©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Increased Expression of Protein Kinase C Activates ERK3 (*)

(Received for publication, November 29, 1995; and in revised form, February 13, 1996)

Samir Sauma Eileen Friedman (§)

From the Laboratory of Gastrointestinal Tumor Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In a prior study, we have shown that stable transfection of expression plasmids for protein kinase Cbeta1 (PKCbeta1) or PKCbeta2 into differentiated colon cancer cells led to elevated levels of PKCbeta1 or PKCbeta2 protein and PKCbeta kinase activities in the transfectants, without altering PKCalpha levels. PKC is not found in these cells, so the major modulation was in PKCbeta. PKCbeta transfectant cells exhibited blocked differentiation, increased growth rate in athymic mice, and restoration of the basic fibroblast growth factor response pathway. In this study, we have extended the analysis of these PKCbeta transfectants to the mitogen-activated protein kinase ERK3. Analysis of cell lysates on the mitogen-activated protein kinase substrate myelin basic protein by in gel kinase assay showed increased activity at 63 kDa, the size of ERK3, in each of two PKCbeta1 and each of two PKCbeta2 transfectants compared with the vector control transfectant. ERK3 was expressed at equal abundance in PKCbeta1, PKCbeta2, and control transfectant cells as demonstrated by Western blotting and by immunoprecipitation with anti-ERK3 monoclonal antibody. However, a >10-fold increase in ERK3 activity in each PKCbeta transfectant was shown by immunoprecipitation with anti-ERK3 monoclonal antibody followed by either immune complex kinase assay or by in gel kinase assay. Thus, while overexpression of transfected PKCbeta does not lead to overexpression of ERK3, it does lead to constitutive activation of ERK3. A causal link between PKCbeta overexpression and ERK3 activation was established because 12-O-tetradecanoylphorbol-13-acetate treatment down-regulated both PKC and ERK3 activities in both PKCbeta1 transfectants. ERK3 activity was found in nuclear and membrane fractions in each PKCbeta transfectant, in contrast to controls, perhaps accounting for constitutive activation of ERK3 in cells with elevated levels of PKCbeta1 or PKCbeta2.


INTRODUCTION

In previous studies, we found that differentiation of colon carcinoma cells down-regulated the abundance and activity of PKCbeta (^1)while not altering levels of other PKC isozymes present in these cells. Each of two independently cloned colon carcinoma lines that display differentiation characteristics of mature fluid-transporting colon enterocytes down-regulated PKCbeta activity and abundance 5-10-fold compared with each of two undifferentiated lines(1) . Neither differentiated line was capable of transmitting mitogenic signals from basic fibroblast growth factor to p57(1) . Both differentiated and undifferentiated lines maintained equal levels of the PKC isozymes alpha, , and , and none of the four lines exhibited either PKC or PKC(1) , limiting at least the major, if not the entire, PKC modulation to the PKCbeta isozyme. We transfected both splice variants of PKCbeta(2) , PKCbeta1 and PKCbeta2, into one of these differentiated colon carcinoma lines, HD3. The HD3 transfectant cells with increased PKCbeta1 and PKCbeta2 expression but unaltered PKCalpha levels were blocked in differentiation, had constitutively activated both the ERK1-related p57 and ERK1, restored the basic fibroblast growth factor response pathway, and had acquired the capacity for rapid growth in athymic mice(3) .

In this study, we have extended our studies of PKCbeta transfectants to the MAP kinase ERK3. ERK3 is structurally related to the more well studied ERK1 and ERK2, with a 43% overall homology(4) , but little is known of the function or activation of ERK3. Two human homologues of the rat erk3 gene have been cloned, one of 63 kDa with a 73% amino acid identity (5) and, more recently, a 97-kDa homologue that has a 98% homology to rat Erk3 through the first 500 amino acids followed by a unique 178-amino acid extension(6) . 97-kDa ERK3 has been shown to have kinase activity on histone H1 when fibroblasts are stimulated with serum or phorbol ester, but not insulin, insulin-like growth factor 1, or epidermal growth factor(6) .

