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J. Biol. Chem., Vol. 279, Issue 21, 22118-22123, May 21, 2004

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Protein Kinase C (PKC) II Induces Cell Invasion through a Ras/Mek-, PKC/Rac 1-dependent Signaling Pathway^*

Jie Zhang, Panos Z. Anastasiadis, Yan Liu, E. Aubrey Thompson, and Alan P. Fields

From the Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224

Received for publication, January 23, 2004 , and in revised form, March 17, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Protein kinase C II (PKCII) promotes colon carcinogenesis. Expression of PKCII in the colon of transgenic mice induces hyperproliferation and increased susceptibility to colon cancer. To determine molecular mechanisms by which PKCII promotes colon cancer, we established rat intestinal epithelial (RIE) cells stably expressing PKCII. Here we show that RIE/PKCII cells acquire an invasive phenotype that is blocked by the PKC inhibitor LY379196. Invasion is not observed in RIE cells expressing a kinase-deficient PKCII, indicating that PKCII activity is required for the invasive phenotype. PKCII induces activation of K-Ras and the Ras effector, Rac1, in RIE/PKCII cells. PKCII-mediated invasion is blocked by the Mek inhibitor, U0126, and by expression of either dominant negative Rac1 or kinase-deficient atypical PKC. Expression of constitutively active Rac1 induces Mek activation and invasion in RIE cells, indicating that Rac1 is the critical downstream effector of PKCII-mediated invasion. Taken together, our results define a novel PKCII Ras PKC /Rac1 Mek signaling pathway that induces invasion in intestinal epithelial cells. This pathway provides a plausible mechanism by which PKCII promotes colon carcinogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Colorectal cancer is the second leading cause of cancer death in the United States (1). Colon carcinogenesis is a complex multistep process involving progressive disruption of intestinal epithelial cell proliferation, differentiation, and survival mechanisms (2). Colon carcinogenesis is driven by environmental factors that modulate cell signaling pathways, and by genetic mutation of transforming oncogenes, and deletion or mutation of DNA repair enzymes and tumor suppressor genes (2). An important challenge is to understand how environmental and genetic factors interact to define colon cancer risk.

Protein kinase C (PKC)¹ is a family of 12 lipid-dependent serine/threonine kinases involved in proliferation, differentiation, and survival (3, 4). Specific, reproducible changes in PKC isozyme expression patterns occur during carcinogen-induced colon carcinogenesis in rodents. Normal rat colonic epithelium contains multiple PKC isozymes, including PKC , , and (5, 6). In azoxymethane-induced rat colonic tumors, the levels of PKC , , and are reduced and the level of PKCII is increased when compared with normal colonic epithelium (5, 6). We recently demonstrated reduced PKC expression and increased PKCII and PKC expression in azoxymethane-induced mouse colon tumors (7, 8). Our subsequent studies provided direct evidence that both PKCII and PKC play critical but distinct roles in colon carcinogenesis (7–10).

We have developed and analyzed transgenic mice that express elevated PKCII in the colonic epithelium (7, 9, 10). Transgenic PKCII mice exhibit hyperproliferation of the colonic epithelium and are prone to carcinogen-induced colon cancer (9, 10). This cancer-prone phenotype results, at least in part, from the imposition of a PKCII-mediated hyperproliferative phenotype (9, 10). To assess the molecular mechanisms by which PKCII promotes colon cancer, we established nontransformed rat intestinal epithelial (RIE) cell lines that overexpress PKCII (RIE/PKCII cells) (10, 11). Genomic analysis of RIE/PKCII cells revealed that PKCII induces the expression of the Cox-2 enzyme and suppresses the expression of the transforming growth factor receptor type II (TGFRII) (11). As a result, RIE/PKCII cells have lost the ability to respond to the growth inhibitory effects of TGF-, and this loss is dependent upon the activity of Cox-2 (11). Therefore, PKCII establishes a novel, pro-carcinogenic PKCII Cox-2 TGFRII signaling pathway that confers resistance to TGF- (11). This pathway is activated in transgenic PKCII mice and accounts, at least in part, for the hyperproliferative phenotype exhibited by these mice (11). Carcinogens induce this pathway, whereas chemopreventive -3 fatty acids inhibit PKCII activity, suppress PKCII-mediated hyperproliferation, and attenuate the cancer-prone phenotype exhibited by transgenic PKCII mice (10, 11).

