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Originally published In Press as doi:10.1074/jbc.M305734200 on November 7, 2003

J. Biol. Chem., Vol. 279, Issue 5, 3300-3307, January 30, 2004
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Requirement of Protein Kinase Cµ Activation and Calpain-mediated Proteolysis for Arachidonic Acid-stimulated Adhesion of MDA-MB-435 Human Mammary Carcinoma Cells to Collagen Type IV*

Sarah B. Kennett, John D. Roberts{ddagger}, and Kenneth Olden

From the Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, June 2, 2003 , and in revised form, October 17, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Arachidonic acid (AA) stimulation of adhesion of human metastatic breast carcinoma cells to collagen type IV depends on the protein kinase C (PKC) pathway(s) and is associated with the translocation of PKCµ from the cytoplasm to the membrane. In the present study, we have further explored the role of PKCµ in AA-stimulated adhesion. PKCµ activation site serines 738/742 and autophosphorylation site serine 910 are rapidly phosphorylated, and in vitro PKCµ kinase activity is enhanced in response to AA treatment. Inhibition of PKCµ activation blocks AA-stimulated adhesion. A phosphorylated, truncated species of PKCµ was detected in AA-treated cells. This 77-kDa protein contains the kinase domain but lacks a significant portion of the regulatory domains. Inhibition of calpain protease activity blocks generation of the truncated protein, promotes accumulation of the activated, full-length protein in the membrane, and blocks the AA-mediated increase in adhesion. p38 MAPK activity is also required for AA-stimulated adhesion. Activation of PKCµ and p38 are independent events. However, inhibition of p38 activity reduces calpain-mediated proteolysis of PKCµ and in vivo calpain activity, suggesting a role for p38 in regulation of calpain activity and a point for cross-talk between the PKC and MAPK pathways. These results support the hypothesis that AA stimulates activation of PKCµ, which is cleaved by calpain at the cell membrane. The resulting truncated kinase, as well as the full-length kinase, may be required for increased cell adhesion to collagen type IV. Additionally, these studies present the first evidence for calpain cleavage of a non-structural protein leading to the promotion of tumor cell adhesion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell-extracellular matrix (ECM)1 adhesion is a necessary component of several important biological processes, including tumor cell metastasis. Studies have shown that increased cellular adhesion to ECM proteins, such as collagen type IV, often increases metastatic potential (1). Cell-ECM interactions are dependent on cell-surface adhesion molecules, such as integrins and selectins, and are influenced by the microenvironment. We are interested in elucidating pathways through which factors in the microenvironment regulate adhesive properties of human tumor cells.

Arachidonic acid (AA) and its precursor, linoleic acid, are common dietary n-6 cis-polyunsaturated fatty acids (PUFA) that may be present in the extracellular microenvironment. Although there is controversy over the effects of fat in the human diet on the development of breast cancer, there is substantial evidence for an effect of cis-PUFAs in animal models of mammary tumorigenesis and metastasis (2–6). AA is also present in an esterified form in cell membrane phospholipids and can be liberated by the actions of phospholipases. Studies have shown that fatty acids or their metabolites can alter the adhesion of breast tumor and other cell lines to components of the ECM (7–9). For example, we have shown previously (10) that AA stimulates adhesion of metastatic human breast carcinoma MDA-MB-435 cells to collagen type IV and also to vitronectin and fibronectin.

In our earlier studies, we have also shown that adhesion to collagen type IV requires {alpha}1{beta}1 and {alpha}2{beta}1 integrins and functional protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) signaling pathways (11, 12). AA stimulation of adhesion is associated with the translocation of PKC{epsilon} and PKCµ from the cytosolic to the membrane fraction of cell lysates, indicative of enzyme activation (11). PKC{epsilon} and PKCµ belong to the calcium-independent, diacylglycerol-responsive novel PKC (nPKC) family (13). However, the lack of a typical pseudosubstrate domain and the addition of an amino-terminal hydrophobic region and large pleckstrin homology domain-containing region within the regulatory domain make PKCµ unique (14). In addition, the PKCµ consensus substrate site is quite different from that of other PKCs (15, 16). Members of the PKC family are regulated, in part, through phosphorylation events, including phosphorylation at an activation site (17). Activation of PKCµ requires phosphorylation at serines 738/742 (mouse homologue PKD serines 744/748) (16, 18). PKCµ can be activated through direct phosphorylation by PKC{epsilon} or PKC{eta} (19, 20). Interestingly, it has been reported that PKCµ, but not PKC{epsilon}, is present in prostate cancer cells in which linoleic acid acts as a pro-cancer agent (21). It has also been reported that activation of PKC{epsilon} is necessary for muscle cell adhesion to fibronectin (22). PKCµ has also been detected in complexes with cortactin and paxillin in {beta}1 integrin-containing breast cancer cell invadopodia into ECM (23).

