©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Alternative Splicing of Protein Kinase C by Insulin (*)

Charles E. Chalfant (1), Harald Mischak (5)(§), James E. Watson (3), Bruce C. Winkler (4), Joanne Goodnight (5), Robert V. Farese (1) (2) (3), Denise R. Cooper (1) (2) (3)(¶)

From the (1) Departments of Biochemistry and Molecular Biology and (2) Internal Medicine, University of South Florida College of Medicine and the (3) James A. Haley Veterans Hospital, Tampa, Florida 33612, the (4) Department of Chemistry, University of Tampa, Tampa, Florida 33606, and the (5) National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin regulates a diverse array of cellular signaling processes involved in the control of growth, differentiation, and cellular metabolism. Insulin increases glucose transport via a protein kinase C (PKC)-dependent pathway in BC3H-1 myocytes, but the function of specific PKC isozymes in insulin action has not been elucidated. Two isoforms of PKC result via alternative splicing of precursor mRNA. As now shown, both isoforms are present in BC3H-1 myocytes, and insulin induces alternative splicing of the PKC mRNA thereby switching expression from PKCI to PKCII mRNA. This effect occurs rapidly (15 min after insulin treatment) and is dose-dependent. The switch in mRNA is reflected by increases in the protein levels of PKCII. High levels of 12-0-tetradecanoylphorbol-13-acetate, which are commonly used to deplete or down-regulate PKC in cells, also induce the switch to PKCII mRNA following overnight treatment, and protein levels of PKCII reflected mRNA increases. To investigate the functional importance of the shift in PKC isoform expression, stable transfectants of NIH-3T3 fibroblasts overexpressing PKCI and PKCII were established. The overexpression of PKCII but not PKCI in NIH-3T3 cells significantly enhanced insulin effects on glucose transport. This suggests that PKCII may be more selective than PKCI for enhancing the glucose transport effects of insulin in at least certain cells and, furthermore, that insulin can regulate the expression of PKCII by alternative mRNA splicing.


INTRODUCTION

Protein kinase C (PKC)() mediates cellular responses elicited by hormones, neurotransmitters, and growth factors. Molecular cloning has revealed that PKC represents a multigene family, to date consisting of 11 different isozymes encoded by 10 distinct genes. Depending on the cofactor requirements, PKCs can be subdivided into three groups: classical PKCs (, I, II, ), which require diacylglycerol, phospholipids, and Ca for full activity; novel PKCs (, , , ), which are phospholipid and diacylglycerol-dependent but Ca-independent; and atypical PKCs (, (), µ), which require only phospholipids (for review, see Refs. 1-3). Activation of classical and novel PKCs involves their translocation from the cytoplasm to various cell membranes and cytoskeletal structures after binding of diacylglycerol, which is generated by agonist-induced hydrolysis of phosphatidylinositol bisphosphate, phosphatidylcholine, a phosphatidylinositol-containing glycan, or from the de novo synthesis of phosphatidic acid (4) .

Insulin activates and translocates PKC in BC3H-1 myocytes, a nonfusing murine cell line with many phenotypic characteristics of muscle cells (5, 6, 7, 8) . Insulin stimulation of glucose uptake in myocytes is inhibited by PKC inhibitors (9) . Treatment of BC3H-1 myocytes overnight with TPA failed to down-regulate insulin-stimulated glucose uptake (6, 7, 8, 10) . The retention of PKC-dependent vinculin phosphorylation following TPA treatment suggested that the remaining apparent PKC (as per hydroxyapatite column purification and immunoreactivity) could explain the continued response of these cells to insulin (6, 7) .