Here, we demonstrate that PKCII induces an invasive phenotype in RIE cells through a signaling pathway that is distinct from the PKCII Cox-2 TGFRII pathway responsible for TGF- resistance. PKCII-mediated invasion is dependent upon Ras, PKC, Rac1, and Mek. Our findings define a novel proinvasive PKCII Ras PKC /Rac1 Mek signaling pathway. Our data indicate that PKCII promotes colon cancer through activation of at least two distinct signaling pathways, one that confers TGF- resistance, and a second that induces invasion, in intestinal epithelial cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents and Antibodies—The PKC-selective inhibitor LY379196 was a kind gift of Dr. James R. Gillig, Lilly Pharmaceutical Co. Puromycin dihydrochloride was purchased from Calbiochem. Polybrene and crystal violet were from Sigma. Anti-PKCII, anti-actin, and horseradish peroxidase-conjugated donkey anti-goat secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. The PhosphoPlus Mek 1/2 antibody was from Cell Signaling Technology. Anti-FLAG epitope antibody was from Sigma. Anti-Rac1 mouse monoclonal antibody was from BD Transduction Laboratories. Peroxidase-labeled goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Kirkegaard and Perry Laboratories.

Construction of a Kinase-deficient Mutant of Human PKCII—A kinase-deficient mutant of human PKCII (kdPKCII) was generated by PCR-based site-directed mutagenesis using the QuikChange XL site-directed mutagenesis kit (Stratagene). Full-length wild type human PKCII was used as a template, and the mutagenic primer, 5'-GGC ACA GAT GAG CTC TAT GCT GTG TGG ATC CTG AAG AAG GAC G-3', was used to replace lysine 372, a residue essential for ATP binding and activity, with a tryptophan residue (12). The kdPKCII mutant was inserted into the pBABE/FLAG/puro retroviral expression vector, and virus stocks were produced using Phoenix-E cells.

Stable Transfection of Cell Lines—RIE cells were maintained in 5% (v/v) fetal bovine serum (Invitrogen) in Dulbecco's modified Eagle's (DME) medium (Invitrogen) in a 5% CO₂ incubator at 37 °C as described previously (10, 11). Populations of RIE cells expressing full-length human PKCII (RIE/PKCII) or kdPKCII (RIE/kdPKCII) were established by transfection with pBabe retrovirus expression vector, which has been described previously (10, 11). Puromycin-resistant, stable transfectants were generated as described at www.stanford.edu/group/nolan/NL-phnxr.html. Expression of PKCII or kdPKCII was confirmed by immunoblot analysis using anti-FLAG and anti-PKCII antibodies.

Retrovirus Production and Transient Transfection—Recombinant retroviruses containing Myc-tagged dominant negative Rac1 (RacN17) or constitutively active Rac1 (RacV12) were generated by excising the Myc-tagged Rac1 constructs from pEXV/Rac vectors (13) with EcoRI and ligating them into the EcoRI site of the LZRS-GFP retrovirus. Each construct was confirmed by sequence analysis. LZRS-GFP-Rac1 retroviruses were used to infect RIE cells and derivative cell lines as described above.