Members of the calpain family of calcium-dependent neutral proteases can activate conventional PKC (cPKC) family members ({alpha}, {beta}, and {gamma}) by limited proteolysis (24, 25). PKC{epsilon} has also been shown to be proteolyzed by calpain in vitro (26). cPKCs are cleaved in the third variable region, releasing the kinase domain from the inhibitory pseudosubstrate domain (25). Cytoskeletal proteins are also calpain substrates, and calpain has been found to co-localize with focal adhesions (27). It is therefore not surprising that calpain inhibition or overexpression affects cell adhesion and migration. However, the effects observed following manipulation of calpain activity appear to be cell type-specific. For example, treatment of Chinese hamster ovary cells with calpain inhibitor results in increased adhesiveness, whereas calpain inhibition results in decreased adhesiveness of mast cells and appears to have no effect on adhesiveness of vascular smooth muscle cells (28–30). Up-regulation of calpain protein expression or mRNA has been observed in metastatic prostate tumors and has been associated with lymph node metastases of malignant renal cell carcinomas (31, 32).

MAPK signaling pathways are activated by a wide variety of stimuli and regulate a plethora of cellular responses. We have reported that of the three MAP kinase subfamilies, the extra-cellular signal-responsive kinases (ERK), the c-Jun amino-terminal kinases (JNK), and the p38 MAP kinases, only the p38 MAP kinase pathway is required for AA-stimulated adhesion of MDA-MB-435 cells to collagen type IV (12). p38 is regulated by phosphorylation that increases upon exposure to AA. Concomitant with the AA-induced increase in p38 phosphorylation, there is an increase in the phosphorylation and activity of the downstream effector, MAPKAPK2, and phosphorylation of the MAPKAPK2 substrate, HSP27 (12). PKCµ has been shown to play a role in the regulation of both the ERK and JNK pathways, but no association has been made between PKCµ and the p38 pathway (19, 20, 33, 34).

In this study, we have examined the involvement of PKCµ in the AA-mediated enhancement of MDA-MB-435 cell adhesion to collagen type IV. We have also investigated the possible involvement of calpain in modulation of breast cancer cell adhesion and in proteolysis of PKCµ. Finally, because we previously found that multiple signaling pathways are required for the AA effect, we have begun to explore potential mechanisms of cross-talk between these pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—PKD/PKCµ, phospho-PKD/PKCµ (Ser-744/748), phospho-PKD/PKCµ (Ser-916), HSP27 G31 monoclonal, and phospho-HSP27 (Ser-82) antibodies were from Cell Signaling Technology (Beverly, MA). Anti-active p38 antibody was from Promega (Madison, WI). Anti-SAPK2a/p38 antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY).

Reagents—Arachidonic acid was from Cayman Chemical Co. (Ann Arbor, MI). Collagen type IV and poly-D-lysine were from BD Biosciences. Gö6976, Gö6983, PD169316, SB202474, calpeptin, benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk), BSA, calpain I (human erythrocyte), and calpain II (rat recombinant) were from Calbiochem. SuperSignal chemiluminescent substrate was from Pierce. MDL-28170 was from Biomol (Plymouth Meeting, PA). 7-Amino-4-chloromethylcoumarin, t-Boc-Leu-Met (t-Boc-Leu-Met-CMAC), was from Molecular Probes (Eugene, OR). Syntide-2 was from Sigma. FuGENE 6 was from Roche Applied Science. [{gamma}-32P]ATP was from ICN (Irvine, CA). pcDNA-PKCµ was from Dr. Franz-Josef Johannes (Institute of Cell Biology and Immunology, University of Stuttgart) (35).

Cell Culture and Preparation—The human mammary adenocarcinoma cell line MDA-MB-435 was obtained from Dr. Janet Price (M.D. Anderson Cancer Center, Houston, TX). Cells were maintained in Eagle's minimal essential medium (Invitrogen), supplemented with 5% fetal bovine serum (Hyclone, Logan, UT), 1 mM sodium pyruvate, 2 mM L-glutamine, and 2x minimal essential medium vitamin solution (Invitrogen). Subconfluent cells were harvested by a brief incubation with Versene (Invitrogen), washed twice with serum-free medium, and resuspended in serum-free medium at 3 x 105 cells/ml (adhesion assay) or 2 x 106 cells/ml. Cells were allowed to equilibrate in serum-free medium for 20 min at 37 °C under 5% CO2.

Adhesion Assays—Adhesion assays were carried out essentially as described (10). 96-Well tissue culture plates were pre-coated with collagen type IV, poly-D-lysine, or BSA. Wells were washed with PBS (137 mM NaCl, 2.7 mM KCl, 1 mM KH2PO4, 7.4 mM Na2HPO4), and nonspecific binding sites were blocked with BSA. Inhibitors were added to cells prepared as described above, and cells were incubated for an additional 30 min. AA (30 µM) or vehicle was added to the cells, and 100 µl of the cell suspension was added to each well. Cells were incubated for 45 min, and non-adherent cells were removed by washing with serum-free medium. Adherent cells were fixed with 6% (v/v) glutaraldehyde and stained with 0.5% (w/v) crystal violet. The dye in adherent cells was solubilized with 1% (w/v) SDS, and the absorbance at 595 nm was determined. After background binding to BSA was subtracted, results were standardized as a percentage of adhesion to poly-D-lysine.

