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First published online 27 January 2009
doi: 10.1242/jcs.035469

Journal of Cell Science 122, 513-523 (2009)
Published by The Company of Biologists 2009
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Research Article

Functional suppression of E-cadherin by protein kinase C{delta}

Chien-Lin Chen and Hong-Chen Chen*

Department of Life Science and Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan

* Author for correspondence (e-mail: hcchen{at}nchu.edu.tw)

Accepted 30 October 2008


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Protein kinase C (PKC) {delta}, a member of the novel PKC subfamily, has been shown to have an important role in cell proliferation, differentiation, apoptosis and cell motility. In this study, we investigated the effect of green fluorescent protein (GFP)-PKC{delta} and GFP-PKC{alpha} on cell-cell junctions of Madin-Darby canine kidney (MDCK) cells and found that only GFP-PKC{delta} suppressed the homophilic interactions between the ectodomains of E-cadherins, accompanied by a weaker cell-cell adhesion. The kinase-deficient mutant of GFP-PKC{delta} retained its localization at cell-cell junctions but failed to suppress the function of E-cadherin. In addition, we demonstrated that the hinge region (residues 280-347) that links the regulatory domain and the catalytic domain of PKC{delta} is essential for both its kinase activity and the targeting of cell-cell junctions. A PKC{delta} mutant with the deletion of amino acids 280-323 within the hinge region, which is catalytically active but defective in the targeting of cell-cell junctions, failed to suppress the function of E-cadherin. Moreover, expression of GFP-PKC{delta} in MDCK cells expedited the detachment of cells from their neighbors and facilitated cell scatter induced by hepatocyte growth factor. By contrast, the GFP-PKC{delta} mutants including the kinase-deficient mutant and the truncated mutant lacking residues 280-323 suppressed hepatocyte-growth-factor-induced cell scattering. Finally, siRNA-mediated knockdown of endogenous PKC{delta} in MDCK cells was found to delay the onset of cell-cell detachment and cell scattering induced by hepatocyte growth factor. Taken together, our results demonstrate that the catalytic activity of PKC{delta} and its localization to cell-cell junctions are necessary for PKC{delta} to suppress the function of E-cadherin, which thereby facilitates scattering of epithelial cells in response to extracellular cues.

Key words: PKC{delta}, E-cadherin, Adherens junction, Tight junction


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Cell-cell adhesion is crucial for the establishment and maintenance of normal tissue architecture. In vertebrates, epithelial cells are joined to each other by a set of intercellular junctions, which consist of gap junctions, tight junctions, adherens junctions and desmosomes. Gap junctions are communicating junctions between cells; they are formed by connexins and can be interspersed with junctional complexes (reviewed by Saez et al., 2003Go). Tight junctions generally encircle cells at the apical end of the lateral membrane, which forms a semipermeable diffusion barrier between cells and regulates epithelial permeability. Tight junctions also act as an intramembranous fence to restrict the intermixing of apical and basolateral membrane components (reviewed by Matter and Balda, 2003Go). Adherens junctions and desmosomes are the main adhesive junctions and are linked to the actin cytoskeleton and intermediate filaments, respectively. Their main receptors are cadherins, which mediate cell-cell adhesions by Ca2+-dependent homophilic interactions. E-cadherin, a 120 kDa glycoprotein, is the predominant cadherin in epithelial tissue. Ca2+ binds to the extracellular domains of E-cadherin, thereby inducing a conformational change that promotes homophilic interaction with E-cadherin in an adjacent cell (reviewed by Gumbiner, 2005Go). The intracellular domain of E-cadherin binds directly to β- or {gamma}-catenin, which, in turn, associates with {alpha}-catenin, which links the complex to the actin cytoskeleton. Loss of E-cadherin expression or function has been shown to be associated with tumorigenesis and tumor progression (reviewed by Thiery, 2003Go).

The protein kinase C (PKC) family of serine/threonine protein kinases has been divided into classical (cPKC), novel (nPKC) and atypical (aPKC) subgroups based on their ability to be activated by Ca2+ and diacylglycerol (DAG). cPKCs ({alpha}, βI, βII, {gamma}) are activated by both Ca2+ and DAG, and the activation of the nPKCs ({delta}, {epsilon}, {eta}, {theta}) is Ca2+ independent but DAG dependent; both of these classes are also activated by phorbol esters. aPKCs ({zeta}, {iota}/{lambda}) are Ca2+-insensitive and do not respond to DAG or phorbol esters (Newton, 1995Go; Nishizuka, 1995Go). All PKC isozymes are composed of an N-terminal regulatory domain and a C-terminal catalytic domain. The regulatory domain contains two key elements: an autoinhibitory sequence (pseudosubstrate) and one or two membrane-targeting modules (C1 and C2 domains). The C1 domain is a cysteine-rich region of approximately 50 residues, which binds DAG or phorbol ester. In cPKCs and nPKCs, it is present as a tandem repeat, named C1A and C1B. The C2 domain is an independent membrane-targeting module found in classical and novel PKCs. The C2 domain in cPKCs binds phosphatidylserine and Ca2+; however, the C2 domain (otherwise called the C2-like domain) in nPKCs does not bind Ca2+ (Newton, 2001Go).

Several PKC isoforms have been found to localize at or close to cell junctions, but little is known about the molecular mechanism by which different PKCs regulate junctional dynamics. As potent activators of cPKC and nPKC, phorbol esters have been shown to induce the disassembly of cell-cell junctions in many different cell lines. PKC{delta} is the most thoroughly studied member of the novel PKC subfamily and is thought to participate in a wide variety of cell functions, including cell proliferation (Ashton et al., 1999Go; Kitamura et al., 2003Go), differentiation (Corbit et al., 1999Go; Pessino et al., 1995Go), apoptosis (Brodie and Blumberg, 2003Go; Kajimoto et al., 2004Go; Zhong et al., 2002Go) and tumor suppression (Lu et al., 1997Go; Reddig et al., 1999Go). Increasing evidence also indicates that PKC{delta} has a positive role in cell motility (Chen et al., 2007Go; Gliki et al., 2002Go; Iwabu et al., 2004Go; Li et al., 2003Go) and the metastatic potential of tumor cells (Kiley et al., 1999Go; Kruger and Reddy, 2003Go; Alonso-Escolano et al., 2006Go; Kharait et al., 2006Go; Villar et al., 2007Go). However, the role of PKC{delta} in intercellular junctions remains obscure. In this study, we aim to explore the role of PKC{delta} in cell-cell junctions using Madin-Darby canine kidney (MDCK) cells as a model.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Expression of GFP-PKC{delta}, but not GFP-PKC{alpha}, suppresses the homophilic interactions between the ectodomains of E-cadherins in MDCK cells
To explore the role of PKC in cell-cell junctions, GFP-PKC{delta} and GFP-PKC{alpha} were stably expressed in MDCK cells (Fig. 1A). Expression of either construct in those cells did not alter their growth (data not shown) or their ability to form cell colonies within which the cells are in contact with each other (Fig. 1B). To examine their effect on adherens junctions and tight junctions, cells stably expressing GFP-PKC{delta} or GFP-PKC{alpha} were co-cultured with parental control MDCK cells and then grown to confluence. The cells were fixed and stained for adherens junctions and tight junctions with anti-E-cadherin and anti-ZO-1, respectively. Neither GFP-PKC{delta} nor GFP-PKC{alpha} affected tight junctions of MDCK cells (Fig. 1C,D). However, the fluorescence intensity of E-cadherin at cell-cell contacts in the cells expressing GFP-PKC{delta}, but not GFP-PKC{alpha}, was much weaker than that in adjacent parental MDCK cells (Fig. 1C,D).