The PKC gene encodes two mRNAs that originate from alternative splicing of the C-terminal exons (11) . The resulting polypeptides, PKCI and PKCII, diverge in the sequence of their C-terminal 50 or 52 amino acids, respectively (11, 12) . Fig. 1provides a schematic of the functional organization of PKC. The expression of different polypeptides and prediction they will occur from the same gene by alternative splicing has been observed for a growing number of genes including fibronectin (13) , the insulin receptor (14) , and a nontransmembrane phosphotyrosine phosphatase (15) . In these cases, alternative splicing patterns are influenced by stages of development, differentiation, growth factors, or disease. The functional significance of alternatively spliced peptides is not fully understood.


Figure 1: Structure of PKCI and -II gene products. A schematic representation of PKC as deduced from cDNA sequence analysis (40) is shown. PKCI and -II are designated as the 671- and 673-amino acid products, respectively. Coding sequences are represented by blocks. C1-C4 represent the conserved regions. V1-V5 are the variable domains. C1 and C2 form the regulatory domain (shaded). Cysteine-rich zinc finger domains are indicated by ovals. The V5, variable region is shaded. Regions corresponding to the PKCI probe used for the RNase protection assay and location of the primers used for RT-PCR analysis are indicated below the scheme. The numbering of nucleotides corresponds to that of Knopf et al. (41).



Differential expression of both PKC messages has been demonstrated in brain, hemopoietic cells, B lymphoblastoid cells, and cardiac myocytes (11, 12, 16, 17) . Immunochemical methods with specific PKCI, -II, and pan-specific antibodies demonstrate that both isoforms are present in these cells and tissues as well as in rat soleus muscle, rat adipocytes, and BC3H-1 murine myocytes (6, 18, 19) . These studies did not, however, evaluate whether expression is regulated in vitro and in vivo. We found that the levels of mRNA for PKCI declined quickly with insulin treatment, and PKCII mRNA levels increased, concomitantly, along with protein levels of PKCII in BC3H-1 myocytes. High levels of TPA mimicked insulin, and PKCII was the major isoform of PKC retained in BC3H-1 myocytes following chronic TPA treatment. To determine if PKCI and -II function differently in response to insulin, both were stably overexpressed in NIH-3T3 fibroblasts, which normally express PKC and only negligible levels of PKC. PKCII overexpression enhanced insulin-stimulated 2-DOG uptake significantly above empty vector control cells or stable transfectants overexpressing PKCI. Thus, one alternatively spliced form may be more specific than the other for increasing glucose transport responses in certain cell types.


MATERIALS AND METHODS

Cell Culture

BC3H-1 myocytes were grown in low glucose Dulbecco's modified Eagle's medium with 10% controlled process serum replacement (Sigma) for 11-14 days on collagen-coated 100-mm plates. Glucose (25 mM) was added to culture media for 24-48 h prior to treatment with varying concentrations of insulin or TPA (Sigma) as indicated prior to isolation of total RNA or cell lysates.

Isolation of RNA, Ribonuclease Protection Analysis (RPA), and RNA Half-life

Total cellular RNA was obtained using a single-step method (20) . For determining mRNA half-life, cells were treated with actinomycin D (5 µg/ml, Sigma) alone or actinomycin D and insulin. For RPA, the PstI fragment of PKCI (BIV5) was subcloned into pGEM-4Z (Fig. 1). Based on predicted splice events (16), PKCI-specific transcripts yield a 716-base pair protected band, while PKCII encoded mRNA is detected as a 566-base pair band. Antisense PKCI was transcribed with T7 polymerase and labeled with [-P]UTP using the Promega RNA transcription kit. Total RNA (10 µg) was hybridized with the P-labeled BIV5 cRNA probe (1.2-5 10 cpm/hybridization) using the RPA II ribonuclease protection assay kit as described (Ambion). Protected RNA fragments were detected following electrophoresis on denaturing polyacrylamide gel (5% polyacrylamide, 8 M urea). The sizes of the probe and protected fragments were verified by 5`-labeled X174 DNA/HinfI ladder. Hybridized fragments were detected by autoradiography.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Generation of single-stranded cDNA templates for RT-PCR was carried out on BC3H-1 myocyte total RNA using SuperScript preamplification kit (Life Technologies, Inc.). The cDNA was aliquoted, and three reactions were amplified: (a) sense primers corresponding to the V3 region of PKC (ATGAAACTGACCGATTTTAACTTCCTG) and antisense primers corresponding to the V5 region of PKCI (AAGAGTTTGTCAGTGGGAGTCAGTTCC), (b) sense primers to V3 region and antisense primers corresponding to the V5 region of PKCII (CGGAGGTCTACAGATCTACTTAGCTCT), and (c) sense and antisense primers for -actin (Clontech). For all primer pairs, cycling conditions were an initial 96 °C melt, 5 min, and then 95 °C, 1 min; 58 °C, 1 min; 72 °C, 3 min. This cycle was repeated 34 times ending with 95 °C, 1 min; 58 °C, 1 min; and 72 °C, 10 min. Specificity of the primers was verified with cDNA for PKCI and PKCII (from Dr. John Knopf, Genetics Institute, Cambridge, MA).