Western Blotting—AA (30 µM) or vehicle was added to cells prepared as described above, and cells were incubated for an additional 1 min to 2 h. Inhibitors were added to the cell suspension, and the cells were incubated for an additional 30 min prior to the addition of AA. Cells were collected, washed with PBS, and lysed in 50 mM Tris-HCl, pH 7.4, 1% (v/v) Nonidet P-40, 0.25% (w/v) deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, 1 mM NaF. Lysates were centrifuged at 16,000 x g for 10 min at 4 °C. The samples were resolved by SDS-PAGE and transferred to Immobilon-P membranes. Membranes were blocked in TBS-T (20 mM Tris, 137 mM NaCl, pH 7.6, plus 0.1% (v/v) Tween 20) with 5% (w/v) milk, incubated with primary antibody for 1 h to overnight, and incubated with an horseradish peroxidase-conjugated secondary antibody for 1 h. Immunoreactivity was detected using SuperSignal chemiluminescent substrate. Blots were stripped by incubation in 100 mM glycine, pH 2.9, for 30 min; they were subsequently rinsed with TBS-T, blocked, and probed.

In Vitro Kinase Assay—Cells were treated and lysed as above. PKCµ was isolated by immunoprecipitation with the PKD/PKCµ antibody (1:100 dilution) for 2 h at 4 °C in lysis buffer. Immune complexes were recovered using protein A-Sepharose. Kinase assays were performed essentially as described by Waldron et al. (36). Immune complexes were washed once with lysis buffer and twice with kinase buffer (30 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol). Syntide-2 substrate peptide (5 µg), [{gamma}-32P]ATP (0.4 µCi), and inhibitors, if applicable, were added in a final volume of 30 µl of kinase buffer. Reactions were incubated for 10 min at 30 °C and stopped by addition of 100 µl of 75 mM H3PO4. Reactions (75 µl) were spotted on Whatman P-81 phosphocellulose, washed with 75 mM H3PO4, dried, and quantitated in a scintillation counter. Results were normalized to levels of PKCµ immunoprecipitated from each sample.

Cell Fractionation—Cell fractionation was carried out as described previously (11). Briefly, cells were treated as above and washed with PBS. Cells were resuspended in lysis buffer (20 mM Tris, pH 7.5, 5 mM dithiothreitol, 250 mM sucrose, 2 mM EDTA, 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin), sonicated with three 10-s pulses at 22.5 kHz, and centrifuged at 100,000 x g for 45 min at 4 °C. Supernatants were collected and saved as the cytoplasmic fraction. Pellets were resuspended in lysis buffer plus 1% Triton X-100, sonicated again, and centrifuged. The supernatants were collected and used as the membrane fraction. PKCµ was examined by Western blotting using equal protein, as described above; loading equal protein resulted in a 2-fold difference in cell equivalents between the cytosolic and membrane fraction, with the greater number of cell equivalents in the membrane fraction.

In Vivo Calpain Assay—Cells were harvested as above, resuspended in serum-free medium, and treated with inhibitors, as appropriate. Cells were incubated for 20 min with 10 µM t-Boc-Leu-Met-CMAC, a cell-permeable fluorogenic calpain substrate. Aliquots (200 µl) of cell suspension were loaded into wells of a 96-well plate, and fluorescence generated through calpain cleavage of t-Boc-Leu-Met-CMAC was analyzed using an Fmax microplate reader (Molecular Devices, Sunnyvale, CA) with a 355 nM excitation filter and a 460 nM emission filter.

In Vitro Calpain Assay—Cells were transfected with pcDNA-PKCµ (2 µg/60-mm dish) using FuGENE. After 48 h, cells were treated and lysed as above, and PKCµ was immunoprecipitated as above. Immune complexes were washed 3 times, with the final wash in 50 mM Tris, pH 7.4. Calpain assays were performed essentially as described by Carragher et al. (37). Calpain I or calpain II (0.05 units) was added to immune complexes in calpain buffer (50 mM Tris, pH 7.4, 10 mM CaCl2, 30 mM NaCl, 5 mM {beta}-mercaptoethanol; 40 µl final volume) in the presence or absence of inhibitors. Reactions were incubated at 30 °C for 30 min and stopped by the addition of SDS sample buffer. Cleavage of PKCµ was analyzed by Western blot as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
AA Promotes Activation of PKCµ in MDA-MB-435 Cells— Subsequent to PKCµ/PKD cloning, many aspects of PKCµ biochemistry have been elucidated, but less has been ascertained about its biological functions. One characteristic of PKCµ is that activation requires phosphorylation at serines 738/742. To investigate the role of PKCµ activation in AA-mediated adhesion, we used an antibody specific for serine 738/742 phosphorylation. With this antibody, we detect multiple species of PKCµ, the origins of which are described below. Treatment of MDA-MB-435 cells with AA led to a rapid increase in serine 738/742 phosphorylation (Fig. 1A). Within 1 min of exposure to AA, phosphorylation of PKCµ increased, and PKCµ phosphorylation was stable for approximately 1 h. AA did not induce a change in the levels of total PKCµ protein during this time course (Fig. 1, A and B).