Figure 1
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  		Fig. 1. Expression of PKC{delta}, but not PKC{alpha}, decreases the immunofluorescence intensity of E-cadherins at cell-cell junctions of MDCK cells using the ECCD-2 anti-E-cadherin antibody. (A) GFP-PKC{alpha} and GFP-PKC{delta} were stably expressed in MDCK cells and their expression levels were analyzed by immunoblotting with anti-GFP. (B) Parental MDCK cells (control) and those stably expressing GFP-PKC{alpha} or GFP-PKC{delta} were grown as colonies. The micrographs were taken under a phase-contrast microscope. Scale bar: 40 µm. Note that the cells stably expressing GFP-PKC{alpha} or GFP-PKC{delta} retain their ability to form colonies within which the cells are in contact with their neighbors. (C) MDCK cells stably expressing GFP-PKC{delta} were co-cultured with parental MDCK cells (control) and then grown to confluence. The cells were fixed and stained for adherens junctions, tight junctions and nuclei with anti-E-cadherin (clone ECCD-2), anti-ZO-1 and DAPI, respectively. (D) MDCK cells stably expressing GFP-PKC{alpha} were co-cultured with parental MDCK cells (control) and then grown to confluence. The cells were fixed and stained as described in C. Dashed lines indicate the boundary between control cells and the cells expressing GFP-PKC. Scale bars: 25 µm.

 

As the total and cell surface levels of E-cadherins were not altered by GFP-PKC{delta} (supplementary material Fig. S1), it is unlikely that the decreased fluorescence intensity of E-cadherin was due to suppression of E-cadherin expression. The rat monoclonal anti-E-cadherin (clone ECCD-2) used in Fig. 1C is known to recognize the extracellular domain of E-cadherin, rendering it possible that GFP-PKC{delta} causes a conformational change on the ectodomain of E-cadherin, which then prevents E-cadherins from detection by the ECCD-2 antibody. To clarify this, a mouse monoclonal antibody (clone 36) that recognizes the cytoplasmic portion of E-cadherins was used. The fluorescence intensity of E-cadherin with the clone 36 antibody did not show differences between control cells and PKC{delta}-overexpressed cells (Fig. 2A), supporting the idea that the conformation of E-cadherins rather than their distribution or expression is affected by PKC{delta}. In addition, we found that the ability of the ECCD-2 antibody, but not the clone 36 antibody, to detect E-cadherin at cell-cell junctions relied on the presence of Ca2+ in the culture medium, as demonstrated by Ca2+-switch assay (Fig. 2B), indicating that the ECCD-2 antibody might actually preferentially recognize `active' E-cadherins when homophilic interactions between their ectodomains are formed. Another rat monoclonal antibody (clone DECMA-1), which recognizes the ectodomain of E-cadherin also relied on the presence of Ca2+ in the culture medium (supplementary material Fig. S2).


Figure 2
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  		Fig. 2. The ECCD-2 anti-E-cadherin antibody preferentially recognizes `active' E-cadherin. (A) MDCK cells stably expressing GFP-PKC{delta} were co-cultured with parental MDCK cells (control) and then grown to confluence. The cells were fixed and stained for E-cadherin with a rat monoclonal antibody (clone ECCD-2) and a mouse monoclonal antibody (clone 36). Scale bar: 25 µm. Note that the ECCD-2 antibody recognizes the extracellular domain of E-cadherin and the clone 36 antibody recognizes the intracellular domain of E-cadherin. (B) Detection of E-cadherin in MDCK cells by immunofluorescence staining with anti-E-cadherin antibodies under three conditions. The cells were grown to confluence (control) or cells were grown to confluence and then treated with 5 mM EGTA in serum-free medium for 30 minutes (Ca2+ depletion). After EGTA treatment, the medium was then replaced with fresh growth medium for 2 hours (Ca2+ repletion). Scale bars: 25 µm.

 
To examine whether decreased fluorescence intensity of E-cadherin detected by the ECCD-2 antibody in the cells expressing GFP-PKC{delta} reflects a weaker cell-cell adhesion, cell dissociation assay and cell aggregation assay were performed. Indeed, the cells expressing GFP-PKC{delta} were much more easily dissociated from their neighbors than the control cells (Fig. 3A). Unlike the control MDCK cells, which tended to form large aggregates in suspension, the cells expressing GFP-PKC{delta} largely remained as single cells or small cell clusters (fewer than 5 cells) in suspension (Fig. 3B). Moreover, the decreased signals of E-cadherins detected by the ECCD-2 antibody was accompanied by decreased accumulation of β-catenin and fewer organized actin filaments at cell-cell contacts, in association with the increased formation of F-actin stress fibers (Fig. 4A), which usually characterize weak cell-cell junctions. These results together indicate that PKC{delta} exerts a suppressive effect on adherens junctions through inhibition of the homophilic interactions of E-cadherins.


Figure 3
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  		Fig. 3. Expression of PKC{delta} weakens cell-cell adhesions. (A) Cell-dissociation assay. MDCK cells stably expressing GFP or GFP-PKC{delta} were allowed to grow as discrete colonies. The cell colonies were scraped in culture medium and suspended by repeated pipetting. Representative images at x100 magnification before (–) and after (+) pipetting are shown. Cell-dissociation index is expressed as the percentage of particles (cell clusters >=3 cells) in total number of cells per dish. Values (means ± s.d.) are from three independent experiments. (B) Cell-aggregation assay. MDCK cells stably expressing GFP or GFP-PKC{delta} were grown to semi-confluence, trypsinized and suspended in culture medium. After 72 hours of constant shaking, cell aggregates (>=10 cells) were enumerated under a phase-contract microscope at x100 magnification. Values (means ± s.d.) are from three independent experiments (n=50). Representative images at 0 hours and after 72 hours of shaking are shown.

 

Figure 4
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  		Fig. 4. The catalytic activity of PKC{delta} is required for suppression of the homophilic interactions between E-cadherins. (A) MDCK cells stably expressing GFP, GFP-PKC{delta} or its mutants (KD and RD) were grown to confluence and stained for E-cadherin (with the ECCD-2 antibody), ZO-1, β-catenin and actin filaments. The kinase-deficient (KD) mutant was generated by substitution of Lys376 with Arg. The RD mutant represents the regulatory domain (a.a. 1-298) of PKC{delta}. Arrowheads indicate the presence of GFP-tagged proteins at cell-cell junctions. Scale bars: 10 µm. The ratio of the fluorescence intensity of E-cadherin at cell-cell junctions (red boxed region) versus adjacent cytoplasm (yellow boxed region) was measured (n=50) (F.I., fluorescence intensity; AJ, adherens junctions; Cyto., cytoplasm). Note that PKC{delta}-mediated suppression of adherens junctions is accompanied by fewer organized β-catenin and F-actin at cell-cell junctions. (B) MDCK cells stably expressing the GFP-PKC{delta} KD mutant were stained with anti-E-cadherin and anti-ZO-1. White lines on confocal X-Y section images represent regions where confocal X-Z section images were taken. The X-Z images reveal that GFP-PKC{delta} colocalizes with E-cadherin, but not ZO-1, at cell-cell junctions. Arrowheads indicate the colocalization of GFP-PKC{delta} and E-cadherin at cell junctions. Scale bars: 5 µm.

 

The catalytic activity of PKC{delta} is essential for its suppression of E-cadherins but dispensable for its localization at cell-cell junctions
To examine the necessity of the catalytic activity of PKC{delta} for its ability to suppress the function of E-cadherin, two PKC{delta} constructs deficient in catalytic activity were generated. One is the kinase-deficient (KD) mutant (K376R); the other is the regulatory domain (RD) of PKC{delta}. Similarly to the wild-type PKC{delta}, a fraction of the KD mutant and the RD construct (residues 1-298) was found to localize at cell-cell junctions. However, both failed to suppress the homophilic interactions between E-cadherins (Fig. 4A), indicating that the catalytic activity of PKC{delta} is required in order for it to be able to suppress the function of E-cadherin. As the PKC{delta} KD mutant was apparently more localized at cell-cell junctions than the wild type, the three-dimensional images for its distribution were reconstituted. The X-Z sections revealed that a fraction of PKC{delta} was indeed localized at cell-cell junctions, where it colocalized with E-cadherins but not ZO-1 proteins (Fig. 4B). These results clearly indicate that the catalytic activity of PKC{delta} is essential for its suppression of E-cadherin but dispensable for the targeting of cell-cell junctions.