Preparation of Recombinant PAK and Rhotekin GST Fusion Proteins—GST·PAK and GST·Rhotekin were used to assay for Rac1 and RhoA activity, respectively. Escherichia coli transformed with the GST·PAK or GST·Rhotekin constructs were grown at 37 °C to an absorbance of 0.6–0.7. Expression of the fusion proteins was induced with isopropyl--D-thiogalactopyranoside (1 mM) for 3 h at 30 °C. Bacteria were harvested by centrifugation at 5000 x g for 10 min. The supernatant was discarded, and the bacterial pellet was suspended in 2 ml of ice-cold Nonidet P-40 lysis buffer (1x phosphate-buffered saline buffer containing 0.2% Nonidet P-40, 25 µg/ml aprotinin, 20 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride), sonicated, and centrifuged at 14,000 x g for 10 min at 4 °C. The supernatant was collected and incubated with glutathione-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C with gentle rocking. The GST·PAK or GST·Rhotekin beads were washed twice in Nonidet P-40 lysis buffer and twice in 1x phosphate-buffered saline buffer.

Ras, Rac1, and RhoA Activity Assays—Ras, Rac1, and RhoA activities were assessed by affinity isolation of GTP-bound Ras, Rac1, or RhoA using binding domains of Raf-1, Pak, and Rhotekin, respectively, essentially as described (14, 15). Briefly, cells were lysed into 500 µl of lysis buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 0.5% Nonidet P-40, 0.2% deoxycholic acid, 5 mM MgCl_2, 2.5 mM NaF, 1 mM sodium vanadate, 25 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) at 4 °C for 5 min. Cellular debris was removed by centrifugation at 20,000 x g for 5 min, and supernatants were transferred to new tubes containing 10 µl of Ras-binding domain of Raf-1 (Raf-1·RBD) coupled to agarose beads (Cytoskeleton, Inc.), GST·Rho-binding domain of PAK (PAK·RBD), or GST·Rhotekin·RBD immobilized on glutathione-Sepharose beads. An aliquot of each supernatant was reserved to determine total Ras, Rac1, RhoA, and actin expression by immunoblot analysis. Following a 45-min incubation at 4 °C, the agarose beads were collected by centrifugation, the supernatant was discarded, and the agarose beads were washed twice in wash buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% Nonidet P-40, 5 mM MgCl_2, 2.5 mM NaF, 1 mM sodium vanadate, 25 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). Bound proteins were solubilized by the addition of 25 µl of SDS sample buffer, resolved by SDS-PAGE, and subjected to immunoblot analysis using Ras, Rac1, or RhoA antibody as appropriate. Cells were incubated for 24 h in serum-free medium and stimulated with 5% fetal bovine serum for the times indicated in the figure legends prior to assay. Ras and Rac1 activity was quantitated by densitometry.

Invasion Assay—Cell invasion was assayed using Matrigel-coated Transwell cell culture chambers (6.5-mm diameter, 8-µm pore size; BD Biosciences). Cells maintained for 24 h in serum-free-medium were trypsinized, resuspended in serum-free DME medium, and placed in the upper chamber of the Transwell insert (5 x 10⁴ cells/well), and DME medium containing 10% fetal bovine serum was added to the lower chamber. After 22 h at 37 °C, 5% CO₂, non-invasive cells in the upper chamber were removed, and invasive cells were fixed in 100% methanol and stained with 0.5% crystal violet in 2% ethanol. Five random microscopic fields at x400 magnification were counted in each filter using a calibrated ocular grid. Experiments were carried out in triplicate. The data are expressed as the average number of cells/field ±S.E. In some cases, the data are presented as -fold change from RIE cells, with the value for RIE cells set as 1. In some cases, cells were treated with LY379196 (50 nM), U0126 (10 µM), or celecoxib (25 µM) for 24 h prior to being subjected to the invasion assay using medium containing the appropriate inhibitor at the concentration indicated above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
PKCII Induces an Invasive Phenotype in RIE Cells—We previously characterized transgenic mice expressing elevated levels of PKCII in the colonic epithelium (7, 9, 10). These mice exhibit colonic hyperproliferation and enhanced colon carcinogenesis (9). To elucidate the molecular mechanisms by which PKCII promotes colon carcinogenesis, we established a cell model system to dissect PKCII-mediated signaling pathways (10, 11). RIE cells are immortalized but not transformed and, like most cells within the intestinal epithelium, express little or no PKCII (Fig. 1A). Therefore, we established RIE cells stably expressing human PKCII by transgenesis (Fig. 1A). Surprisingly, RIE/PKCII cells acquire a highly invasive phenotype (Fig. 1B). Treatment of RIE and RIE/PKCII cells with the selective PKC inhibitor LY379196 (16) blocked invasion of RIE/PKCII cells while having no effect on RIE cells (Fig. 1C). Unlike RIE/PKCII cells, RIE cells expressing a kinase-deficient PKCII mutant (RIE/kdPKCII) do not exhibit enhanced invasion (Fig. 1D). Taken together, these results demonstrate that PKCII induces an invasive phenotype that is dependent upon PKCII activity.