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  		FIG. 1.
AA activates PKCµ in MDA-MB-435 cells. MDA-MB-435 cells were untreated (1st lane) or treated with 30 µM AA (2nd to 6th lanes) or vehicle (EtOH, 7th to 9th lanes) for the indicated times (1–90 min). Proteins from whole cell lysates (50 µg) were analyzed by SDS-PAGE and immunoblotting. A, blot was probed with an antibody that recognizes phosphorylated PKCµ activation site serines 738/742 (phospho-PKD/PKCµ (Ser-744/748)). B, blot was probed with an antibody that recognizes autophosphorylated PKCµ serine 910 (phospho-PKD/PKCµ (Ser-916)). A and B, blots were stripped and probed with a total PKCµ antibody.

 
The activation site serines 738/742 are predominantly transphosphorylated, in this instance probably by PKC{epsilon} (36). However, PKCµ contains additional phosphorylation sites, including an autophosphorylation site at serine 910 (PKD serine 916) (38, 39). Phosphorylation at serine 910 has been correlated with and used as a marker for PKCµ activity (38, 40, 41). A serine 910 phosphorylation-specific antibody revealed AA-induced autophosphorylation of PKCµ that is stimulated as rapidly as activation site phosphorylation (Fig. 1B).

To further establish AA as a modulator of PKCµ activity, we examined the activity of PKCµ purified from MDA-MB-435 cells. PKCµ from AA-stimulated cells showed an increase in in vitro kinase activity within 2 min of AA treatment. The enhancement of activity peaked at an average of 207% of the activity of PKCµ from vehicle-treated cells at ~5 min and extended to at least 10 min. In addition, treatment with higher concentrations of AA led to further stimulation of PKCµ (data not shown). These results, along with the translocation of PKCµ, support the conclusion that AA activates PKCµ.

Effect of PKCµ Inhibitors on AA-mediated Adhesion to Collagen Type IV—PKC inhibitors Gö6976 and Gö6983 inhibit the activity of PKCµ in vitro at vastly different concentrations. Gö6976 inhibits recombinant PKCµ from baculovirus-infected cells in the nanomolar range, whereas Gö6983 inhibits PKCµ in the micromolar range; similar concentrations inhibit in vitro activity of PKCµ purified from MDA-MD-435 cells (42) (data not shown). The capacity of these inhibitors to block PKCµ activity in MDA-MB-435 cells was examined by treating the cells with inhibitor and then stimulating with AA. The results in Fig. 2 demonstrate that both inhibitors block PKCµ activity, as measured by autophosphorylation. These inhibitors did not affect levels of total PKCµ protein (see Fig. 7B). Subsequently, we examined the abilities of Gö6976 and Gö6983 to alter AA-stimulated adhesion of MDA-MB-435 cells to collagen type IV. Cells were incubated with inhibitor for 30 min prior to treatment with AA and plating on collagen type IV. Both inhibitors blocked the AA-mediated increase in adhesion (Fig. 3). A significant decrease in the response to AA was obtained with 20 nM Gö6976, and nearly complete inhibition was seen at 80 nM. Similar levels of inhibition of adhesion required 10–20 µM Gö6983. The inhibitor solvent (Me2SO) did not decrease the AA-stimulated adhesion (data not shown).



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  		FIG. 2.
PKC inhibitors Gö6976 and Gö6983 block PKCµ activity. MDA-MB-435 cells were pretreated with PKC inhibitor Gö6976 (160 nM) or Gö6983 (20 µM) for 30 min. Pretreated cells or cells not exposed to inhibitor (–) were incubated with 30 µM AA for 5 min. Proteins were analyzed by SDS-PAGE and immunoblotting for autophosphorylated PKCµ.

 



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  		FIG. 7.
Activation of PKCµ and p38 are independent events. Cells were pretreated with PKCµ inhibitor Gö6976 (160 nM) or Gö6983 (20 µM), p38 inhibitor PD169316 (10 µM), non-functioning p38 inhibitor analogue SB202474 (10 µM), or vehicle (DMSO) for 30 min prior to exposure to AA. Pretreated or control (–) cells were incubated with 30 µM AA (2nd to 7th lanes) or vehicle (1st lane, EtOH) for 5 min. Proteins were examined by SDS-PAGE and immunoblotting. A, blot was divided and probed with an active (phosphorylated) p38 antibody and with a phospho-HSP27 antibody. The blots were then stripped and probed for total p38 or total HSP27. B, blot was probed with autophosphorylation-specific PKCµ antibody. The blot was then stripped and probed for total PKCµ.

 



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  		FIG. 3.
PKCµ inhibition prevents the AA-mediated enhancement of MDA-MB-435 cell adhesion to collagen type IV. Cells were pretreated with the indicated concentrations of PKC inhibitor Gö6976 (A) or Gö6983 (B) for 30 min prior to exposure to 30 µM AA or vehicle (EtOH) and analysis for adhesion to collagen type IV. Shown is the AA-induced fold increase in adhesion relative to adhesion of vehicle-treated cells. *, values are significantly different from uninhibited control (p < 0.05, Student's t test).