The hinge region of PKC{delta} is critical for regulation of both its kinase activity and targeting of cell-cell junctions
To identify the domain(s) of PKC{delta} responsible for its localization at cell-cell junctions, the PKC{delta} mutants with a deletion of its C2 domain (named as the {Delta}C2 mutant, with deletion of residues 1-123), C1A domain (named as the {Delta}C1A mutant, with deletion of residues 159-208), C1B domain (named as the {Delta}C1B mutant, with deletion of residues 232-280) or hinge region (named as the {Delta}H mutant, with deletion of residues 280-347) were generated and stably expressed in MDCK cells (Fig. 5A). Deletion of the C2 domain, C1A domain or C1B domain did not affect the catalytic activity of PKC{delta} towards myelin basic protein (MBP), which serves as an exogenous substrate in the in vitro kinase assay (Fig. 5B). However, deletion of the hinge region of PKC{delta} severely impaired its kinase activity (Fig. 5B). The PKC{delta} {Delta}C2, {Delta}C1A and {Delta}C1B mutants retained their localization at cell-cell junctions, which correlates with suppression of the homophilic interactions between E-cadherins (Fig. 6). By contrast, the PKC{delta} {Delta}H mutant, which failed to localize at cell-cell junctions, was unable to suppress the function of E-cadherin (Fig. 6). Therefore, the hinge region of PKC{delta}, which links the regulatory domain and the catalytic domain of the enzyme, is crucial for regulation of both its catalytic activity and targeting of cell-cell junctions.


Figure 5
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  		Fig. 5. The hinge region of PKC{delta} is required for its catalytic activity. (A) The diagram depicts the domain structure of GFP-PKC{delta} and its mutants used in this study. The mutants with deletion of the C2 domain ({Delta}C2; a.a. 1-123), C1A domain ({Delta}C1A; a.a. 159-208), C1B domain ({Delta}C1B; a.a. 232-280) or hinge region ({Delta}H; a.a. 280-347) were generated and stably expressed in MDCK cells. KD, kinase-deficient mutant; RD, regulatory domain. (B) GFP-PKC{delta} and its mutants were immunoprecipitated by anti-GFP antibody. The immunocomplexes were subjected to immunoblotting with anti-GFP antibody or an in vitro kinase assay using MBP as a substrate. The 32P-incorporated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The phosphorylation of MBP was measured and expressed as a percentage relative to the level induced by GFP-PKC{delta} wild type, which is defined as 100%. Values (means ± s.d.) are from three independent experiments. IVK, in vitro kinase assay.

 

Figure 6
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  		Fig. 6. The hinge region of PKC{delta} is required for the targeting of cell-cell junctions. MDCK cells stably expressing the GFP-PKC{delta} mutants ({Delta}C1A, {Delta}C1B, {Delta}C2 and {Delta}H) were grown to confluence and then stained for E-cadherin (with the ECCD-2 antibody), ZO-1, β-catenin and F-actin. Arrowheads indicate the presence of the GFP-tagged proteins at cell-cell junctions. Boxed regions represent the areas selected for measurement of the ratio of the fluorescence intensity of E-cadherin at cell-cell junctions versus adjacent cytoplasm. Scale bars: 10 µm. The ratio of the fluorescence intensity of E-cadherin at cell-cell junctions versus adjacent cytoplasm was measured (n=50) (F.I., fluorescence intensity; AJ, adherens junctions. Cyto., cytoplasm). Values are mean ± s.d. Note that E-cadherin suppression by the {Delta}C1A, {Delta}C1B or {Delta}C2 mutant is associated with fewer organized β-catenin and actin filaments at cell-cell junctions.

 

Localization of PKC{delta} to cell-cell junctions is required for suppression of the homophilic interactions between E-cadherins
In all the experiments performed up to this point, all of the PKC{delta} constructs capable of suppressing adherens junctions, including the wild-type PKC{delta} and its deleted mutants ({Delta}C2, {Delta}C1A and {Delta}C1B), localized at cell-cell junctions. To examine whether the localization of PKC{delta} to cell-cell junctions is required in order for it to be able to suppress adherens junctions, two more PKC{delta} mutants with deletions within the hinge region were generated (Fig. 7A), one with a deletion of the amino acid 280-323 (designated as {Delta}H280-323) and the other with a deletion of the amino acid 324-347 (designated as {Delta}H324-347). The {Delta}H280-323 mutant retained a kinase activity comparable to that of the wild-type PKC{delta} (Fig. 7B), but failed to localize at cell-cell junctions (Fig. 7C). In contrast, the {Delta}H324-347 mutant had a much lower kinase activity than the wild type (Fig. 7B), but retained its localization at cell-cell junctions (Fig. 7C). Nevertheless, both {Delta}H280-323 and {Delta}H324-347 mutants failed to suppress the homophilic interactions between E-cadherins (Fig. 7C). Together, these results not only support the notion that both the catalytic activity and cell-junction localization of PKC{delta} are required in order for it to be able to suppress the functions of E-cadherin, but also underscore the significance of residues 280-323 of PKC{delta} in its targeting of cell-cell junctions.


Figure 7
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  		Fig. 7. Localization of PKC{delta} to cell-cell junctions is essential for adherens junction suppression. (A) The diagram depicts the domain structures of GFP-PKC{delta} mutants ({Delta}H, {Delta}H280-323 and {Delta}H324-347) with deletion in the hinge region. (B) GFP-PKC{delta} or its mutants were stably expressed in MDCK cells and their kinase activities were analyzed by an in vitro kinase assay using MBP as a substrate. The 32P-incorporated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The phosphorylation of MBP was measured and expressed as a percentage of the level induced by GFP-PKC{delta} wild-type, which is defined as 100%. Values (means ± s.d.) are from three independent experiments. IVK, in vitro kinase assay. (C) MDCK cells stably expressing GFP-PKC{delta} or its mutants ({Delta}H, {Delta}H280-323 and {Delta}H324-347) were grown to confluence. The cells were stained for E-cadherin and nuclei. Arrowheads indicate the presence of the GFP-tagged proteins at cell-cell contacts. Scale bar: 10 µm. The ratio of the fluorescence intensity of GFP-tagged proteins at cell-cell junctions (red boxed region) versus adjacent cytoplasm (yellow boxed region) was measured (n=50) and is shown below the micrographs (F.I., fluorescence intensity; AJ, adherens junctions; Cyto., cytoplasm). (D) MDCK cells stably expressing GFP-PKC{delta} or its mutants, {Delta}C1A, {Delta}C1B and {Delta}H280-323, were sparsely cultured, treated with 100 nM PMA for 20 minutes, and stained with DAPI. Arrowheads indicate the presence of GFP-tagged proteins at the plasma membrane. Scale bar: 10 µm. The ratio of the fluorescence intensity of GFP-tagged proteins at the plasma membrane (red boxed region) versus adjacent cytoplasm (yellow boxed region) was measured (n=50) and is shown beneath the micrographs. Note that GFP-PKC{delta} and {Delta}H280-323, but not the {Delta}C1A or {Delta}C1B mutants, translocate to the plasma membrane upon PMA treatment.

 

PKC{delta} targets cell-cell junctions and cellular membranes through different domains
As shown in Fig. 6, the PKC{delta} mutants {Delta}C2, {Delta}C1A and {Delta}C1B retained their localization at cell-cell junctions, indicating that C2, C1A and C1B domains are not required for the targeting of PKC{delta} to cell-cell junctions. Instead, we found that the hinge region, in particular residues 280-323, of PKC{delta} was essential for its localization at cell-cell junctions (Fig. 7C). However, similarly to wild-type PKC{delta}, the {Delta}H280-323 mutant was able to translocate to the plasma membrane upon phorbol-12-myristate-13-acetate (PMA) stimulation (Fig. 7D), suggesting that residues 280-323 of PKC{delta} are not required for its translocation to the plasma membrane. The C1A domain of PKC{delta} has been reported to bind to diacylglycerol with high affinity (Stahelin et al., 2004Go) and is thought to be crucial for the association of PKC{delta} with the plasma membrane. Indeed, the PKC{delta} {Delta}C1A mutant failed to associate with the plasma membrane upon PMA stimulation (Fig. 7D). Consistent with previous reports (Kajimoto et al., 2004Go; Schultz et al., 2004Go), the PKC{delta} {Delta}C1B mutant was defective in localizing at the Golgi complex (Fig. 7D). Interestingly, the PKC{delta} {Delta}C1B mutant also failed to translocate to the plasma membrane upon PMA stimulation. Therefore, our results demonstrate that PKC{delta} targets cell-cell junctions and the plasma membrane through different domains.