View larger version (21K): [in this window] [in a new window]   FIG. 1. PKCII induces an invasive phenotype in RIE cells. A, overexpression of human PKCII in RIE cells. RIE cells were stably transfected with either a control pBABE retrovirus expression vector (RIE) or a pBABE vector containing the full-length human PKCII cDNA (RIE/PKCII). Lysates from each cell line were subjected to immunoblot analysis using antibodies to PKCII and actin as described under "Experimental Procedures." B, RIE/PKCII cells acquire an invasive phenotype. The invasive potential of RIE and RIE/PKCII cells was determined using Matrigel-coated Transwell chambers as described under "Experimental Procedures." The data represent invasiveness relative to RIE cells and are the mean of triplicate determinations ± S.E. *, p < 0.02. C, LY379196 blocks invasion of RIE/PKCII cells. RIE and RIE/PKCII cells were assessed for invasion in the absence or presence of 30 nM LY379196 as described under "Experimental Procedures." Values represent the mean number of invading cells in triplicate determinations ±S.E. *, p < 0.05 versus RIE cells; **, p < 0.05 versus RIE/PKCII cells treated with Me2SO (DMSO). D, expression of kinase-deficient PKCII (kdPKCII) does not induce an invasive phenotype in RIE cells. RIE, RIE/PKCII, and RIE/kdPKCII cells were assayed for invasion as described in C above. Values represent the mean number of invading cells in triplicate determinations ± S.E. *, p < 0.03 versus RIE cells; **, p < 0.05 versus RIE/PKCII cells.

PKCII Activates K-Ras in RIE Cells—

View larger version (38K): [in this window] [in a new window]   FIG. 2. PKCII-mediated invasion requires Mek activity but not Cox-2. A, RIE and RIE/PKCII cells were subjected to immunoblot analysis for K-Ras, H-Ras, and actin as described under "Experimental Procedures." B, RIE and RIE/PKCII cells were incubated in the absence of serum for 24 h to induce quiescence and then stimulated with 5% fetal bovine serum (FBS) for the indicated times. Cells were lysed and assayed for Ras activity using the Ras-binding domain of Raf-1 as an affinity ligand. Ras-GTP and total Ras were detected by immunoblot analysis. Data are representative of three independent experiments. C, RIE and RIE/PKCII cells were incubated in the absence of serum for 24 h as described in A or maintained in serum-containing medium. Cell lysates were subjected to immunoblot analysis for phospho-Mek, total Mek, and actin. D, RIE and RIE/PKCII cells were incubated in the presence of the Mek inhibitor U0126, the Cox-2 inhibitor celecoxib, or diluent control and assayed for invasion as described under "Experimental Procedures." Values represent the mean number of invading cells in triplicate determinations ± S.E. *, p < 0.05 versus RIE cells; **, p < 0.05 versus RIE/PKCII in the absence of U0126; ***, p < 0.05 versus RIE cells and not significantly different from RIE/PKCII cells.