ERK1 and ERK2 are activated by phosphorylation on tyrosine and threonine in a TEY site in subdomain VII (7, 8, 9, 10) by a well known pathway through MEK and MEK kinase (summarized in (11) ). In contrast, ERK3 has an SEG site at the homologous site in subdomain VII (4) and may be activated in vivo through different MEKs and MEK kinases or other kinases. We have asked in this study whether elevated levels of PKCbeta1 and PKCbeta2, which block differentiation in colon cancer cells(3) , alter the activation or abundance of ERK3.


EXPERIMENTAL PROCEDURES

Materials

I-Protein A and [-P]ATP were obtained from DuPont NEN; myelin basic protein and TPA were from Sigma; protein A-Sepharose was from Pharmacia Biotech Inc.; and Immobilon-P polyvinylidene difluoride transfer paper was from Millipore Corp. Anti-pan-ERK M antiserum and anti-ERK3 monoclonal antibody were purchased from Transduction Laboratories, and sheep anti-mouse IgM was from BIODESIGN International.

Cell Culture

Cells were maintained in Dulbecco's modified Eagle's medium containing 7% fetal bovine serum as described (12) .

Partial Purification of PKC from PKCbeta Transfectants

The protocol was essentially that used before(1) . Briefly, log-phase cultures from each line were washed twice in phosphate-buffered saline, swelled for 15 min on ice, and then homogenized in hypotonic buffer B (10 mM Hepes (pH 7.5) containing 20 µg/ml aprotinin, 20 µg/ml leupeptin, 20 µg/ml phenylmethylsulfonyl fluoride, 5 mM EGTA, 2 mM EDTA, and 2 mM dithiothreitol). A membrane pellet was prepared by centrifugation of the homogenate at 100,000 times g for 1 h at 4 °C. The supernatant was used to prepare the cytosolic PKC preparation, which was absorbed to DE52 columns and eluted in 0.1 M NaCl after washing, exactly as described(1) . The membrane pellet was extracted with 0.5% Triton X-100 in buffer B for 30 min on ice and pelleted at 10,000 times g for 10 min to remove debris. The supernatant was the membrane extract.

Kinase Assays in MBP-containing Polyacrylamide Gels

The method was adapted from one previously used(1) . Log-phase cells were lysed in buffer A (20 mM Tris-HCl (pH 7.5) containing 10 µg/ml aprotinin, 20 µg/ml leupeptin, 24 µg/ml phenylmethylsulfonyl fluoride, 1 mM EGTA, 2 mM EDTA, 1 mM NaF, 100 µM Na(3)VO(4), and 2 mM benzamide), boiled in Laemmli sample buffer for 2 min, and then electrophoresed in a 7.5% SDS-polyacrylamide gel (0.5 mm times 5 cm) containing 0.5 mg/ml MBP. After fixing the gel with four changes of 20% 2-propanol in 50 mM Tris-HCl buffer (pH 8.0) for 2 h, SDS was removed by washing the gel for 2 h in several gel volumes of 50 mM Tris-HCl (pH 8.0) containing 5 mM 2-mercaptoethanol, with frequent changes. The MBP kinases were then redenatured with 6 M guanidine HCl for 2 h and then renatured by 10 washes of 20 min each in several gel volumes of 50 mM Tris-HCl (pH 8.0) containing 0.04% Tween 40 and 5 mM 2-mercaptoethanol. After preincubation for 1 h with 5 ml of 40 mM Hepes (pH 8.0) containing 2 mM mercaptoethanol and 10 mM MgCl(2), phosphorylation of MBP within the gel was carried out by incubating the gel at room temperature for 1 h in 5 ml of 40 mM Hepes (pH 8.0) containing 25 µCi of [-P]ATP, 40 µM ATP, 0.5 mM EGTA, and 10 mM MgCl(2) and then washing the gel in 5% (w/v) trichloroacetic acid containing 1% sodium pyrophosphate several times until the radioactivity reached background levels.

Immunodetection

Extracts were adjusted to equal protein concentrations in SDS sample buffer, heated for 5 min at 100 °C, and then electrophoresed in 10% SDS-acrylamide gels. Proteins separated in the gel were transferred by electrophoresis to a polyvinylidene difluoride membrane exactly as detailed(1) . Proteins were detected using either a 1:2000 dilution of anti-pan-ERK antibody (0.25 µg/ml) or a 1:250 dilution of anti-ERK3 mAb (1 µg/ml) followed by I-protein A and autoradiography.