 
Phosphorylated PKCµ Is Subject to Limited Proteolysis by Calpain at the Cell Membrane—The predominant serine 738/742-phosphorylated form of PKCµ that is detected after stimulation with AA is smaller than the full-length protein (Fig. 4). Given previous reports of calpain proteolysis of cPKC isoforms and PKC{epsilon} and the stability of this form of PKCµ (Fig. 1A), we examined the hypothesis that the truncated PKCµ is generated through limited proteolysis by calpain (24–26). MDA-MB-435 cells were treated with a cell-permeable calpain inhibitor, either calpeptin (Fig. 4) or MDL-28170 (Fig. 5), prior to AA exposure. In the absence of calpain inhibitor, a 77-kDa activation site phosphorylated protein was detected, and the amount of phosphorylated protein significantly increased upon addition of AA (Fig. 4). In the presence of calpain inhibitor, the full-length, phosphorylated protein was detected, and AA treatment increased the levels of phosphorylated, full-length PKCµ. Concomitant with the increase in full-length, phosphorylated protein observed when AA-treated cells were pretreated with calpain inhibitor, there was a decrease in the level of proteolyzed, phosphorylated PKCµ, indicating that the truncated protein was produced via calpain cleavage of the full-length protein (Fig. 4).



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  		FIG. 4.
Proteolysis of phosphorylated PKCµ by calpain results in a 77-kDa PKCµ isoform. MDA-MB-435 cells were incubated with calpain inhibitor calpeptin (100 µg/ml) for 30 min prior to addition of AA. Cells were untreated or exposed to 30 µM AA or vehicle (EtOH) for 5 min. Proteins were resolved by SDS-PAGE and analyzed by immunoblotting for activation site-phosphorylated PKCµ. Molecular weights were calculated relative to molecular weight markers.

 



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  		FIG. 5.
Inhibition of calpain activity leads to retention of activated, full-length PKCµ in the cell membrane. MDA-MB-435 cells were treated with calpain inhibitor MDL-28170 (300 µM) for 30 min prior to AA exposure or untreated (–). Cells were treated with 30 µM AA for 2 min or 10 min and then separated into cytosolic (C) and membrane (M) fractions. Proteins (50 µg) from each fraction were analyzed by SDS-PAGE and immunoblotting. The blot was probed with antibody that detects activation site-phosphorylated PKCµ.

 
We have previously shown translocation of total PKCµ from the cytosolic fraction to the membrane fraction in lysates of MDA-MB-435 cells treated with AA (11). Cell fractionation revealed that proteolyzed, activated PKCµ was localized predominantly in the cytosolic fraction of cells treated with AA (Fig. 5). Pretreatment of cells with calpain inhibitor allowed detection of phosphorylated, full-length PKCµ; this form was also localized to the cytosolic fraction. When calpain inhibitor-treated cells were exposed to AA, full-length, activated PKCµ accumulated in the membrane, and levels of the proteolyzed PKCµ decreased relative to the levels observed in cells not pretreated with calpain inhibitor (Fig. 5). These results indicate that activated, full-length PKCµ translocates to the membrane where it is converted to the 77-kDa form through limited proteolysis by calpain.

Calpain Activity Is Required for the AA-mediated Enhancement of MDA-MB-435 Cell Adhesion to Collagen Type IV— Calpain is active in MDA-MB-435 cells following AA stimulation; however, calpain activity may be unrelated to the increased adhesion that is triggered by AA. To determine whether calpain is required for enhancement of MDA-MB-435 cell adhesion, we analyzed cells that had been pretreated with calpain inhibitor for adhesion to collagen type IV upon exposure to AA. The addition of calpain inhibitor MDL-28170 decreased the AA-induced enhancement of cell adhesion to collagen type IV in a dose-dependent manner, with a significant decrease in adhesion seen at 75 µM (Fig. 6A). The concentrations of calpain inhibitor used in these assays did not affect the ability of vehicle-treated cells to adhere to collagen type IV nor the ability of AA- or vehicle-treated cells to adhere to poly-D-lysine (Fig. 6A and data not shown). As shown in Fig. 6B, the decrease in adhesion to collagen type IV parallels the increase in full-length, phosphorylated PKCµ that accumulates when cells that have been pretreated with calpain inhibitor are stimulated with AA. In fact, an approximate 50% increase in full-length, phosphorylated PKCµ corresponds to an approximate 50% decrease in AA-stimulated adhesion.



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  		FIG. 6.
Calpain activity is required for the AA-induced increase in adhesion of MDA-MB-435 cells to collagen type IV. Cells were pretreated with the indicated concentrations of calpain inhibitor MDL-28170 for 30 min. AA (30 µM) or vehicle (EtOH) was added to the cells. A, the cells were assayed for adhesion to collagen type IV. Shown is the AA-induced fold increase in adhesion relative to adhesion of vehicle-treated cells. *, values are significantly different from uninhibited control (p < 0.05). B, the cells were lysed, and proteins were examined by SDS-PAGE and immunoblotting for activation site-phosphorylated PKCµ and total PKCµ. The relative amounts of full-length, activated PKCµ were determined by densitometry and are expressed as a percent of the full-length, activated PKCµ observed at the highest concentration of calpain inhibitor. The corresponding levels of AA-induced adhesion are expressed as a percent of the maximal level of adhesion observed in cells not treated with calpain inhibitor.