We recently demonstrated that PKCII induces Cox-2 and suppresses TGFRII in RIE/PKCII cells and that Cox-2 activity is required for PKCII-mediated resistance to TGF- and suppression of TGFRII expression (11). However, treatment of RIE and RIE/PKCII cells with the selective Cox-2 inhibitor celecoxib had no effect on the invasive behavior of either RIE or RIE/PKCII cells, demonstrating that PKCII-mediated invasion does not require Cox-2 activity (Fig. 2D).

PKC II Activates Rac1, but Not RhoA, in RIE Cells—Oncogenic Ras-mediated invasion requires the GTPase activity of Rac1 (13, 23). Interestingly, RIE/PKCII cells exhibit elevated levels of GTP-bound, active Rac1 when compared with RIE cells (Fig. 3A). Densitometric analysis of multiple experiments demonstrated that RIE/PKCII cells contain 5-fold higher Rac1 activity than RIE cells without an increase in the total levels of Rac1 (Fig. 3B). RIE and RIE/PKCII cells exhibit a similar, low level of RhoA activity in the absence of serum (Fig. 3C). Furthermore, treatment with serum leads to activation of RhoA to a similar extent in RIE and RIE/PKCII cells. Therefore, PKCII induces activation of Rac1 but not of the related GTPase RhoA in RIE/PKCII cells. LY379196 reduces Rac1 activity in RIE/PKCII cells to levels comparable with those observed in RIE cells while having no effect on Rac1 activity in RIE cells (Fig. 4A). Furthermore, although RIE/PKCII cells exhibit elevated Rac1 activity, RIE/kdPKCII cells exhibit Rac1 activity indistinguishable from RIE cells (Fig. 4B). Taken together, these results demonstrate that PKCII-mediated Rac1 activation, like cell invasion, requires PKCII activity.

View larger version (16K): [in this window] [in a new window]   FIG. 3. PKCII activates Rac1 in RIE cells. A, RIE and RIE/PKCII cells were grown in DME medium containing 5% fetal bovine serum for 2 days to a confluence of 70%. Cells were lysed and assayed for Rac1 activity (top panel), total Rac1 (middle panel), and actin (lower panel) as described under "Experimental Procedures." B, Rac1 activity is plotted for RIE and RIE/PKCII cells relative to RIE cells. Data represent the mean ± S.E. from four independent determinations. *, p < 0.002 versus RIE cells. C, RIE and RIE/PKCII cells were maintained in serum-free medium for 24 h to induce quiescence. Some cultures were then treated with 5% fetal bovine serum for 24 h. Lysates were assayed for RhoA activity (top panel), total RhoA (middle panel), and actin (lower panel). Data are representative of three independent experiments.

View larger version (30K): [in this window] [in a new window]   FIG. 4. PKCII-mediated invasion requires Rac1 and PKC. A, RIE and RIE/PKCII cells were assayed for Rac1 activity (top panel), total Rac1 (middle panel), and actin (lower panel) in the absence and presence of 50 nM LY379196. Data are representative of three independent experiments. B, RIE, RIE/PKCII, and RIE/kdPKCII cells were assayed for Rac1 activity (top panel), total Rac1 (middle panel), and actin (lower panel). Data are representative of three independent experiments. C, RIE, RIE/PKCII, RIE/RacN17, and RIE/PKCII/RacN17 cells were assayed for invasion. *, p < 0.05 versus RIE cells; **, p < 0.05 versus RIE/PKCII cells in the absence of RacN17. D, RIE, RIE/PKCII, RIE/kdPKC, and RIE/PKCII/kdPKC cells were assayed for invasion. *, p < 0.05 versus RIE cells; **, p < 0.05 versus RIE/PKCII cells in the absence of kdPKC. The data represent invasiveness relative to RIE cells and are the mean of triplicate determinations ± S.E.