Immunoprecipitation

Cells were homogenized in buffer A containing protease and phosphatase inhibitors, and a cytosol fraction was made as described above. 10 µg of mouse anti-Erk3 mAb were first incubated with 500 µg of cytosol protein for 3 h at 4 °C with rocking, and then 5 µg of sheep anti-mouse IgM was added for an additional incubation of 2 h and bound to protein A-Sepharose (50 µl of a 10% slurry) for 2 h. The immunoprecipitates bound to protein A-beads were washed three times in buffer A and once in 10 mM Hepes (pH 7.2) and then boiled in SDS sample buffer, and the supernatants were analyzed either by Western blotting with anti-Erk3 antibody or by the in gel kinase assay on immobilized MBP as described above.

Immune Complex Kinase Reaction

Immunoprecipitations were performed as described above, pelleted using a microcentrifuge at 12,000 times g for 5 min, washed six times with buffer A, and then incubated for 1 h at room temperature in 50 µl of 40 mM Hepes (pH 8.0) containing 0.5 mg/ml MBP, 25 µCi of [-P]ATP, 40 µM ATP, and 10 mM MgCl(2). Assays were terminated by spotting 15-µl aliquots onto a 1.5-cm^2 piece of Whatman P-81 phosphocellulose paper. The papers were washed eight times by shaking for 5 min in 300 ml of 1% phosphoric acid followed by transfer into vials containing 5 ml of scintillation fluor and quantitated for radioactivity in a Beckman LS6000IC counter.

Cell Fractionation

The method was adapted from Chen et al.(13) . After two washes with phosphate-buffered saline, cells were scraped into 0.5 ml of hypotonic lysis buffer (1 mM EGTA, 1 mM EDTA, 10 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 2 mM MgCl(2), 10 mM KCl, 1 mM dithiothreitol, 40 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). The cell suspension was incubated on ice for 20 min to allow swelling. The cells were then homogenized in a Dounce homogenizer (20 strokes), and the homogenate was loaded onto 1 ml of 1 M sucrose in lysis buffer and centrifuged at 1600 times g for 10 min to pellet nuclei. The supernatant above the sucrose cushion was centrifuged at 150,000 times g for 30 min to prepare the cytosol fraction, and the pellet was solubilized in hypotonic lysis buffer containing 0.5% Nonidet P-40 and 0.1% sodium deoxycholate and centrifuged at 10,000 times g for 5 min to remove insoluble material. Nonidet P-40 and sodium deoxycholate were added to the cytosol fraction to give equal concentrations of these detergents in all fractions.


RESULTS

ERK3 Activated in PKCbeta1 and PKCbeta2 Transfectants

In our earlier study(3) , four PKCbeta transfectants (PKCbeta1-1, PKCbeta1-2, PKCbeta2-2, and PKCbeta2-3) were isolated that exhibited elevated activity and abundance of PKCbeta1 and PKCbeta2, respectively, compared with the vector control transfectant. PKCalpha levels were not altered, and PKC was not expressed in these transfectants. Lysates of both PKCbeta1 and both PKCbeta2 transfectants and one empty vector transfectant line were analyzed for activation of MAP kinases by an in gel kinase assay with immobilized MBP as the substrate. A major band of activity was seen at roughly 63 kDa in each PKCbeta transfectant, but not in the control cells (Fig. 1), suggesting either activation of a kinase or increased abundance of a kinase. Longer exposure of the gel was necessary to detect p57 or p44/ERK1 ( (3) and data not shown). ERK3 was immunoprecipitated from both PKCbeta and control transfectant lines with monoclonal antibody. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blotting with anti-ERK3 and demonstrated that each PKCbeta transfectant and the control transfectant expressed equal amounts of the p63 form of ERK3 (Fig. 2). Larger molecular mass forms of ERK3 (6) were not detected in these cells. Western blotting of total cell lysates with mAb to ERK3 also demonstrated equal amounts of ERK3 in PKCbeta and control transfectants (Fig. 3), as did a similar experiment with a pan-ERK antiserum (data not shown). Immunoprecipitation with a pan-ERK antiserum from S-prelabeled cells also demonstrated equal abundance of a 63-kDa MAP kinase in PKCbeta and control transfectant cells(3) . Thus, overexpression of PKCbeta1 or PKCbeta2 did not lead to overexpression of ERK3.