 
Active PKCµ Is Not Required for Activation of p38 MAPK and Active p38 MAPK Is Not Required for Activation of PKCµ—We reported previously (12) that in addition to the PKC pathway, p38 MAPK is required for the AA-induced increase in adhesion. Given the recent reports (19, 20, 33, 34) of PKCµ effects on other MAPK pathways, we examined the effects of PKCµ inhibition on activation of p38 during AA stimulation. Cells were treated with inhibitor followed by AA, and cell lysates were analyzed for p38 activity by immunoblotting with an antibody that recognizes active (phosphorylated) p38. Pretreatment with the p38 inhibitor PD169316 led to inhibition of AA-stimulated p38 activation and to inhibition of phosphorylation of the down-stream target HSP27, whereas pretreatment with the PKC inhibitor Gö6976 or Gö6983 did not affect p38 activation nor HSP27 phosphorylation (Fig. 7). Similarly, the non-functional analogue of the p38 inhibitor SB202474 did not affect p38 activation (Fig. 7). In a parallel experiment, we examined the potential effects of inhibition of p38 activity on the activation of PKCµ. p38 inhibitor PD169316 pretreatment of cells did not affect AA-induced autophosphorylation of PKCµ, whereas the PKC inhibitor Gö6976 or Gö6983 blocked PKCµ activity, indicating that activation of PKCµ and activation of p38 are independent events (Fig. 7).

Active p38 Is Required for Full Calpain Activity and Complete Proteolysis of PKCµ—The PKCµ and p38 pathways appear not to interact upstream from activation of these kinases. However, a downstream step in the adhesion process where signals from each pathway could merge is the cleavage of PKCµ by calpain. To examine this possibility, we analyzed the effects of p38 inhibition on cleavage of phosphorylated PKCµ. Incubation of cells with p38 inhibitor PD169316 prior to AA exposure resulted in a significant increase in the level of serine 738/742 phosphorylated, full-length PKCµ and a decrease in the level of phosphorylated, truncated PKCµ, compared with cells not exposed to inhibitor or cells treated with the non-functional analogue SB202474 (Fig. 8A). The amount of full-length, activated PKCµ detected in the presence of p38 inhibitor is not equivalent to the amount of full-length, phosphorylated protein observed when calpain is directly inhibited by using MDL-28170; however, these results indicate that active p38 plays a role in regulating PKCµ cleavage by calpain. In vitro calpain assays were used to examine the specificity of the p38 inhibitor. Both purified calpain I and purified calpain II proteolyze purified PKCµ in vitro (Fig. 8B). In the presence of calpain inhibitor MDL-28170, proteolysis is blocked; however, in the presence of p38 inhibitor PD169316 or the non-functional analogue SB202474, PKCµ is proteolyzed by calpain (Fig. 8B). These results indicate that the p38 inhibitor is not directly inhibiting calpain but is blocking the ability of p38 to regulate calpain.



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  		FIG. 8.
p38 activity is required for complete processing of activated PKCµ by calpain. A, MDA-MB-435 cells were treated with p38 inhibitor PD169316 (10 µM), non-functioning p38 inhibitor analogue SB202747 (10 µM), or calpain inhibitor MDL-28170 (300 µM) for 30 min prior to AA exposure. Inhibitor-treated or untreated cells were incubated with 30 µM AA (+) or vehicle (–, EtOH) for 5 min. Proteins were analyzed by SDS-PAGE and immunoblotting. The blot was divided and probed for activation site-phosphorylated PKCµ and active p38. The p38 blot was stripped and probed for total p38. B, MDA-MB-435 cells were transfected with vector (1st lane) or pcDNA-PKCµ (2nd to 6th lanes). PKCµ was purified via immunoprecipitation and examined in calpain cleavage assays in the presence (+, 3rd to 6th lanes) or absence (–, 1st and 2nd lanes) of recombinant calpain I (top) or calpain II (bottom). Inhibitors were added to the assays at the concentrations used above. Reactions were analyzed by SDS-PAGE and immunoblotting for total PKCµ.

 
Further evidence for a role of p38 in modulation of calpain activity was obtained by using an in vivo assay for calpain activity with a fluorogenic substrate. The cell-permeable calpain substrate, t-Boc-Leu-Met-CMAC, emits fluorescent light upon cleavage. Incubation of MDA-MB-435 cells with calpain inhibitor or with p38 inhibitor prior to addition of calpain substrate reduced the resulting fluorescence relative to control cells (Table I). Calpain inhibitor reduced fluorescence to levels approaching those seen in cells containing no substrate. Inhibition of p38 reduced calpain activity by 57%, whereas addition of the non-functioning analogue of the 38 inhibitor led to only a 23% reduction, suggesting that p38 is one regulator of calpain activity in MDA-MB-435 cells (Table I).


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  		TABLE I
Calpain activity in MDA-MB-435 cells is dependent on active p38