Western Blot Analysis

Cell lysates (50 µg) from BC3H-1 myocytes and NIH-3T3 cells overexpressing PKCI and II were analyzed as described previously (21, 22) . PKCI antibodies were obtained from Research and Diagnostic Antibodies, from Drs. Susan Jaken and Susan Kiley (W. Alton Jones Cell Science Center, Lake Placid, NY), or from Dr. Yusaf Hannun (Duke University Medical Center, Durham, NC). PKCII antibodies were provided by Dr. D. Kirk Ways (East Carolina University, Greenville, NC) or Drs. Jaken and Kiley. Following incubation with primary antibody, blots were developed as described previously (23) or with the ECL kit (Amersham Corp.). The PKC antisera were characterized previously (16, 17) . Specificity of PKCII antisera obtained from Dr. D. Kirk Ways for PKCII in BC3H-1 myocytes was performed by preabsorption of antisera with peptides.

Overexpression of PKCI and -II in NIH 3T3 Cells

NIH-3T3 cells were stably transfected as described with MTHII (24) or MV7I (25) using Lipofectin (Life Technologies, Inc.). Stable transfectants were selected in the presence of 750 µg/ml G418 (Life Technologies, Inc.), and the more than 100 clones obtained were cultured in bulk cultures. In the case of PKCII, data from one stable clone is also shown with the stable bulk transfected cells. Stable transfectants are grown in high glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Overexpression was evaluated by Western blot analysis and by PKC activity toward histone III-S (Sigma) as described previously (21) . 2-[1,2-H]Deoxy-D-glucose uptake-NIH-3T3 cells overexpressing PKCI and -II were grown in high glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Sigma) in 24-well plates until >80% confluent. Prior to 2-[H]DOG uptake, cells are switched to Dulbecco's modified Eagle's medium without serum overnight. 2-[H]DOG uptake is assayed as described previously (21) . 2-Deoxyglucose uptake refers to transport across the plasma membrane operating in tandem with phosphorylation by hexokinase.


RESULTS

PKCI mRNA Switches to PKCII mRNA in the Presence of Insulin

To simultaneously examine PKCI and -II mRNA in BC3H-1 myocytes, we used a cRNA probe spanning the alternative splice site for PKCI and -II for RPA (Fig. 1). When BC3H-1 myocytes were treated overnight with increasing concentrations of insulin, cellular expression switched PKC mRNA from the PKCI to PKCII mRNA as demonstrated by the appearance of the shorter, 566-base pair, protected fragment (Fig. 2A). This suggested that insulin regulated alternative splicing of PKC mRNA. This phenomenon was dose-dependent and was apparent at 0.2 nM insulin. Initially, PKCI was the primary mRNA detected by this assay. In the presence of insulin, PKCII mRNA became the primary splice product. A time course showed that the PKCI to PKCII mRNA shift was complete after 15 min (Fig. 2B).