PKC{delta} modulates cell scattering induced by hepatocyte growth factor
Disruption of cell-cell junctions is a prerequisite for epithelial cells to scatter. To examine whether PKC{delta}-mediated suppression of E-cadherin facilitates the scatter of epithelial cells, MDCK cells stably expressing GFP-PKC{delta} or its mutants (KD and {Delta}H280-323) were allowed to grow as colonies and then treated with hepatocyte growth factor (HGF) to trigger scattering. Expression of wild-type PKC{delta} in MDCK cells expedited the detachment of the cells from their neighbors and led to a more apparent `scattered' phenomenon upon HGF stimulation (Fig. 8). By contrast, the KD mutant and the {Delta}H280-323 mutant suppressed the scattering of the cells (Fig. 8), suggesting a dominant-negative effect exerted by both mutants. Accordingly, the selective PKC{delta} inhibitor rottlerin abolished HGF-induced scatter of MDCK cells (data not shown). To examine the role of PKC{delta} in the suppression of adherens junctions further, a stable MDCK cell line that expresses PKC{delta}-specific siRNA upon addition of doxycycline was used (Chen et al., 2007Go). The extent of PKC{delta} knockdown was estimated to be 70% 72 hours after doxycycline addition (Fig. 9A), which delayed the onset of cell-cell detachment (Fig. 9B) and cell scattering induced by HGF (Fig. 9C). Moreover, PKC{delta} knockdown retained the fluorescence intensity of E-cadherin at cell-cell junctions in response to HGF stimulation (Fig. 9D,E), suggesting that PKC{delta} knockdown might strengthen adherens junctions of MDCK cells and delay the disruption of adherens junctions induced by HGF. Together, our results strongly suggest a role for PKC{delta} in the suppression of adherens junctions, which might thereby facilitate scattering of epithelial cells.


Figure 8
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  		Fig. 8. Overexpression of GFP-PKC{delta} expedites HGF-induced scatter of MDCK cells. (A) MDCK cells stably expressing GFP, GFP-PKC{delta} wild-type, the KD mutant or the {Delta}H280-323 mutant were allowed to grow as colonies and then treated with 20 ng/ml HGF to induce cell scattering. Time-lapse micrographs were taken by cooled CCD under a differential interference contrast microscope. Representative images at the indicated time points are shown. Scale bar: 20 µm. (B) At various lengths of time after HGF stimulation, the percentage of scattered colonies in all counted colonies was determined. A colony is judged as `scattered' when the half of the cells have lost contact with their neighbors and exhibit a fibroblast-like phenotype. Values (means ± s.d.) are from three independent experiments (n=100).

 

Figure 9
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  		Fig. 9. siRNA-mediated knockdown of endogenous PKC{delta} in MDCK cells delays the disruption of cell-cell adhesions induced by HGF. (A) The MDCK cells that stably express PKC{delta}-specific siRNA upon addition of doxycycline (Dox) were incubated with (+) or without (–) doxycycline (Dox) for 72 hours. The expression level of PKC{delta} was analyzed by immunoblotting with anti-PKC{delta}. (B) 72 hours after induction of PKC{delta}-specific siRNA, the cells were treated with 20 ng/ml HGF. The time-lapse micrographs were taken by cooled CCD under a differential interference contrast microscope. Representative images at the indicated time points are shown. Scale bar: 20 µm. (C) Quantitative result of HGF-induced cell scattering. At various lengths of time after HGF stimulation, the percentage of scattered colonies in all counted colonies was determined. Values (means ± s.d.) are from three independent experiments (n=100). (D) 72 hours after induction of PKC{delta}-specific siRNA, the cells were treated with 20 ng/ml HGF and then stained for E-cadherin with the ECCD-2 antibody at the indicated time points. White lines represent the regions of interest used to measure fluorescence intensity. (E) 2 hours and 4 hours after HGF treatment, the profiles of the E-cadherin fluorescence intensity along the white lines drawn in D were shown in line graphs (blue; without PKC{delta} siRNA induction; red, with PKC{delta} siRNA induction; a.u., arbitrary units). The line-scan measurements of the fluorescence intensity were recorded by Zeiss LSM imaging software.

 

   Discussion
Top
 Summary
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
In this study, we used MDCK cells as a model to examine the role of PKC{delta} in cell-cell junctions. Expression of GFP-PKC{delta}, but not GFP-PKC{alpha}, caused a decreased immunofluorescence intensity of E-cadherins at cell-cell contacts with the ECCD-2 monoclonal antibody, which is known to recognize the extracellular domain of E-cadherin (Fig. 1C). However, when another monoclonal anti-E-cadherin (clone 36) recognizing the intracellular portion of E-cadherin was used, the immunofluorescence intensity of E-cadherins at cell-cell contacts of the cells expressing GFP-PKC{delta} was as strong as that of the control cells (Fig. 2A), suggesting that PKC{delta} might cause a conformational change of E-cadherin on its ectodomain rather than its expression or distribution. Moreover, we demonstrated that the ability of the ECCD-2 antibody to detect E-cadherin is dependent on the presence of Ca2+ under the condition with which E-cadherin is retained on cell-cell junctions (Fig. 2B), supporting the notion that the ECCD-2 antibody preferentially recognizes the `active' form of E-cadherin. Therefore, the decreased intensity of E-cadherin at cell-cell junctions by immunofluorescence staining with the ECCD-2 antibody is likely to reflect fewer homophilic interactions between E-cadherin ectodomains.

The decreased immunofluorescence intensity of E-cadherin with the ECCD-2 antibody at cell-cell junctions was associated with characteristics of weak adherens junctions, manifested by decreased accumulation of β-catenin and fewer organized actin filaments at those sites, in association with the increased formation of F-actin stress fibers (Fig. 4A). Accordingly, we found that the cell-cell contacts of the cells expressing GFP-PKC{delta} were much more easily disrupted than those of the control cells (Fig. 3A). Moreover, unlike control MDCK cells, which tended to form large cell aggregates in suspension, the cells expressing GFP-PKC{delta} had a defect in this regard (Fig. 3B). These results together indicate that overexpression of PKC{delta} suppresses the function of E-cadherin in MDCK cells.

We conclude that the ability of PKC{delta} to suppress the function of E-cadherin requires both its catalytic activity and localization to cell-cell junctions. This conclusion was mainly supported by our results derived from two PKC{delta} mutants, KD and {Delta}H280-323. The KD mutant localized at cell-cell junctions but was deficient in catalytic activity (Fig. 4); however, the {Delta}H280-323 mutant retained its catalytic activity but failed to localize at cell-cell junctions (Fig. 7). Nevertheless, both failed to suppress the homophilic interactions between E-cadherins, indicating that the catalytic activity of PKC{delta} and its targeting of cell-cell junctions are both essential for its ability to suppress the function of E-cadherin. Moreover, these results also indicate that the catalytic activity of PKC{delta} is dispensable for its localization at cell-cell junctions and underscore the importance of residues 280-323 within the hinge region for the targeting of cell-cell junctions.

The PKC{delta} {Delta}H mutant with a deletion of the entire hinge region (a.a. 280-347) lost its catalytic activity (Fig. 5B) and ability to localize at cell-cell junctions (Fig. 6), indicating that the hinge region of PKC{delta} is important for the regulation of both events. Moreover, the results derived from the PKC{delta} {Delta}H280-323 and {Delta}H324-347 mutants indicate that amino acids 280-323 and 324-347 within the hinge region are crucial for PKC{delta} in the targeting of cell-cell junctions and to retain its catalytic activity, respectively (Fig. 7). As the RD construct (residues 1-298) of PKC{delta} was able to localize at cell-cell junctions (Fig. 4A), we speculate that a stretch of 19 amino acids (280-298) of PKC{delta} might be the most critical determinant for its targeting of cell-cell junctions. Several residues located in the hinge region of PKC{delta}, including Ser299, Ser302, Ser304, Tyr311 and Tyr332, have been shown to be phosphorylated under certain circumstances (Konishi et al., 2001Go; Durgan et al., 2007Go). Some of these phosphorylation events are already known to regulate the catalytic activity of PKC{delta} (Konishi et al., 2001Go; Durgan et al., 2007Go). However, it is not clear whether any of these phosphorylation events could modulate the targeting of PKC{delta} to cell-cell junctions.