PKCII-mediated Invasion Requires Rac1 and the Atypical PKC Isozyme, PKC—

RIE cells transfected with a constitutively active Rac1 mutant (RIE/RacV12 cells) exhibit an invasive phenotype similar to that observed in RIE/PKCII cells (Fig. 5A). Furthermore, although invasion of RIE/PKCII cells is blocked by LY379196, RIE/PKCII cells expressing RacV12 exhibit an invasive phenotype that is not blocked by LY379196 (Fig. 5B). Thus, Rac1 functions downstream of PKCII and is sufficient to induce invasion. RIE/RacV12 cells exhibit elevated levels of phospho-Mek (Fig. 5C), consistent with reports that Rac1 can activate Mek (29, 30). Furthermore, invasion of RIE/RacV12 cells is blocked by U0126, indicating that active Mek is required for RacV12-mediated invasion (Fig. 5D). Taken together, these results demonstrate that Rac1 is the relevant downstream target for PKCII-mediated invasion and that active Rac1 is both necessary and sufficient to induce an invasive phenotype in RIE cells through activation of Mek.

View larger version (26K): [in this window] [in a new window]   FIG. 5. PKCII-mediated invasion requires Rac1 and PKC. A, RIE and RIE/RacV12 cells were assayed for invasion. *, p < 0.05 versus RIE cells. B, RIE, RIE/PKCII, and RIE/PKCII/RacV12 cells were assayed for invasion in the absence (-) and presence (+) of 30 nM LY379196. *, p < 0.05 versus RIE cells and not different from each other; **, p < 0.05 versus RIE/PKCII cells in the absence of LY379196. C, RIE and RIE/RacV12 cells were subjected to immunoblot analysis from phospho-Mek, Mek, and actin. D, RIE and RIE/RacV12 cells were assayed for invasion in the presence or absence of 10 µM U10126 [GenBank] . *, p < 0.05 versus RIE cells; **, p < 0.05 versus RIE/RacV12 cells in the absence of U0126. For A, B, and D, values represent the mean of triplicate determinations ± S.E.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have used rat intestinal epithelial cells as a model of the normal colonic epithelium to investigate the molecular mechanisms by which PKCII promotes colon carcinogenesis (10, 11). Expression of PKCII in RIE cells induces several aspects of the transformed phenotype, including the acquisition of TGF- resistance and an invasive phenotype (Fig. 6). RIE cells, like normal colonic epithelial cells, are growth-inhibited by TGF- (9). However, RIE/PKCII cells no longer respond to TGF- (9). We have demonstrated that PKCII induces a TGF--resistant state in RIE cells through activation of a novel PKCII Cox-2 TGFRII signaling axis (11). We also demonstrated that this pathway operates in the colonic epithelium of transgenic PKCII mice in vivo and that dietary -3 fatty acids exert their chemopreventive effects on colon carcinogenesis, at least in part, through inhibition of this pathway (11). Cox-2 is frequently overexpressed in colon cancers (31) and colon cancer cell lines (32, 33) and has been implicated in multiple functions critical to colon carcinogenesis including angiogenesis (34), tumor cell proliferation (35, 36), invasiveness (37), and metastatic potential (38–41). Our data are consistent with the role of Cox-2 in the loss of TGF- responsiveness, which is observed in the vast majority of colon cancers and colon cancer cell lines (42, 43). PKCII-mediated induction of Cox-2 provides a plausible mechanism by which Cox-2 expression and TGF- resistance is induced in the early stages of colon carcinogenesis. PKCII may also be responsible for maintaining Cox-2 expression and TGF- resistance in established colon cancer cell lines and colon cancers.

View larger version (16K): [in this window] [in a new window]   FIG. 6. Schematic showing the two PKCII-mediated signaling pathways responsible for TGF- resistance and invasion in RIE cells. Lines with arrows denote a signaling pathway. A line with a circled plus sign denotes the transcriptional activation of the gene by PKCII; a line with a circled minus sign denotes the suppression of gene expression by PKCII.