Figure 1: In gel kinase assay showing major band of activity at 63 kDa in each PKCbeta transfectant compared with vector control cells. Molecular mass markers of 44, 71, 110, and 200 kDa are indicated by dashes.




Figure 2: Immunoprecipitation of equal amounts of ERK3 protein from each of two PKCbeta1 and each of two PKCbeta2 transfectants and one vector control transfectant cell line. Antibody was not used in excess. Immunoprecipitates were analyzed by Western blotting with anti-ERK3 antibody and I-protein A. The arrow marks the position of ERK3. The lighter bands are immunoglobulin heavy chain and light chain. Molecular mass markers of 30, 44, 70, 110, and 200 kDa indicated at the right. V, vector control transfectant cell line.




Figure 3: Western blotting of cell lysates identifies equal levels of ERK3 protein in each of two PKCbeta1 and each of two PKCbeta2 transfectants and one vector control transfectant cell line using anti-ERK3 mAb. Detection was with I-protein A following secondary antibody. The area around the 63-kDa band only is shown. The arrow indicates ERK3 protein. V, vector control transfectant cell line.



ERK3 was immunoprecipitated from each PKCbeta and control transfectant, and ERK3 activation was then analyzed in two ways. An immune complex kinase experiment was performed using MBP as the substrate. Both PKCbeta1 and both PKCbeta2 transfectant lines exhibited 13-fold more ERK3 activity than the vector control in triplicate assays (Fig. 4). ERK3 immunoprecipitates were also analyzed by in gel kinase assay on immobilized MBP. This assay allows one to visualize the kinase reaction product at the molecular mass of the kinase, confirming the identify of the MAP kinase immunoprecipitated by anti-ERK3 mAb as the p63 form of ERK3 (Fig. 5). Kinase product was only visualized in immunoprecipitates from the PKCbeta transfectant lines and was at least 10-fold control levels. Similar activation of ERK3 in PKCbeta transfectants was also observed using the pan-ERK antiserum in two additional immunoprecipitation experiments (data not shown). Therefore, these experiments show that increased levels of PKCbeta1 and PKCbeta2 correlate in each of four transfectant lines with activation of ERK3, but not with an increase in ERK3 abundance.


Figure 4: Immune complex kinase reaction demonstrates 13-fold increased activity of ERK3 in each of two PKCbeta1 and each of two PKCbeta2 transfectants compared with one vector control transfectant cell line. Means ± S.E. of three separate determinations are shown. The actual total cpm incorporated per PKCbeta transfectant ranged from 12,000 to 13,000. Vec, vector control transfectant cell line.




Figure 5: Immunoprecipitation of ERK3 followed by analysis of ERK3 activity on in gel kinase assay on MBP showing detectable activity of ERK3 only in PKCbeta1-1 and PKCbeta2-2 transfectants, but not in vector control transfectants. Molecular mass markers of 44, 71, 110, and 200 kDa are indicated by dashes. V, vector control transfectant.