Figure 2: RNase protection analysis of PKCI and II mRNA in BC3H-1 myocytes after insulin treatment. PanelA, RNase protection analysis of BC3H-1 myocytes following treatment with insulin. Glucose (25 mM) was added to culture media prior to treatment with varying concentrations of insulin overnight prior to isolation and analysis of total RNA (10 µg) as described under ``Materials and Methods.'' Y1 and Y2 refer to the undigested and digested probe. Panel B, cells were treated with insulin (200 nM) for 15 min to 1 h prior to isolation of total RNA. The data shown are representative of more than five separate experiments.



Demonstration of PKCI and PKCII mRNA Switch by RT-PCR

To establish that insulin regulated the expression of PKCI and -II mRNA using another method, the alternative splice products were examined by RT-PCR (Fig. 1). This assay allows for the relative evaluation of PKCI and -II mRNA, but it is not quantitative. The amplification spans several splice sites in mature PKC mRNA and eliminates amplification of any precursor mRNA forms. As observed with the RPA, the switch from PKCI to PKCII mRNA was complete after 15 min following insulin treatment (Fig. 3). With this assay, both PKCI and -II mRNA were detected in unstimulated BC3H-1 myocytes, but only minor traces of PKCI mRNA could be detected in cells that had been incubated with insulin for up to 60 min.


Figure 3: RT-PCR analysis of BC3H-1 myocyte PKCI and PKCII mRNA. Total RNA (1 µg) was subjected to RT-PCR. Polymerase chain reaction fragments were fractionated on agarose gels and transferred to nylon for verification of products by Southern blot using a P-labeled full-length PKCI/II cDNA probe as shown. The data shown are representative of three separate experiments.



Determination of Insulin Effect on PKCI mRNA Stability

The rapid disappearance of the PKCI mRNA in the presence of insulin suggests that another event accompanying the switch to PKCII mRNA is the destabilization of PKCI mRNA. To evaluate this, cells were treated with actinomycin D alone or with insulin for varying times. In the presence of insulin, PKCI mRNA was decreased by 75% after 15 min, demonstrating that the effect of insulin on PKCI mRNA was to destabilize the message (Fig. 4). In the absence of insulin, PKCI message was only detected for up to 1 h in cells treated with actinomycin D. This indicates that message stability may play some role in regulating differential expression of PKCI mRNA.


Figure 4: Message stability of PKCI mRNA in BC3H-1 myocytes. Total RNA from BC3H-1 myocytes previously treated with 5 µg/ml actinomycin D or actinomycin D with insulin (200 nM) for 15 min was analyzed by RPA with the BIV5 probe cut with BbuI. Only PKC I is evaluated with this probe. Autoradiograms were analyzed by densitometry.



Phorbol Ester Mimicked Insulin in Switching the Splice Product at Concentrations Exceeding 1 µM

To determine whether the activation and subsequent down-regulation of PKC was involved in PKC alternative splicing, PKC was directly activated with TPA. Under these conditions, we have shown that BC3H-1 myocytes appear to retain PKC immunoreactive material (7, 8) . TPA treatment increased PKCI mRNA levels by 50% at lower concentrations, which is not surprising, as TPA induces the basal promoter of PKC (26) . However, at higher concentrations of TPA, 1-5 µM, alternative splicing of PKCI to PKCII was induced (Fig. 5).


Figure 5: Effect of TPA on PKCI and PKCII mRNA. Total RNA from BC3H-1 myocytes cultured overnight in the presence of 1 nM to 5 µM TPA or 0.01% dimethyl sulfoxide (control vehicle) was assayed by RPA using the BIV5 probe. The effect of TPA was noted in three separate experiments.