Cells were pretreated with calpain inhibitor MDL-28170 (300 µM), p38 inhibitor PD169316 (10 µM), or the non-functioning analogue of the p38 inhibitor SB202474 (10 µM) for 30 min. Cell-permeable fluorescent calpain substrate (t-BOC-Leu-Met-CMAC, 10 µM) was added to pretreated or untreated (-) cells, and cells were allowed to incubate for an additional 20 min. Fluorescence resulting from calpain cleavage was detected using an Fmax microplate reader. Results are representative of three experiments performed in triplicate. RFU, relative fluorescence units.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In these studies we have examined the signal transduction pathways that relay the information required for cis-PUFA promotion of breast cancer cell adhesion to ECM. We had shown previously (11) that PKC pathways were involved and that PKC{epsilon} and PKCµ were probable mediators of this signaling. Biological functions of PKCµ have not yet been well documented, but PKCµ is involved in transport through the Golgi and in regulation of apoptosis, DNA synthesis, and some MAPK signaling pathways (20, 35, 40, 41, 43–45). In this report, we have further implicated PKCµ in regulation of the adhesion process, which is an important aspect of the metastatic cascade. We show that PKCµ rapidly becomes activated upon exposure of MDA-MB-435 breast cancer cells to AA and present the first evidence of a requirement for PKCµ in AA-enhanced cell adhesion to ECM. We show for the first time that phosphorylated PKCµ is a target for calpain-mediated proteolysis and that calpain activity is also required for AA-enhanced breast cancer cell adhesion to ECM. Finally, we present data that suggest the regulation of calpain is partially controlled by p38 MAPK.

In response to AA, PKCµ is rapidly phosphorylated at activation site serines and acquires the capacity for autophosphorylation. These events lead to increased PKCµ kinase activity, as measured by in vitro assays. PKC inhibitors that block activity of PKCµ inhibit the enhancement of MDA-MB-435 cell adhesion imparted by AA. The inhibitor Gö6976 affects cPKCs as well as PKCµ; however, we have no evidence for activation of cPKCs by AA (42). Gö6976 does not affect other nPKCs, such as PKC{epsilon}, at low concentration (reviewed in Ref. 38). Therefore, these results indicate that active PKCµ is required for adhesion. The specificity and concentration requirements of Gö6983 in vivo have been less well documented. This inhibitor is active on PKCµ in vitro only at micromolar concentrations, whereas it is active on cPKCs and some other nPKCs in vitro at nanomolar concentrations; PKC{epsilon} was not examined in that study (42). The high concentration of Gö6983 required for inhibition of adhesion of the MDA-MB-435 cells in response to AA implicates PKCµ as responsible for signal transduction. Immunoblotting for autophosphorylated PKCµ reveals that this concentration of Gö6983 does, indeed, block in vivo activity of PKCµ. However, it remains possible that the block in PKCµ activity is due to inhibition of activation of PKCµ through a block in the activity of PKC{epsilon}. PKCµ is an in vitro substrate for PKC{epsilon}; PKCµ and PKC{epsilon} form co-immunoprecipitable complexes in vivo, and overexpression of PKC{epsilon} or co-expression of PKC{epsilon} with the upstream phosphoinositide-dependent kinase leads to activation of PKCµ in HEK293 cells (20, 46).

PKCµ that has been activated by phosphorylation at activation site serines appears to translocate to the cell membrane and become rapidly proteolyzed. In some cell types, activated calpain localizes to the cell membrane (47, 48). Inhibition of calpain blocks PKCµ proteolysis. Calpain cleaves cPKCs, releasing a truncated, active kinase, which has been termed PKM (25, 49). More recently, calpain has been shown to cleave PKC{epsilon} in vitro, also resulting in release of a catalytically active fragment (26). Experiments have suggested that the activated form of cPKC is the preferred target for calpain-mediated proteolysis (24, 25). A consensus cleavage site for calpain has not been defined. Amino acid preferences determined by examination of calpain cleavage of peptides and histones do not account for calpain cleavage of all substrates; however, these studies have also suggested that calpain recognizes higher order structures (50). The region containing the calpain cleavage site of cPKCs does not have an exact PKCµ counterpart. cPKCs are cleaved in the V3 "hinge region" between the C2 calcium-binding domain region and the C3 kinase domain region (25). However, amino-terminal to the PKCµ kinase domain is the pleckstrin homology domain rather than a classical V3 region (14). Based on the size of the truncated protein, it appears that PKCµ is cleaved in or near the second cysteine-rich region. Given that calpain recognizes inter-domain regions of its protein substrates, we speculate that the calpain cleavage site for PKCµ resides at the amino or carboxyl terminus of the second cysteine-rich "zinc finger" region and that the truncated protein would contain the entire kinase domain. PKD mutants that lack one or both cysteine-rich domains are constitutively active kinases (51). There is some discrepancy between these findings and results of experiments performed with PKCµ; however, different experimental systems were employed (52). In addition, a 14-3-3 protein binding domain, which has been associated with inhibition of PKCµ in T-cells, would be removed upon truncation (53). These findings lead us to believe that the calpain-proteolyzed PKCµ is catalytically active. Our hypothesis is supported by the report that PKCµ that has been proteolyzed by caspase-3 is a constitutively active kinase. Caspase-3 cleaves PKCµ at amino acid 378, between the second cysteine-rich region and the pleckstrin homology domain, generating a smaller 60-kDa protein (54). As has been reported for caspase inhibitors, we have found the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk) (caspases 1, 3, 4, and 7) to be nonspecific in vitro, i.e. this inhibitor will block calpain I or calpain II activity (55).2 However, the appearance of the 77-kDa phosphorylated form of PKCµ/PKD in both MCF-7 cells, which lack caspase-3, and in NIH-3T3 cells, in which PKD lacks the caspase-3 cleavage site, indicates that the 77-kDa isoform is a distinct species (56).2