The hinge regions of PKC{alpha} and PKC{epsilon} are reported to be important for the targeting of cell-cell contacts of rat pituitary GH3B6 cells (Quittau-Prevostel et al., 2004Go). In particular, a three-residue motif located in the hinge region of PKC{alpha} and PKC{epsilon} (GDE and GEE, respectively) has been demonstrated to be crucial, but not sufficient, for the targeting of cell-cell junctions (Quittau-Prevostel et al., 2004Go). In addition to the hinge region, the C2 domain of PKC{alpha} has also been shown to be required for the targeting of cell-cell junctions (Quittau-Prevostel et al., 2004Go). As the GD(E)E motif is absent in the hinge region of PKC{delta} and the C2 domain of PKC{delta} is not required for the targeting of cell-cell junctions (Fig. 6), it is likely that the mechanism for PKC{delta} in targeting cell-cell junctions is different from those for PKC{alpha} and PKC{epsilon}.

A role for PKCs has been implicated in cell-cell junctions. However, whether PKC isozymes exert a redundant or specific effect on different junctional structures remains to be clarified. In this report, we demonstrate that a fraction of PKC{delta} localizes to cell-cell junctions, where it exerts a suppressive effect on the function of E-cadherin. Accordingly, it has recently been reported that activation of PKC{delta} is involved in the regulation of peripheral actin organization and cell-cell contacts in the epithelium (Oh et al., 2007Go). In addition, PKC{delta} has been implicated as modulating tight junctions (Mullin et al., 1998Go; Eckert et al., 2005Go). Unlike PKC{delta}, the function of PKC{alpha} at cell-cell junctions is thought to modulate desmosomal adhesion of epithelial cells in response to wounding (Wallis et al., 2000Go) and to increase endothelial permeability by the disruption of VE-cadherin junctions (Sandoval et al., 2001Go). As for PKC{epsilon}, this has been shown to reduce gap junction permeability (Doble et al., 2000Go; Miura et al., 2007Go), weaken tight junctions (D'Souza et al., 2007Go) and suppress adherens junctions (Imamdi et al., 2004Go). PKC{gamma} was recently reported to have a role in the regulation of gap junctions (Akoyev and Takemoto, 2007Go). In epithelial cells, atypical PKCs, such as PKC{zeta} and PKC{lambda}, colocalize with Par6 and Par3 at cell-cell contacts and the Par6-aPKC-Par3 trimolecular complexes are important for the formation of tight junctions, leading to epithelial cell polarity (Etienne-Manneville and Hall, 2003Go). Therefore, different PKC isozymes might have preferred, but overlapping, targets at cell-cell junctions, where they coordinately regulate the junctional structures.

As the scatter of epithelial cell colonies possesses characteristics of epithelial-mesenchymal transition, such as the loss of epithelial cell-cell junctions and the acquisition of a motile mesenchymal cell phenotype, the scatter assay has been used for studying epithelial-mesenchymal transition and for detecting factors able to induce migratory behavior of cells (Lai et al., 2000Go; Liang and Chen, 2001Go). Here we show that increased expression of PKC{delta} in MDCK cells expedited cell-cell detachment and facilitated their scatter upon HGF stimulation (Fig. 8). By contrast, expression of the PKC{delta} KD and {Delta}H280-323 mutants (Fig. 8) or suppression of endogenous PKC{delta} (Fig. 9) in MDCK cells delayed the onset of cell-cell detachment and cell scattering. The facilitation of the scatter response of MDCK cells by PKC{delta} is likely to be a combined consequence of its negative effect on adherens junctions and positive effect on cell motility. We have recently demonstrated that increased expression of PKC{delta} in MDCK cells promotes lamellipodia formation and cell motility, at least partially, through its phosphorylation on adducin (Chen et al., 2007Go). The loss of cell-cell junctions is a crucial event in cancer progression and is commonly associated with an increased aggressiveness of a tumor. Our studies therefore support a positive role for PKC{delta} in the malignant progression of tumors. Actually, elevated expression of PKC{delta} has been detected in certain tumors (Chen et al., 2001Go; Nabha et al., 2005Go; Villar et al., 2007Go) and has been found to increase the motility and metastatic potential of tumor cells (Kiley et al., 1999Go; Alonso-Escolano et al., 2006Go; Kharait et al., 2006Go; Villar et al., 2007Go).

The results of this study suggest that PKC{delta} probably suppresses adherens junctions through interference of homophilic interactions between E-cadherin ectodomains. How does PKC{delta} exert such a suppressive effect on E-cadherin? It is possible that PKC{delta} directly phosphorylates the cytoplasmic domain of E-cadherin and thereby causes a conformational change on the ectodomains, which disfavors the homophilic binding of E-cadherin. Alternatively, PKC{delta} may phosphorylate molecules involved in the assembly of adherens junctions, such as {alpha}-catenin and β-catenin. The phosphorylation of these molecules by PKC{delta} might weaken the adherens junction assembly and thereby suppress the interactions between E-cadherin ectodomains. Recently, {alpha}-adducin, a membrane cortical skeletal protein known to promote spectrin-actin complexes (Bennett et al., 1988Go; Gardner and Bennett, 1987Go), has been reported to colocalize with β2-spectrin at cell-cell contacts and to have a crucial role in stabilizing the lateral membrane and, in particular, E-cadherin-mediated adherens junctions of bronchial epithelial cells (Abdi and Bennett, 2008Go). PKC{delta} selectively phosphorylates adducin at its Ser726 within the myristoylated alanine-rich C kinase substrate (MARCKS) domain (Chen et al., 2007Go), whose phosphorylation has been demonstrated as abolishing adducin activity in vitro, leading to the dissociation of spectrin and actin complexes in activated platelets and some other types of cells (Matsuoka et al., 1998Go; Barkalow et al., 2003Go). Therefore, we propose that PKC{delta} phosphorylates adducin at cell-cell contacts and thereby prevents the assembly of a spectrin-actin network at the lateral membrane, leading to weakness of adherens junctions. Experiments are in progress to test these possibilities.


   Materials and Methods
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Materials
Rat monoclonal anti-E-cadherin (clone ECCD-2), mouse monoclonal anti-ZO-1 (clone ZO1-1A12), fetal bovine serum and Lipofectamine were purchased from Invitrogen. Mouse monoclonal anti-E-cadherin (clone 36 for immunofluorescence staining and clone 34 for immunoblotting) was purchased from BD Transduction Laboratories. Rat monoclonal anti-E-cadherin (DECMA-1) was purchased from Abcam (Cambridge, UK). Polyclonal anti-PKC{delta} (C20) was purchased from Santa Cruz Biotechnology. Monoclonal anti-GFP was purchased from Roche. Mouse monoclonal anti-β-catenin (clone 14) was purchased from BD Transduction Laboratories. Monoclonal anti-tubulin, recombinant, MBP, tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin, ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and protein-A-Sepharose were purchased from Sigma. PMA, G418 sulfate and doxycycline were purchased from Calbiochem (San Diego, CA). The mounting medium with 4',6-diamidino-2-phenylindole (DAPI) was purchased from Vector Laboratories (Burlingame, CA). Plasmid pEGFP-N1-PKC{delta} was kindly provided by P. M. Blumberg (National Institutes of Health, Bethesda, MD) and has been previously described (Wang et al., 1999Go). Plasmid pEGFP-N1-PKC{alpha} was kindly provided by D. Joubert (Institut de Génomique Fonctionnelle, Montpellier, France) and has been previously described (Quittau-Prevostel et al., 2004Go). Plasmid pSRD-PKC{delta}-RD was kindly provided by J. S. Brugge (Harvard Medical School, Boston, MA) and has been previously described (Miranti et al., 1999Go). Plasmid pTB701-PKC{delta} ({Delta}C1B)-GFP was kindly provided by N. Saito (Kobe University, Kobe, Japan) and has been previously described (Kajimoto et al., 2004Go).