Demonstration of Increased PKCII Protein Levels with Insulin and TPA Treatment

To determine whether the shift from PKCI to PKCII mRNA following insulin treatment was reflected at the protein level, lysates from insulin-treated BC3H-1 myocytes were analyzed. Very low levels of PKCII protein (a single 82 kDa band) were detected in untreated cells, but levels increased more than 5-fold after 30 min of treatment and more than 12-fold following 24 h of insulin treatment (Fig. 6). The induction of PKCII by insulin was apparent using antibodies from Drs. Jaken and Kiley and Dr. Ways. PKCI, the predominant PKC mRNA and protein form in resting BC3H-1 myocytes, diminished to half of its initial level with insulin treatment. PKCI was detected as a doublet at 74-76 kDa and sometimes as another doublet at 80-82 kDa in whole cell lysates with antibodies from Drs. Jaken and Kiley and Dr. Hannun, Life Technologies, Inc. (affinity purified), and Research and Diagnostic Antibodies. A PKCII immunoreactive band (at 80-82 kDa) was demonstrated in BC3H-1 myocytes following overnight treatment with 5 µM TPA with antisera from Dr. Ways (Fig. 7) and Drs. Jaken and Kiley (data not shown). PKCI levels (at 74-76 kDa) were diminished by more than 80% following TPA treatment as detected with antibody from Life Technologies, Inc. that recognizes the (V3) region.() The greater relative diminution of PKCI following treatment with TPA versus insulin may reflect the ability of TPA to activate PKCI while insulin may preferentially stimulate PKCII under these conditions, and insulin may not enhance the proteolytic turnover of PKCI to the same extent as TPA. Thus, the switch from PKCI to PKCII mRNA is paralleled by the increased expression of PKCII.


Figure 6: Effect of insulin on PKCI and PKCII protein expression in BC3H-1 myocytes. BC3H-1 myocytes were treated with insulin (200 nM) as indicated prior to the addition of Laemmli lysis buffer to washed cell monolayers. Results of densitometric scans are shown with insets of the Western blot data using PKCI (lower band analyzed) and PKCII antibodies from Drs. Susan Jaken and Sue Kiley as described under ``Materials and Methods.'' Similar patterns for PKCI (a doublet) and PKCII were noted in three separate passages of cells with three different antibodies to PKCI and two antibodies to PKCII.




Figure 7: Effect of phorbol esters on PKCI and PKCII protein expression in BC3H-1 myocytes. BC3H-1 myocytes were treated with vehicle (A) or 5 µM TPA (B) overnight. Cytosolic (75 and 60 µg of protein for PKCI and PKCII, respectively) and membrane (150 and 40 µg of protein for PKCI and PKCII, respectively) fractions were prepared for Western blot analysis using alkaline phosphatase detection as described previously (21). Antibody against PKC hinge region (Life Technologies, Inc.) was used here to detect PKCI as the lower, 74 kDa, band. (This antibody does not detect PKCII unless chemiluminesence is used for detection.) PKCII antibody was from Dr. D. Kirk Ways. Similar decreases in PKCI and increases in the upper PKCII band were detectable using the Life Technologies, Inc. PKC antibody with ECL detection. The 80 kDa marker is indicated by .



The increase in PKCII protein with insulin occurs rapidly, and its induction may play an important role in glucose transport. The functional significance of this finding is difficult to address in the BC3H-1 myocytes due to the expression of PKC and other PKC isoforms (6). There is also the potential for counter-regulation of these PKC isoforms and glucose transporter levels with insulin. We found that the insulin effect on glucose uptake was enhanced in TPA-treated BC3H-1 myocytes even though acute TPA stimulatory effects were no longer observed. For example, we found 789 ± 20 cpm of 2-[H]DOG uptake/well for basal uptake versus 1749 ± 35 cpm for insulin (200 nM), and, following 1 µM TPA treatment overnight, we found 1161 ± 69 cpm for basal versus 3609 ± 139 cpm for insulin-treated cells.