Calpain activity is required for the AA-mediated enhancement of MDA-MB-435 cell adhesion to collagen type IV. Calpain inhibitor blocks the increase in adhesion of cells that have been treated with AA but does not affect the adhesive capacity of cells that have been treated with vehicle nor the ability of cells to bind to poly-D-lysine. Therefore, the decrease in adhesion due to calpain inhibition is specific for the AA-stimulated signaling pathway and not a result of nonspecific alterations in calpain-mediated effects on cytoskeletal proteins. Adhesion of human umbilical vein endothelial cells to prothrombin substrate is also blocked by treatment with either PKC inhibitor or calpain inhibitor; however, the calpain target is unknown (57). In addition, T-cell adhesion to fibronectin that has been induced by T-cell receptor cross-linking or 12-O-tetradecanoyl-phorbol-13-acetate treatment is sensitive to calpain inhibition. In this case, the decrease in adhesion has been linked to a block in proteolysis of protein-tyrosine phosphatase 1B (58).

PKCµ regulates the ERK and JNK pathways in some cell types but has not yet been reported to affect p38 (19, 20, 33, 34). Given our previous observations that p38 is activated on exposure to AA and that both the p38 and PKC pathways are required for AA-enhanced adhesion, we examined these pathways for cross-talk (11, 12). Activation of either kinase is not dependent on activity of the other, but proteolysis of PKCµ by calpain is regulated, in part, by active p38. In MDA-MB-435 cells, active p38 is required for at least a portion of calpain activity. This represents one step in the AA-stimulated adhesion process where signals from each pathway could merge. Calpain can be phosphorylated in vitro; however, it is not known how this modification affects activity (27, 59).

In this report, we have presented evidence to support our hypothesis that PKCµ is involved in AA-stimulated adhesion of metastatic human mammary carcinoma cells to collagen type IV, and we have introduced calpain as a mediator of PKCµ signaling. In our current model (Fig. 9), AA stimulates activation of PKC{epsilon} via signaling by a lipoxygenase metabolite of AA. Active PKC{epsilon} then phosphorylates PKCµ at activation site serines. PKCµ translocates to the cell membrane where it is proteolyzed by calpain into a catalytically active 77-kDa molecule that is localized to the cytosol. p38 may play a role in funneling signals from AA to the PKC pathway through regulation of calpain activity. p38, which is also activated via AA signaling, may also mediate adhesion through activation of MAPKAPK2 and phosphorylation of HSP27, or through interactions with cytoskeletal proteins at focal adhesions.3



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  		FIG. 9.
Model of the signaling pathways involved in the AA-induced increase in mammary tumor cell adhesion to ECM proteins. AA or its metabolite, hydroxyeicosatetraenoic acid (HETE), induces activation of PKC{epsilon}, which, in turn, activates PKCµ. Activated PKCµ translocates to the cell membrane, where it undergoes proteolysis by calpain to become an active cytosolic kinase. Calpain is regulated, in part, by p38, which is also activated through AA signaling. Each of these enzymes, PKCµ, p38, and calpain, may be involved in the activation of {beta}1 integrins and cytoskeletal rearrangements required for cell adhesion to ECM.

 

Few in vivo PKCµ substrates are known, and the targets for PKCµ in this system have not yet been identified. We speculate that both the full-length and the truncated PKCµ isoforms may act as important signaling molecules in the adhesion process. Both full-length and truncated cPKC isoforms are catalytically active, and there is precedent for distinct activities of each kinase as described for PKC{alpha}/PKM (60). PKC{alpha} and PKM function in alternatively activated signal transduction pathways that lead to similar phosphorylation events but have different signaling requirements (60). We expect that identification of the targets of PKCµ will further the understanding of both the AA-stimulated adhesion process and the biological functions of PKCµ. As we progress toward defining the signal transduction mechanisms that regulate AA-mediated increases in cell adhesion to ECM, and potentially, mechanisms that regulate metastasis, we hope to uncover steps that could be targeted for inhibition.


   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. Back

{ddagger} To whom correspondence should be addressed: P.O. Box 12233, Mail Drop C2-14, Research Triangle Park, NC 27709. Tel.: 919-541-5023; Fax: 919-541-0146; E-mail: roberts1{at}niehs.nih.gov.

1 The abbreviations used are: ECM, extracellular matrix; AA, arachidonic acid; PK, protein kinase; cPKC, conventional protein kinase C; nPKC, novel protein kinase C; MAP, mitogen-activated protein; MAPK, MAP kinase; PUFA, polyunsaturated fatty acid; ERK, extracellular signal-responsive kinase; JNK, c-Jun amino-terminal kinase; BSA, bovine serum albumin; t-Boc, t-butoxycarbonyl; CMAC, 7-amino-4-chloromethylcoumarin; PBS, phosphate-buffered saline. Back

2 S. B. Kennett, J. D. Roberts, and K. Olden, unpublished data. Back

3 M. D. George, S. K. Akiyama, T. Smith, K. Olden, and J. D. Roberts, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Drs. B. Alex Merrick and Elizabeth Murphy, both of the National Institute of Environmental Health Sciences, for critically reviewing this manuscript and Dr. Steven K. Akiyama of the National Institute of Environmental Health Sciences for ongoing scientific discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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