Down-regulation of PKC Levels by TPA Treatment Decreases ERK3 Activity

Both PKCbeta1 transfectant lines and the vector control were treated for 24 h with 100 ng/ml TPA or the diluent, 0.1% dimethyl sulfoxide. PKCbeta was partially purified by DE52 chromatography from the cytosols of each treated line (see ``Experimental Procedures''). Total calcium/phosphatidylserine-dependent PKC activities consisted of the beta1, beta2, and alpha isoforms as is not present. A 20% decrease in total calcium/phosphatidylserine-dependent activities (data not shown) was induced by 24 h of TPA treatment and was not further decreased by 48 h of TPA treatment. Down-regulation of PKC activity decreased the p63 activities in both PKCbeta1 transfectant lines (Fig. 6). To confirm that the TPA-down-regulated p63 activity was ERK3, ERK3 was immunoprecipitated from TPA- and diluent-treated PKCbeta1-1 transfectant cells, and an immune complex kinase assay was performed, using MBP as the substrate. Decreased ERK3 activity was seen in TPA-treated PKCbeta1-1 cells compared with diluent-treated control cells (4299 ± 34 cpm versus 5341 ± 53 cpm, triplicate assays). Thus, the decrease in p63 seen in TPA-treated PKCbeta1 transfectants (Fig. 6) was correlated with a decrease in ERK3 activity immunoprecipitated with specific mAb, and both decreases were roughly 20%.


Figure 6: Down-regulation of PKC activity in each of two PKCbeta2 transfectants inhibits ERK3 MBP kinase activity in in gel kinase assay. Cells were treated with either 100 ng/ml TPA or 0.1% dimethyl sulfoxide, the diluent. The arrow indicates ERK3 activity. Molecular mass markers of 44, 71, 110, and 200 kDa are indicated by dashes. Vec, vector control transfectant cell line.



It has been reported that PKCbeta2 found in colonocytes is not down-regulated efficiently by TPA, in contrast to other PKC isozymes (14) . We were not able to decrease p63 activity, as measured by in gel kinase assay, or ERK3 activity, as assayed by immune complex kinase assay after immunoprecipitation, by TPA treatment of PKCbeta2 transfectant cells. Since neither activity was affected, however, this experiment again correlated ERK3 activity with p63 activity (data not shown).

Constitutive Activation of ERK3 in Each PKCbeta Transfectant Line Correlates with Nuclear Localization

Other investigators have shown that treatment of serum-deprived HeLa cells with serum or TPA led to activation of p44/ERK1 as shown by increased tyrosine and threonine phosphorylation of p44 and translocation of the activated p44 to the nucleus(13) . Since we had established that the p63 activity seen in cell lysates from PKCbeta transfectants was ERK3, we used the simple, quantitative in gel kinase assay to determine whether constitutively activated ERK3 in PKCbeta transfectants would be found in the nucleus without growth factor treatment. Protein from cytosolic, nuclear, and membrane fractions was assayed proportionally to the total amount of protein in each fraction. Each of the four PKCbeta transfectant lines exhibited more ERK3 activity in the nuclear fractions than was seen in the vector control in duplicate experiments, on immobilized MBP by the in gel kinase reaction (Fig. 7). The PKCbeta transfectants constitutively displayed a characteristic of mitogen-treated control cells since treatment of vector control cells with a mitogenic concentration of epidermal growth factor caused an appearance of p63/ERK3 activity in the nuclear fraction (data not shown). ERK3 and two larger, uncharacterized MBP kinases were also present in membrane fractions from each of the four PKCbeta transfectant lines (Fig. 8). In these experiments, only membrane and nuclear fractions, not cytosols, were analyzed proportionally to the total amount of protein in each fraction, so larger amounts of proteins from these fractions could be size-fractionated. Treatment of the parental HD3 cells with growth-inhibiting concentrations of transforming growth factor beta1 decreased the activity of the larger MBP kinases found in the membrane fraction in Fig. 8, linking modulation of these activities with growth modulation(15) . Therefore, elevating the abundance and therefore the activities of PKCbeta1 and PKCbeta2 by transfection led to activation of several kinases including ERK3, several of which may play roles in growth control.


Figure 7: PKCbeta transfectants contain activated ERK3 kinase in nuclear fractions. In gel kinase assay was carried out on MBP of cytosolic (C), nuclear (N), and membrane (M) fractions from PKCbeta transfectants and vector controls (CONT). Molecular mass markers of 44, 71, 110, and 200 kDa are indicated by dashes. The arrow indicates activated ERK3 kinase.




Figure 8: PKCbeta transfectants contain activated ERK3 activities in their nuclear and membrane fractions. Unidentified MBP kinases of 105 and 130 kDa are also seen in the membrane fractions of only PKCbeta transfectants. Molecular mass markers of 44, 72, 110, and 200 kDa are indicated at the left. The arrow indicates activated ERK3 activities. Vec, vector control transfectant cell line.