Potential Functional Significance of the Switch from PKCI to PKCII

The observation that PKC is an insulin responsive PKC isozyme infers that the switch from PKCI to PKCII expression induced by insulin or high concentrations of phorbol ester may be of functional significance for glucose uptake. If PKCII protein expression reflects an increase in the alternative expression of PKCII mRNA, one might predict that insulin-stimulated glucose transport will be increased when PKCII is increased. Although we found that insulin effects on glucose uptake were retained and enhanced by chronic TPA treatment, it was also evident that another PKC isozyme, PKC, was down-regulated in myocytes (6, 7) . To avoid the effects of down-regulating PKC and other isozymes in BC3H-1 myocytes, we used NIH-3T3 cells overexpressing PKCI and PKCII to examine the effects of increased PKCII. PKC and negligible amounts of PKC are expressed in parent NIH-3T3 cells used here (27) , thus allowing us to assess the role of PKC isoforms without encountering significant endogenous PKC as detected in BC3H-1 myocytes. Western blot analysis of transfected NIH-3T3 cells demonstrated the stable overexpression of PKCI and PKCII (Fig. 8). PKC activity as determined with histone III-S as substrate was increased 2-fold in cytosol and 3-4-fold in membranes compared with empty vector control cell lines for both isozymes.


Figure 8: Characterization of PKCI and PKCII protein overexpression in stably transfected NIH-3T3 cells by Western blot analysis. Cell lysates (50 µg) from NIH-3T3 cells overexpressing PKCI and PKCII were prepared as described previously (7). Antisera to PKCI was from Research and Diagnostics, and antiserum for PKCII was from Dr. D. Kirk Ways. LanesA, C, and E denote the empty vector control cell lines; laneB represents transfected cells overexpressing PKCI; laneD is from transfected cells overexpressing PKCII; and laneF is a clonal line (clone 10) selected from the batch transfection of PKCII.



We previously found in adipocytes and BC3H-1 myocytes that PKC contributes to insulin-stimulated glucose transport (28, 29) . In NIH-3T3 cells, we found that 1 and 10 nM insulin increases glucose uptake by 10 and 30%, respectively, in parental or control cells (). This effect of insulin is statistically significant and may reflect a ``PKC component'' of glucose transport in NIH-3T3 cells. Cells overexpressing PKCI did not respond differently than control cells in that insulin-stimulated 2-DOG uptake remained at 10 and 30% above basal uptake. Cells overexpressing PKCII demonstrated enhanced, dose-dependent (1-10 nM), insulin-stimulated 2-DOG uptake of 60 and 100%, respectively, above basal uptake. This enhanced response was also observed in the clonal line overexpressing PKCII as well as in stable bulk cultures of PKCII transfectants. The insulin effect is PKC-dependent as indicated by inhibition of 2-DOG uptake with GF 109203X (Calbiochem) and CGP 41251 (Ciba Giegy), specific PKC inhibitors (30, 31) . These results indicate that PKCII but not PKCI is capable of further enhancing insulin-stimulated glucose transport in NIH-3T3 cells and further suggests a physiological significance in the regulation of PKC alternative splicing.


DISCUSSION

To our knowledge, this is the first report of a peptide hormone regulating alternative splicing of mRNA within minutes. Furthermore, the protein product of the alternatively spliced mRNA turns out to be a relevant intracellular signaling component for the hormone, insulin. This data sheds new light on the details of insulin signaling as well as PKC isozyme function. The alternatively spliced PKC exon detected here corresponds to the C-terminal variable region, V5, which is believed to determine substrate specificity (1) .