Plasmid construction
To construct pEGFP-PKC{delta} RD (a.a. 1-298), the corresponding DNA fragment was amplified by polymerase chain reaction (PCR) with the primers: forward, 5'-ATGGCACCCTTCCTG-3' and reverse, 5'-AATGTCCAGGAATTGCTC-3', using pSRD-PKC{delta} RD as the template and ligated in-frame to pEGFP-N2 vector via the SmaI site. To construct the pEGFP-PKC{delta} {Delta}C2 construct (a.a. 124-674), the corresponding DNA fragment was amplified by PCR with the primers forward 5'-ATGGATGGGGATTGCAAA-3' and reverse 5'-AATGTCCAGGAATTGCTC-3' and ligated in frame to pEGFP-N2 vector via the SmaI site. To construct GFP-PKC{delta} {Delta}C1A (with deletion of residues 159-208), two DNA fragments corresponding to residues 1-158 and 209-674 of PKC{delta} were obtained by PCR using pEGFP-N1-PKC{delta} as the template and ligated to pEGFP-N3 vector via the EcoRI and BamHI sites. A similar strategy was used to construct the {Delta}H mutant (with deletion of a.a. 280-347), {Delta}H280-323 (with deletion of a.a. 280-323) and {Delta}H324-347 (with deletion of a.a. 324-347) in pEGFP-N3 vector. For the DNA fragment encoding residues 1-158, the primers used were: forward, 5'-GGCGAATTCATGGCACCCTTCCTGCGC-3' and reverse, 5'-GGAGAATTCGTTCTTGATGTAGTGGATCTT-3'; residues 1-279: forward, 5'-GGCGAATTCATGGCACCCTTCCTGCGC-3' and reverse, 5'-GGAGAATTCACACAGGTTGGCCACCTT-3'; residues 1-323: forward, 5'-GGCGAATTCATGGCACCCTTCCTGCGC-3' and reverse, 5'-GGAGAATTCACTCCCAGAGACTTCTGG-3'; residues 209-674: forward, 5'-GGCGAATTCACTGGCACTGCCACCAATAGC-3' and reverse, 5'-GATGGATCCAATGTCCAGGAATTGCTC-3'; residues 324-674: forward, 5'-GGCGAATTCGACATCCTAGACAACAAC-3' and reverse, 5'-GGCGAATTCACTGGCACTGCCACCAATAGC-3'; residues 348-674: forward 5'-GGCGAATTCACCTTCCAAAAAGTCCTT-3' and reverse, 5'-GGCGAATTCACTGGCACTGCCACCAATAGC-3'. The restriction enzyme sites are underlined. Mutagenesis was carried out using a QuikChange site-directed mutagenesis kit (Stratagene). For the PKC{delta} KD (K376R) mutant, the mutagenic primer, 5'-GATAAGTACTTTGCAAATCAGGTGTCTGAAGAAGGACG-3' was used, where the position of the substituted codon is underlined. The desired mutations were confirmed by dideoxy DNA sequencing, a service provided by the Biotechnology Center of National Chung Hsing University, Taiwan.

Cell culture and transfections
MDCK cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air. To generate MDCK cells stably expressing GFP-PKC{alpha}, GFP-PKC{delta}, or its mutants, MDCK cells were transfected with plasmids encoding GFP-PKC constructs using Lipofectamine. Two days after transfection, the cells were selected in the medium containing 0.5 mg/ml G418. Ten days later, neomycin-resistant cells were pooled and analyzed for the expression of GFP-tagged proteins by immunoblotting with the monoclonal anti-GFP. A MDCK cell line that expresses PKC{delta}-specific siRNA upon addition of doxycycline was established in our laboratory and has been previously described (Chen et al., 2007Go).

Cell dissociation assay and cell aggregation assay
For the cell dissociation assay, MDCK cells were allowed to grow as discrete colonies by seeding at 2x103 per 60 mm dish. When the colonies contained ~20 cells, they were collected in serum-free medium by scraping and passed through a micropipette 30 times under constant force. The number of cell particles (Np) containing more than three cells was measured by using a hemocytometer. The cell suspension containing cell particles was subsequently subjected to centrifugation and trypsinization for cell number (Nc) measurement. The cell dissociation index was expressed as Np/Nc x 100%. For the cell aggregation assay, MDCK cells were trypsinized, suspended in DMEM with 10% serum at 106 cells/ml, and subjected to a constant rotation at 4 r.p.m. at 37°C. 72 hours later, the cell suspension was transferred to a 60 mm dish and the number of cell particles containing more than ten cells was measured under a phase contrast microscopy at x100 magnification.

Immunoprecipitation and immunoblotting
Cells were lysed in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol and 1 mM Na3VO4) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitory units/ml aprotinin and 20 µg/ml leupeptin). Lysates were centrifuged for 10 minutes at 4°C to remove debris, and the protein concentrations were determined using the Bio-Rad protein assay (Hercules, CA). For immunoprecipitation, aliquots of cell lysates were incubated with 0.4 µg monoclonal anti-GFP for 1.5 hours at 4°C. Immunocomplexes were collected on protein-A-Sepharose beads. For monoclonal antibodies, protein-A-Sepharose beads were coupled with rabbit anti-mouse IgG (1 µg) before use. The beads were washed three times with 1% Nonidet P-40 lysis buffer, boiled for 3 minutes in SDS sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose (Schleicher and Schuell). Immunoblotting was performed with appropriate antibodies using the Amersham Biosciences enhanced chemiluminescence system for detection. Chemiluminescent signals were detected and quantified using the Fuji LAS-3000 luminescence image system.

In vitro protein kinase assay
To perform in vitro kinase assays for GFP-PKC{delta}, the immunoprecipitates by the monoclonal anti-GFP were washed 3 times with 1% NP-40 lysis buffer and once with 25 mM Tris buffer. Kinase reactions were carried out in 40 µl kinase buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM CaCl2, 1 mM dithiothreitol, 1.25 µg phosphatidylserine) containing 10 µCi of [{gamma}-32P] ATP (3000 Ci mmol–1; PerkinElmer Life Sciences) and 5 µg MBP at 25°C for 20 minutes. Reactions were terminated by addition of SDS sample buffer, and the 32P-incorporated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The radioisotope activity was quantified using a phosphoimager system (Pharmacia).

Laser-scanning confocal fluorescent microscopy
In most cases, equal numbers of MDCK cells expressing various GFP-PKC constructs were grown on 12-mm coverslips for 3 days until a monolayer was formed. For calcium switch assay, MDCK cells were grown to confluence, washed three times with DMEM in the absence of serum, and then incubated in DMEM with 5 mM EGTA for 30 minutes (Ca2+ depletion). For Ca2+ repletion, the cells were washed with DMEM and incubated in DMEM with 10% serum for 2 hours. For PMA stimulation, MDCK cells were sparsely plated on coverslips for 18 hours and then treated with 100 nM PMA for 20 minutes.

For immunofluorescence staining, the cells were fixed in phosphate-buffered saline containing 4% paraformaldehyde for 15 minutes, permeabilized in phosphate-buffered saline containing 0.2% Triton X-100 for 15 minutes, and stained with primary antibodies at 4°C overnight, followed by TRITC-conjugated or Cy5-conjugated secondary antibodies (Jackson ImmunoReseach Laboratories) at 4 µg/ml for 120 minutes. The primary antibodies used in immunofluorescence staining were diluted before use: anti-E-cadherin (1:200), anti-ZO-1 (1:200) and anti-β-catenin (1:200). 2 µM TRITC-conjugated phalloidin was used to stain actin filaments. Coverslips were mounted in anti-fading solution with DAPI and viewed using a Zeiss LSM510 laser-scanning confocal microscope image system with a Zeiss x63 Plan-Apochromat objective. Wavelengths of 488 nm, 543 nm and 633 nm were used to excite GFP, TRITC and Cy5, respectively. Beam path filters (BP 505-530 nm and BP 560-615 nm) and a long path filter (LP 650 nm) were used to acquire images for the emission from GFP, TRITC and Cy5, respectively, in a multi-track channel mode.