PKCII induces the activity of the Ras effector, Rac1, a critical downstream effector of Ras transformation (13). Rac1 is essential for Ras-mediated changes in the actin cytoskeleton that induce invasion (14) and is required for PKCII-mediated invasion. Expression of a constitutively active Rac1 mutant, RacV12, is sufficient to induce invasion in RIE cells in the absence of PKCII. Furthermore, invasion in RIE/PKCII cells transfected with RacV12 is no longer blocked by LY379196, demonstrating that active Rac1 is sufficient to induce PKCII-independent invasion. Interestingly, RacV12 induces activation of Mek, the activity of which is required for RacV12-induced invasion. Taken together, these data provide compelling evidence that both Rac1 and Mek are critical effectors of PKCII-induced invasion. The proinvasive activity of PKCII can be explained through the activation of Ras, which is capable of activating both Rac1 and Mek. The fact that RacV12 can also activate Mek in the absence of PKCII suggests that Rac1 is the critical effector of invasion. The relative contribution of the canonical Ras/Raf pathway, and of Rac1, in the activation of Mek-dependent invasion will require further experimentation.

Our data are consistent with earlier reports that PKC isozymes are important regulators of cytoskeletal function (24, 44–46). PKCII has been shown to interact with the actin cytoskeleton (44). Here we demonstrate that PKCII activates Rac1, and for the first time, demonstrate the functional importance of PKC-mediated activation of Rac1. We recently demonstrated that like Rac1, the atypical PKC isozyme, PKC, is required for Ras-mediated transformation (8). PKC resides between Ras and Rac1 in a pathway required for Ras-mediated invasion and anchorage-independent growth (8). PKCII-mediated invasion is also dependent upon PKC, providing further evidence for the involvement of Ras in PKCII-induced invasion. PKC resides in a complex with Rac1 and Par6 that regulates the polarity of epithelial cells (25–27). Our present data indicate that this complex is also involved in the process of cellular invasion through upstream input from PKCII and activated Ras. Future studies will be aimed at determining the role of PKC/Par6/Rac1 complexes in transformation and invasion.

Taken together, our data are consistent with a model in which PKCII induces invasion through activation of a PKCII Ras PKC/Rac1 Mek signaling pathway (Fig. 6). Our data have important implications for the role of PKCII in colon carcinogenesis. We previously demonstrated that induction of PKCII expression occurs very early in colon carcinogenesis, prior to acquisition of oncogenic mutations such as activated Ras. PKCII activates cellular Ras, suggesting that PKCII activation represents a phenocopy of an activated ras allele. Thus, the pathway delineated in the present report could explain the critical role of PKCII in early carcinogenesis, prior to acquisition of a Ras mutation. An important question is whether PKCII is required to maintain the transformed phenotype of colon cancer cells harboring a Ras mutation or whether it becomes dispensable in the presence of oncogenic Ras. We have found that many human colon cancer cell lines express elevated levels of PKCII regardless of Ras mutational status, suggesting that PKCII plays an important function in the transformed phenotype of colon cancer cells even in the presence of oncogenic Ras. In this regard, PKCII may be required to maintain the TGF--resistant state present in virtually all colon cancer cell lines and colon cancers or to maintain other aspects of the transformed phenotype not yet identified. Future studies will investigate the role of PKCII in transformed colon cancer cells.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: The Mayo Clinic Comprehensive Cancer Center, Griffin Cancer Research Bldg., Rm. 312, 4500 San Pablo Rd., Jacksonville, FL 32224. Tel.: 904-953-6109; Fax: 904-953-0277; E-mail: fields.alan{at}mayo.edu.

¹ The abbreviations used are: PKC, protein kinase C; RIE, rat intestinal epithelial; Mek, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; TGF-, transforming growth factor ; TGFRII, TGF- receptor type II; PAK, p21-activated kinase; DME, Dulbecco's modified Eagle's; GFP, green fluorescent protein; GST, glutathione S-transferase; RBD, Ras-binding domain; kd, kinase-deficient.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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