The regulation of alternative splicing involves a switch in mRNA processing (11) . Here we demonstrated a change in alternatively spliced products using a probe that overlaps the C4 or catalytic region and the alternative splice site. By selecting a region of PKC spanning the area of the mRNA, which is alternatively spliced, both PKCI and PKCII mRNA are detected in the same sample following RNase digestion. Probes directed solely to the V5 region or downstream untranslated region can hybridize with precursor PKCI mRNA in addition to alternatively spliced mRNA, and the detection of a splicing event is not obvious. We further supported our observation by amplifying mRNA by RT-PCR. The ability to detect PKCII mRNA in untreated cells using RT-PCR is attributed to the superior sensitivity of the RT-PCR method to amplify low message levels. Using both methods, we found that PKCI mRNA levels quickly diminish with insulin treatment and, in contrast, PKCII mRNA levels increase.

One explanation for the decrease in PKCI mRNA may be that insulin diminishes message stability. Although this was found to be the case in experiments where cells were treated with actinomycin D, message destabilization does not explain why PKCII mRNA levels are then increased, and it is clear that alternative splicing to the default product, PKCII, is occurring. One scheme of alternative splicing for PKC is that retention of the PKCII exon results in PKCII mRNA. When this exon is excised, PKCI is encoded, and the PKCI polyadenylation site was used by both PKCI and PKCII transcripts (11) . A likely role for insulin might involve regulation of splicing factors required for the mRNA splicing complex, and the factors involved in alternative pre-mRNA splicing are possibly induced by insulin (32) . One such factor was isolated from regenerating liver (33). It is of interest that insulin regulates alternative splicing of insulin receptor isoforms in hepatic FAO cells similar to the time course we see in the BC3H-1 myocyte for PKC splicing (34) . Changes in alternative splicing of the insulin receptor is also reported in a diabetic patient (14) .

These studies further demonstrate the expression of PKCI and PKCII by BC3H-1 myocytes. Using two techniques for detecting mRNA and six different antibodies, we found mRNA and protein PKC levels are regulated by insulin and phorbol ester. Recent studies from other labs failed to detect PKCI or PKCII in BC3H-1 myocytes (35) . Moreover, as will be reported later, PKCI was not only fully down-regulated by overnight TPA treatment, but it was acutely translocated by TPA (as well as by insulin). The failure of Stumpo et al.(35) to detect PKCI in untreated BC3H-1 myocytes may be due to differences in antisera, detection techniques or cell culture. The chemiluminesence method used here may be more sensitive for detecting PKCI. In our studies, PKCI appears at a lower apparent molecular mass, 74-76 kDa, than PKCII, which migrates at 80-82 kDa. PKCI and II isoforms were often detected as doublets. The difference in apparent molecular masses between the two isozymes may reflect a number of cellular phosphorylations that PKC may undergo in the processing of newly translated protein or limited proteolysis of PKCI as suggested (36) . In addition, we frequently change our culture stocks, and cells are serum-starved prior to experiments.

Our PKC protein analyses of myocyte lysates are similar to studies with IM-9 and BJA-B cell lines, in which PKCII migrated as an upper 77 kDa and lower 74 kDa band (16) . PKC I migrated at a lower molecular mass in that study. The upper band did not down-regulate with long term phorbol ester treatment (16) . In our studies, a similar upper band was induced with TPA and insulin in BC3H-1 myocytes and is consistent with a switch from PKCI to PKCII. In these studies it was not possible to compare the amounts of PKCI with PKCII. However, the relative changes in PKCI and PKCII with insulin and phorbol ester reflect changes related to mRNA levels.

In our studies, we are able to demonstrate that increasing levels of PKCII protein accompany the switch of mRNA products. In this case, we have linked the functional significance of alternative mRNA splicing of PKCI and II to glucose transport. Glucose transport is stimulated by insulin in a number of tissues and cells (37) . There appear to be PKC and PKC-dependent components in rat adipocytes demonstrated with antisense oligonucleotides to down-regulate these isozymes (29) . In other studies, insulin-dependent glucose transport was restored by introducing PKC and PKC enzymes into PKC-depleted adipocytes (28). For the present studies, we stably transfected NIH-3T3 cells to overexpress PKCI and PKCII. Overexpression of PKCII but not PKCI further enhanced insulin-dependent increases in glucose transport. Whether this same relationship between PKCI and PKCII is relevant to other cell types must await further studies. It may be of interest that both BC3H-1 myocytes and NIH-3T3 cells, murine cell lines, express GLUT1-type glucose transporters.