To compare the extent of adherens junction localization in different GFP-PKC{delta} constructs, the ratio of the fluorescence intensity between adherens junctions and the cytoplasm for each construct was measured. To do this, the pixels from an area (12 µm x 3 µm) on adherens junctions and its adjacent area (12 µm x 3 µm) on the cytoplasm were obtained using Zeiss LSM image software. The ratio of fluorescence intensity (adherens junction versus cytoplasm) for each construct was calculated from 50 cells. A similar method was used to measure the ratio of the fluorescence intensity between the cell membrane and the cytoplasm.

To analyze the fluorescent distribution of the E-cadherin staining after HGF stimulation, the line-scan measurements of E-cadherin fluorescence intensity were conducted along the lines drawn across the regions of interests by Zeiss LSM image software.

Cell-scatter assay and time-lapse microscopy
Cell-scatter assays were performed as described previously (Lai et al., 2000Go; Chen, 2005Go). Briefly, MDCK cells were allowed to grow as colonies on glass coverslips. When the colonies contained about 20 cells, the growth medium containing 10% serum was replaced by fresh medium containing 2% serum and 20 ng/ml HGF. At various lengths of time after HGF stimulation, the cell colonies were fixed and stained with Giemsa stain. Digital images of the colonies were taken under a microscope and the percentage of scattered colonies in all counted colonies (n=100) was determined. A colony is judged as `scattered' when the half of the cells in it have lost contact with their neighbors and exhibit a fibroblast-like phenotype. For time-lapse microscopy, cells on the microscope stage were maintained at 37°C in a humid CO2 atmosphere in a micro-cultivation system (model POC-R, Zeiss) with temperature and CO2 control devices (tempcontrol 37-2 digital and CTI controller 3700 digital, Zeiss). The effect of HGF on cell scattering was monitored under differential interference contrast optics on an inverted Zeiss microscope (Axiovert 100) using a Zeiss x20 LD Achroplan objective. Time-lapse sequential micrographs were captured every 3 minutes using a cooled CCD camera (CoolSNAP fx, Roper Scientific) and analyzed by Meta Imaging SeriesTM software (version 4.5) from Universal Imaging Corporation (West Chester, PA).


   Footnotes
 
We thank P. M. Blumberg for pEGFP-PKC{delta}, D. Joubert for pEGFP-PKC{alpha}, J. S. Brugge for pSRD-PKC{delta}-RD and N. Saito for pTB701-PKC{delta} ({Delta}C1B)-GFP. This work was supported by the National Science Council, Taiwan, Grants NSC96-2320-B-005-002 and NSC96-3112-B-005-001.

Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/4/513/DC1


   References
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

Abdi, K. M. and Bennett, V. (2008). Adducin promotes micron-scale organization of β2-spectrin in lateral membrane of bronchial epithelial cells. Mol. Biol. Cell 19, 536-545.[Abstract/Free Full Text]

Akoyev, V. and Takemoto, D. (2007). ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43. Cell. Signal. 19, 958-967.[CrossRef][Medline]

Alonso-Escolano, D., Medina, C., Cieslik, K., Radomski, A., Jurasz, P., Santos-Martinez, M. J., Jiffar, T., Ruvolo, P. and Radomski, M. W. (2006). Protein kinase C{delta} mediates platelet-induced breast cancer cell invasion. J. Pharmacol. Exp. Ther. 318, 373-380.[Abstract/Free Full Text]

Ashton, A. W., Watanabe, G., Albanese, C., Harrington, E. O., Ware, J. A. and Pestell, R. G. (1999). Protein kinase C delta inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27(Kip1). J. Biol. Chem. 274, 20805-20811.[Abstract/Free Full Text]

Barkalow, K. L., Italiano, J. E., Jr, Chou, D. E., Matsuoka, Y., Bennett,V. and Hartwig, J. H. (2003). Alpha-adducin dissociates from F-actin and spectrin during platelet activation. J. Cell Biol. 161, 557-570.[Abstract/Free Full Text]

Bennett, V., Gardner, K. and Steiner, J. P. (1988). Brain adducin: a protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction. J. Biol. Chem. 263, 5860-5869.[Abstract/Free Full Text]

Brodie, C. and Blumberg, P. M. (2003). Regulation of cell apoptosis by protein kinase C delta. Apoptosis 8, 19-27.[CrossRef][Medline]

Chen, H. C. (2005). Cell scatter assay. In Cell Migration: Developmental Methods and Protocols (ed. J. L. Guan), pp. 69-78. Totowa, NJ: Humana Press.

Chen, C. L., Hsieh, Y. T. and Chen, H. C. (2007). Phosphorylation of adducin by protein kinase C{delta} promotes cell motility. J. Cell Sci. 120, 1157-1167.[Abstract/Free Full Text]

Chen, J. J. W., Peck, K., Hong, T. M., Yang, S. C., Sher, Y. P., Shih, J. Y., Wu, R., Cheng, J. L., Roffler, S. R., Wu, C. W. et al. (2001). Global analysis of gene expression in invasion by a lung cancer model. Cancer Res. 61, 5223-5230.[Abstract/Free Full Text]

Corbit, K. C., Foster, D. A. and Rosner, M. R. (1999). Protein kinase C delta mediates neurogenic but not mitogenic activation of mitogen-activated protein kinase in neuronal cells. Mol. Cell. Biol. 19, 4209-4218.[Abstract/Free Full Text]

Doble, B. W., Ping, P. and Kardami, E. (2000). The {epsilon} subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation. Circ. Res. 86, 293-301.[Abstract/Free Full Text]

D'Souza, T., Indig, F. E. and Morin, P. J. (2007). Phosphorylation of claudin-4 by PKC{epsilon} regulates tight junction barrier function in ovarian cancer cells. Exp. Cell Res. 313, 3364-3375.[CrossRef][Medline]

Durgan, J., Michael, N., Totty, N. and Parker, P. (2007). Novel phosphorylation site markers of protein kinase C delta activation. FEBS Lett. 581, 3377-3381.[CrossRef][Medline]

Eckert, J. J., McCallum, A., Mears, A., Rumsby, M. G., Cameron, I. T. and Fleming, T. P. (2005). Relative contribution of cell contact pattern, specific PKC isoforms and gap junctional communication in tight junction assembly in the mouse early embryo. Dev. Biol. 288, 234-247.[CrossRef][Medline]

Etienne-Manneville, S. and Hall, A. (2003). Cell polarity: Par6, aPKC and cytoskeletal crosstalk. Curr. Opin. Cell Biol. 15, 67-72.[CrossRef][Medline]

Gardner, K. and Bennett, V. (1987). Modulation of spectrin-actin assembly by erythrocyte adducin. Nature 328, 359-362.[CrossRef][Medline]

Gliki, G., Wheeler-Jones, C. and Zachary, I. (2002). Vascular endothelial growth factor induces protein kinase C (PKC)-dependent Akt/PKB activation and phosphatidylinositol 3'-kinase-mediates PKC delta phosphorylation: role of PKC in angiogenesis. Cell Biol. Int. 26, 751-759.[CrossRef][Medline]

Gumbiner, B. M. (2005). Regulation of cadherin-mediated adhesion in morphogenesis. Nat. Rev. Mol. Cell. Biol. 6, 622-634.[Medline]

Imamdi, R., de Graauw, M. and van de Water, B. (2004). Protein kinase C mediates cisplatin-induced loss of adherens junctions followed by apoptosis of renal proximal tubular epithelial cells. J. Pharmacol. Exp. Ther. 311, 892-903.[Abstract/Free Full Text]

Iwabu, A., Smith, K., Allen, F. D., Lauffenburger, D. A. and Wells, A. (2004). Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C delta-dependent pathway. J. Biol. Chem. 279, 14551-14560.[Abstract/Free Full Text]

Kajimoto, T., Shirai, Y., Sakai, N., Yamamoto, T., Matsuzaki, H., Kikkawa, U. and Saito, N. (2004). Ceramide-induced apoptosis by translocation, phosphorylation, and activation of protein kinase C{delta} in the Golgi complex. J. Biol. Chem. 279, 12668-12676.[Abstract/Free Full Text]

Kharait, S., Dhir, R., Lauffenburger, D. and Wells, A. (2006). Protein kinase C{delta} signaling downstream of the EGF receptor mediates migration and invasiveness of prostate cancer cells. Biochem. Biophys. Res. Commun. 343, 848-856.[Medline]

