised version received October 28, 1991

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