Functional differences between PKCI and -II have also been demonstrated in other systems. PKCI was found to be more efficient in inactivating glycogen synthase kinase-3 by phosphorylation than PKCII (38) . In cardiac myocytes the redistribution of PKCI from the perinuclear membrane to the nucleolus while PKCII translocated from the cytoplasm to the perinuclear and plasma membrane following TPA and norepinephrine treatment was shown (39) . These studies demonstrate that not only do the catalytic potentials of PKCI and-II differ, but their ability to translocate to distinct intracellular compartments is also distinct.

The present studies indicate that the ability of cells to respond to signals from insulin may depend upon the presence of one PKC isoform versus the other, and their expression is regulated by insulin. Insulin stimulation of cells leads to increases in diacylglycerol and, hence, activates several PKC isozymes. Our data suggest that this rather nonspecific activation of PKC isozymes is further fine tuned by the control of PKC isozyme expression via alternative splicing of PKC. The rapidity of the switching observed in BC3H-1 myocytes infers that this process has functional consequences under varying physiological states. Although it is recognized that developmental stages or cell types can express different PKC transcripts (11, 12, 16) , this study indicates that a cell can alter expression of one splice product versus another in response to a peptide hormone to alter metabolic pathways.

  
Table: Effect of insulin on 2-deoxyglucose uptake in NIH-3T3 cells stably transfected to overexpress PKCI and PKCII

Heterogeneous pools of stably transfected cells were grown in 24-well plates until >80% confluent. NIH-II-clone 10 denotes one clone from the heterogeneous pool of transfectants. Prior to 2-[H]DOG uptake, cells are switched to Dulbecco's modified Eagle's medium without serum overnight (21). Data are the mean and S.E. of picomoles uptake for the number of separate cell passages assayed (n). Results of two-way ANOVA at p < 0.05 are indicated by * for the effect of insulin on 2-DOG uptake and ** for the effect of the PKCII vector on the insulin effect compared with vector control.



FOOTNOTES

*
This work was supported by the Medical Research Service of the Department of Veterans Affairs (to D. R. C., and R. V. F.), by a National Science Foundation Grant 9318124 (to D. R. C.), by the American Heart Association, Florida affiliate (to D. R. C.), and by National Institutes of Health Grant DK 38079 (to R. V. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Inst. for Clinical Molecular Biology and Tumor Genetics, GSF, Marchionistrasse 25, D-81377 Munich, Germany.

To whom correspondence should be addressed: Research Service 151, J. A. Haley Veterans Hospital, 13000 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-972-2000 (ext. 7017); Fax: 813-972-7623; E-mail: dcooper@com1.med.usf.edu.

The abbreviations used are: PKC, protein kinase C; TPA, 12-0-tetradecanoylphorbol-13-acetate; 2-DOG, 2-deoxyglucose; RPA, ribonuclease protection analysis; RT-PCR, reverse transcriptase polymerase chain reaction.

Antibody from Life Technologies, Inc. detected only lower molecular mass forms of PKC, presumably PKCI, when blots were developed and visualized using alkaline phosphatase. This antibody detected higher molecular mass forms of PKCII as well as lower molecular mass PKCI when Amersham ECL was used for visualization.


ACKNOWLEDGEMENTS

We thank Dr. Walter Kolch, Institute for Clinical Molecular Biology and Tumor Genetics, GSF, Munich, Germany; Dr. J. F. Mushinski, National Cancer Institute, National Institutes of Health, Bethesda, MD; and Dr. Duane Eichler, University of South Florida, Tampa, FL for thoughtful and critical reading of the manuscript.


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