Kiley, S., Clark, K. J., Goodnough, M., Welch, D. R. and Jaken, S. (1999). Protein kinase {delta} involvement in mammary tumor cell metastasis. Cancer Res. 59, 3230-3238.[Abstract/Free Full Text]

Kitamura, K., Mizuno, K., Etoh, A., Akita, Y., Miyamoto, A., Nakayama, K. and Ohno, S. (2003). The second phase activation of protein kinase C delta at late G1 is required for DNA synthesis in serum-induced cell cycle progression. Genes Cells 8, 311-324.[Abstract]

Konishi, H., Yamauchi, E., Taniguchi, H., Yamamoto, T., Matsuzaki, H., Takemura, Y., Ohmae, K., Kikkaawa, U. and Nishizuka, Y. (2001). Phosphorylation sites of protein kinase C {delta} in H2O2-treated cells and its activation by tyrosine kinase in vitro. Proc. Natl. Acad. Sci. USA 98, 6587-6592.[Abstract/Free Full Text]

Kruger, J. S. and Reddy, K. B. (2003). Distinct mechanisms mediate the initial and sustained phases of cell migration in epidermal growth factor receptor-overexpressing cells. Mol. Cancer Res. 1, 801-809.[Abstract/Free Full Text]

Lai, J. F., Kao, S. C., Jiang, S. T., Tang, M. J., Chan, P. C. and Chen, H. C. (2000). Involvement of focal adhesion kinase in hepatocyte growth factor-induced scatter of Madin-Darby canine kidney cells. J. Biol. Chem. 275, 7474-7480.[Abstract/Free Full Text]

Li, C., Wernig, F., Leitges, M., Hu, Y. and Xu, Q. (2003). Mechanical stress-activated PKCdelta regulates smooth muscle cell migration. FASEB J. 17, 2106-2108.[Medline]

Liang, C. C. and Chen, H. C. (2001). Sustained activation of extracellular signal-regulated kinase stimulated by hepatocyte growth factor leads to integrin alpha2 expression that is involved in cell scattering. J. Biol. Chem. 276, 21146-21152.[Abstract/Free Full Text]

Lu, Z., Hornia, A., Jiang, Y. W., Frankel, P., Zang, Q. and Foster, D. A. (1997). Tumor promotion by depleting cells of protein kinase C delta. Mol. Cell. Biol. 17, 3418-3428.[Abstract]

Matsuoka, Y., Li, X. and Bennett, V. (1998). Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons. J. Cell Biol. 142, 485-497.[Abstract/Free Full Text]

Matter, K. and Balda, M. S. (2003). Signaling to and from tight junctions. Nat. Rev. Mol. Cell. Biol. 4, 225-236.[CrossRef][Medline]

Miranti, C. K., Ohno, S. and Brugge, J. S. (1999). Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc. J. Biol. Chem. 274, 10571-10581.[Abstract/Free Full Text]

Miura, T., Yano, T., Naitoh, K., Nishihara, M., Miki, T., Tanno, M. and Shimamoto, K. (2007). {delta}-Opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-{epsilon}-mediated phosphorylation of connexin 43. Am. J. Physiol. Heart Circ. Physiol. 293, H1425-H1431.[Abstract/Free Full Text]

Mullin, J. M., Kampherstein, J. A., Laughlin, K. V., Clarkin, C. E. K., Miller, R. D., Szallasi, Z., Kachar, B., Soler, A. P. and Rosson, D. (1998). Overexpression of protein kinase C-{delta} increases tight junction permeability in LLC-PK1 epithelia. Am. J. Physiol. 275, C544-C554.[Medline]

Nabha, S. M., Glaros, S., Hong, M., Lykkesfeldt, A. E., Schiff, R., Osborne, K. and Reddy, K. B. (2005). Upregulation of PKC-{delta} contributes to antiestrogen resistance in mammary tumor cells. Oncogene 24, 3166-3176.[CrossRef][Medline]

Newton, A. C. (1995). Protein kinase C: structure, function, and regulation. J. Biol. Chem. 270, 28495-28498.[Free Full Text]

Newton, A. C. (2001). Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem. Rev. 101, 2353-2364.[CrossRef][Medline]

Nishizuka, Y. (1995). Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 9, 484-496.[Abstract]

Oh, M. A., Kang, E. S., Lee, S. A., Lee, E. O., Kim, Y. B., Kim, S. H. and Lee, J. W. (2007). PKC{delta} and cofilin activation affects peripheral actin reorganization and cell-cell contact in cells expressing integrin {alpha}5 but not its tailess mutant. J. Cell Sci. 120, 2717-2730.[Abstract/Free Full Text]

Pessino, A., Passalacqua, M., Sparatore, B., Patrone, M., Melloni, E., Pontremoli, S. and Pessino, A. (1995). Antisense oligodeoxynucleotide inhibition of delta protein kinase C expression accelerates induced differentiation of murine erythroleukaemia cells. Biochem. J. 312, 549-554.[Medline]

Quittau-Prevostel, C., Delaunay, N., Collazos, A., Vallentin, A. and Joubert, D. (2004). Targeting of PKC{alpha} and {epsilon} in the pituitary: a highly regulated mechanism involving a GD(E)E motif of the V3 region. J. Cell Sci. 117, 63-72.[Abstract/Free Full Text]

Reddig, P. J., Dreckschmidt, N. E., Ahrens, H., Simsiman, R., Tseng, C. P., Zou, J., Oberley, T. D. and Verma, A. K. (1999). Transgenic mice overexpressing protein kinase C delta in the epidermis are resistant to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 59, 5710-5718.[Abstract/Free Full Text]

Saez, J. C., Berthoud, V. M., Branes, M. C., Martinez, A. D. and Beyer, E. C. (2003). Plasma membrane channels formed by connexins: their regulation and functions. Physiol. Rev. 83, 1359-1400.[Abstract/Free Full Text]

Sandoval, R., Malik, A. B., Minshall, R. D., Kouklis, P., Ellis, C. A. and Tiruppathi, C. (2001). Ca2+ signaling and PKC{alpha} activate increased endothelial permeability by disassembly of VE-cadherin junctions. J. Physiol. 533, 433-445.[Abstract/Free Full Text]

Schultz, A., Ling, M. and Larsson, C. (2004). Identification of an amino acid residue in the protein kinase C C1b domain crucial for its localization to the Golgi network. J. Biol. Chem. 279, 31750-31760.[Abstract/Free Full Text]

Stahelin, R. V., Digman, M. A., Medkova, M., Ananthanarayanan, B. and Rafter, J. D., Melowic, H. R. and Cho, W. (2004). Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase C{delta}. J. Biol. Chem. 279, 29501-29512.[Abstract/Free Full Text]

Thiery, J. P. (2003). Epithelial-mesemchymal transitions in development and pathologies. Cur. Opin. Cell Biol. 15, 740-746.[CrossRef][Medline]

Villar, J., Arenas, M. I., MacCarthy, C. M., Blanquez, M. J., Tirado, O. M. and Notario, V. (2007). PCPH/ENTPD5 expression enhances the invasiveness of human prostate cancer cells by a protein kinase C{delta}-dependent mechanism. Cancer Res. 67, 10859-10868.[Abstract/Free Full Text]

Wallis, S., Lloyd, S., Wise, I., Ireland, G., Fleming, T. P. and Garrod, D. (2000). The {alpha} isoform of protein kinase C is involved in signaling the response of desmosomes to wounding in cultured epithelial cells. Mol. Biol. Cell 11, 1077-1092.[Abstract/Free Full Text]

Wang, Q. J., Bhattacharyya, D., Garfield, S., Nacro, K., Marquez, V. E. and Blumberg, P. M. (1999). Differential localization of protein kinase C delta by phorbol esters and related compounds using a fusion protein with green fluorescent protein. J. Biol. Chem. 274, 37233-37239.[Abstract/Free Full Text]

Zhong, M., Lu, Z. and Foster, D. A. (2002). Downregulating PKC delta provides a PI3K/Akt-independent survival signal that overcomes apoptotic signals generated by c-Src overexpression. Oncogene 21, 1071-1078.[CrossRef][Medline]


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