Constitutive activation of ERK3 might be related to the enhanced levels of ERK3 found in nuclear and membrane fractions of PKCbeta transfectant cells. The nuclear and membrane location of some of the ERK3 activity makes it unlikely that the MBP kinase activity immunoprecipitated with both ERK3-specific mAb and pan-ERK antiserum ( Fig. 4and Fig. 5) was a 63-kDa proteolytic fragment of PKCbeta contaminating both immunoprecipitations.


DISCUSSION

One of properties of the p44/p42 isoforms is their capacity for activation by the phorbol ester class of PKC activators (11, 16) . We have shown here and in an earlier study (3) that overexpression of one PKC isoform, PKCbeta, in colon cancer cells blocks differentiation and activates not only the well studied MAP kinase ERK1, but also ERK3 and the ERK1-related p57. Thus, permutation in only one PKC isozyme, PKCbeta, activates several MAP kinases, each of which may have different sites of action, either within the nucleus to activate different sets of transcription factors or at the cytoskeleton to phosphorylate microtubule-associated proteins, destabilizing microtubules and thus cytoskeletal structure and organization(11, 16) . Several MAP kinases have been isolated from various species from Drosophila to Xenopus to human, and these can be divided into different families depending on DNA sequence homologies. MAP kinase families are characterized by high homology between family members; for example, ERK1 exhibits 90% sequence homology to ERK2(4) . However, these ``classical'' MAP kinases exhibit much lower sequence homology to other MAP kinases. ERK1 has 43% homology to the ERK3 family(4) ; 40% homology to Jun kinases and p38, which are activated by cellular stress such as protein synthesis inhibitors, osmotic shock, or UV light(11, 17, 18) ; and 40% homology to ERK5(19) , a newly cloned, larger MAP kinase. These different MAP kinase families are expressed preferentially in various cell types, suggesting that they may have different roles in proliferation, differentiation, or embryonic development. Little is known about the activation or role in cell proliferation or differentiation of any of the ERK3 family members. 97-kDa ERK3 has been shown to have kinase activity on histone H1 when fibroblasts are stimulated with serum or phorbol ester, but not insulin, insulin-like growth factor 1, or epidermal growth factor(6) . ERK1 and ERK2 and the Jun kinases are activated by phosphorylation on tyrosine and threonine in a TXY site in subdomain VII(7, 8, 9, 10, 11, 17, 18) by a well known pathway through specific MEKs and MEK kinases(11) . In contrast, ERK3 has an SEG site at the homologous site in subdomain VII (4) and may be activated by other kinases in vivo.

There are at least 12 PKC members classified into three groups: Ca-, phosphatidylserine-, and diacylglycerol-dependent conventional PKC isoforms (alpha, betaI, betaII, and ); Ca-independent novel PKC isoforms (, , µ, , and ); and Ca- and diacylglycerol-independent, phosphatidylserine-dependent atypical PKC isoforms (, , and ). Roles for PKC isoforms may depend on cell type, and the ratios of the different PKC isozymes within a cell also may alter a cell's response to PKC activators. Craven and DeRubertis (20) have found that treatment of rats with a colon carcinogen led to a relative increase in PKCbeta expression, together with a decrease in PKCalpha expression in colon epithelial cells, leading to an increased PKCbeta/PKCalpha ratio. Marian and co-workers (21) have measured PKC isozyme protein levels in normal human colonocytes, human benign colon tumors, and human malignant colon tumors and found a decrease in PKC isozyme levels in all benign and malignant tumor cells, with much less decrease in PKCbeta, leading to an enrichment of PKCbeta levels in colon tumors relative to the other isozymes detectable in colon tumor tissue: alpha, , , , and . The benign tumor cells with increased PKCbeta/PKCalpha ratio were responsive to TPA, a mitogen for colon adenoma cells(21, 22) . The loss in most PKC isozymes on the protein level in colon tumor tissue is consistent with earlier reports that colon tumors express less total PKC mRNA than normal tissue (23) and, more recently, less PKCbeta and less PKC mRNAs(24) .

We found in earlier studies that differentiation of colon carcinoma cells to fluid-transporting enterocytic-like cells led to a 5-10-fold decrease in PKCbeta abundance and activity, with no change in abundance of the other PKC isozymes detectable, alpha, , and (1) . These studies, like the studies cited above, point to a role for enhanced levels of PKCbeta relative to the other isozymes in colon tumor progression. Transfection of either PKCbeta isoform, PKCbeta1 or PKCbeta2, into such differentiated colon carcinoma cells restored the undifferentiated phenotype and proliferative response to basic fibroblast growth factor and allowed more rapid growth in athymic mice (3) . The mechanism, at least in part, for this blocked differentiation was a constitutive activation of both ERK1 and the ERK1-related p57(3) . In the current study, a third MAP kinase, ERK3, was found also to be constitutively activated by overexpression of PKCbeta1 or PKCbeta2. ERK3 was found associated with the nucleus and membrane in PKCbeta transfectant cells. Nuclear location may allow the activated ERK3 to act as a transcription factor kinase, while membrane location may allow association with the microtubule cytoskeleton. ERK1 and ERK2 are associated with the microtubule cytoskeleton in NIH3T3 fibroblasts(25) . MAP kinases are known to phosphorylate microtubule-associated proteins, causing microtubule instability(11) , perhaps leading to the cellular and nuclear shape changes that occur during S phase and mitosis.

Our studies have correlated increased expression of PKCbeta1 and PKCbeta2 with increased proliferation in colon cancer due to activation of multiple MAP kinases. Others have found constitutive activation of MAP kinases in human renal cancers(26) . In a series of 25 cases of paired normal kidney tissue and renal carcinomas, constitutive activation of MAP kinases, as determined by the appearance of phosphorylated forms of ERK2, was found in 48% of the cases(26) . Others have found PKCbeta to be elevated in activity in invasive gastric cancers(27) . PKCbeta2 is required for the proliferation of K562 human erythroleukemia cells (28) . Overexpression of PKCbeta2 in HL-60 promyelocytic leukemic cells enhances their proliferation and makes them resistant to TPA-induced differentiation(29) . PKCbeta2 translocates to the nuclear envelope and phosphorylates lamin B in cells treated with mitogenic stimuli, but not with differentiation inducers like TPA(29) . Thus, proliferation of HL-60 cells is correlated with nuclear translocation of PKCbeta2 and its phosphorylation of nuclear envelope lamin B, part of the nuclear membrane breakdown that occurs when cells enter mitosis. It would be of interest to determine whether nuclear forms of ERK3 could be responsible for this phosphorylation. Thus, overexpression of PKCbeta2 has, in several cell types, been associated with increased proliferation and blocked differentiation.

The role of PKCbeta1 is more problematic. Other investigators have shown that expression of elevated levels of PKCbeta1 in uncloned HT29 colon carcinoma cells (30) and SW480 colon carcinoma cells (31) inhibited tumorigenicity and cell growth. The uncloned HT29 line and the SW480 line are both undifferentiated and highly tumorigenic lines. In our studies, PKCbeta1 was overexpressed following stable transfection of a poorly tumorigenic, differentiated cell line with low PKCbeta levels (1) , and the transfectants simply had restored PKCbeta1 levels to those characteristic of undifferentiated parental cells(3) , not significantly above this level as in the other cited studies. In this and earlier studies, we have shown that at least part of the mechanism of action of PKCbeta in colon cancer cells is constitutive activation of multiple MAP kinases. It will now be necessary to determine the spectrum of substrates for the ERK1-like p57, ERK3, and ERK1 in colon carcinoma cells in both the nucleus and cytoskeleton. In this way, it may be possible to identify the proximal effectors of PKCbeta.


FOOTNOTES

*
This work was supported by NCI Grants RO1 CA45783 and RO1 CA67405 (to E. 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.

§
To whom correspondence should be addressed: Dept. of Pathology, SUNY Syracuse Health Science Center, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464-7148; Fax: 315-464-7130.

(^1)
The abbreviations used are: PKC, protein kinase C; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MEK, mitogen-activated kinase kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; MBP, myelin basic protein; mAb, monoclonal antibody.


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