Abstract

Protocadherins are a major subfamily of the cadherin superfamily, but little is known about their functions and intracellular signal transduction. We cloned a novel human protocadherin gene, containing seven EC domains, and identified functional aspects of this gene. The gene was predominantly expressed in l iver, k idney and c olon tissues, and was thus designated Protocadherin LKC . The expression of Protocadherin LKC is markedly reduced in cancers arising from these tissues at both transcriptional and protein levels. To investigate the effects of Protocadherin LKC expression in colon cancer, we introduced the gene into colon cancer cell line HCT116, which does not express this gene. Significantly, Protocadherin LKC expression induced contact inhibition of cell proliferation although it did not affect growth rate. When grown to post-confluence in monolayer cells cultures, Protocadherin LKC -expressing HCT116 no longer formed multiple cell layers and showed the typical paving stone morphology of normal epithelial cells. Furthermore, expression of Protocadherin LKC suppressed tumor formation of HCT116 cells in a nude mouse model. In addition, we identified a protein, hMAST205 (microtubule-associated serine/threonine kinase-205 kDa), which interacted with Protocadherin LKC; the interaction occurring between the PDZ domain of hMAST205 and C-terminal tail of Protocadherin LKC. Our results suggest that Protocadherin LKC, which directly binds PDZ protein, is a molecular switch for contact inhibition of epithelial cells in the liver, kidney and colon tissues.

Introduction

Cadherins play an important role in the communication between adjacent cells. There are at least 80 members of the cadherin superfamily in mammalians including classic cadherins, desmogleins, desmocollins, protocadherins, CNRs, fats, seven-pass transmembrane cadherins, T-cadherin and Ret tyrosine kinase ( 1–3 ). They are characterized by a unique domain called the cadherin motif or EC domain, containing negatively charged DXD, DRE and DXNDNAPXF peptide sequence motifs, which are Ca 2+ -dependent homophilic-binding domains ( 4 ). The EC domains are tandemly repeated in the extracellular segment of all members of the cadherin superfamily, with the number of EC domains varying considerably among the members ( 1 ). Although the presence of EC domains is a hallmark of this molecular superfamily, the amino acid sequences of the other domains, in particular the cytoplasmic domain, significantly vary among the members ( 2 ). Over the past few years, >60 members of protocadherins have been identified and protocadherins are currently a major subfamily of the cadherin superfamily ( 3,5 ). Previously cloned protocadherins members have up to seven EC domains, a single transmembrane region and divergent and distinct cytoplasmic proteins, which also exhibit cell-to-cell adhesion activity, but the adhesion mechanism is thought to be distinct from that of classical cadherins ( 6–8 ). Protocadherins are thought to have other important activities, although the major functions of each protocadherin have not been well elucidated.

The expression levels of cadherins appear to be tightly regulated during development, and each tissue or cell type shows a characteristic pattern of cadherin expression ( 3 ). Down regulation of cadherin expression or functional alteration mainly caused by mutations have been observed in human malignancies, resulting in aggravation of cancer cell invasion and metastasis ( 9 ), suggesting that at least few cadherins act as tumor suppressors ( 10 ). Mutations of E-cadherin are reported in familial sporadic gastric cancers and colorectal cancers ( 10–13 ) and down regulation of E-cadherin was observed in many epithelial cancers ( 14–16 ). T-cadherin/H-cadherin/cadherin-13 is truncated, lacks both the transmembrane and cytoplasmic regions and has also been implicated in tumor suppression ( 17,18 ). Gain-of-function mutations in the Ret gene are associated with multiple endocrine neoplasia ( 19,20 ).

Mutation of the Drosophila fat gene causes hyperplastic, tumor-like overgrowth of larval imaginal discs, defects in differentiation and morphogenesis and death during the pupal stage ( 21,22 ). Thus, the Drosophila fat gene functions as a tumor suppressor gene. Recently, mammalian homologs of the Drosophila fat gene were isolated. Among mammalian species, not only the EC domain but also the cytoplasmic domain of fat genes was highly conserved. The cytoplasmic domain of fat contains a C-terminal peptide motif to bind PDZ domain. PDZ domains are ∼80 amino acid motifs that mediate protein–protein interactions at the plasma membrane ( 23,24 ). PDZ proteins have single or multiple PDZ domain(s), and often contain other types of protein–protein interaction modules, protein kinase and phosphatase domains ( 25,26 ). Although PDZ proteins are found in many different cellular structures, each PDZ protein is generally restricted to specific subcellular compartments, such as synapses; cell-to-cell contacts; or the apical, basal or lateral cell surface ( 27 ). PDZ proteins serve to localize receptors and cytoplasmic signal molecules to specialized membrane sites ( 23 ). In addition, PDZ proteins act as scaffolding to organize other proteins into large supramolecular complexes ( 27,28 ). Consistent with such subcellular compartments and recruitment of the signal molecules, many PDZ proteins play important roles in transduction of receptor–ligand signal and the assembly of the signaling machinery at the plasma membranes.

In this study, we isolated a new human protocadherin that has moderate similarity to the Drosophila tumor suppressor fat and designated it Protocadherin LKC (liver, kidney and colon) based on its tissue-specific expression pattern. The expression of Protocadherin LKC was significantly decreased in liver, kidney and colon cancers. The overexpression of Protocadherin LKC in a colon cancer cell line restored the property of contact inhibition and suppressed tumor formation in nude mice. Furthermore, we identified a Protocadherin LKC -interacting protein, hMAST205 ( h uman m icrotubule- a ssociated s erine/ t hreonine kinase- 205 kDa), using the yeast two-hybrid system. We demonstrate that this interaction occurs between the C-terminal tail of Protocadherin LKC and the PDZ domain of hMAST205. We believe this to be the first report of a PDZ protein interacting directly with a cadherin.

Materials and methods

Protocadherin LKC cDNA cloning

The sequence information of the hypothetical protein FLJ20383 (KAIA2948 clone) allowed the synthesis of two specific primers for 5′ RACE; 5′-R1 (5′-GGAAACGTGGGCCTGTGGTCATTAATG-3′), and 5′-R2 (5′-TCTGTGGCCACAACCTGCACCGCCAT-3′). Using an adult kidney Marathon-Ready cDNA library (Clontech Laboratories, Palo Alto, CA), 5′ nested PCR was performed according to the instructions provided by the manufacturer. The PCR fragments were ligated into a pCR2.1-TOPO cloning vector (Invitrogen, Carlsbad, CA). pCR2.1-LKC carrying the full-length protocadherin LKC cDNA was constructed by subcloning the Cla I– Not I LKC cDNA fragment from KAIA2948 into the Cla I– Not I site in this plasmid. The nucleotide sequence was confirmed by sequencing several clones to avoid errors introduced during the PCR reaction.

Northern blot analysis

Total cellular RNA was extracted by the acid-guanidinium thiocyanate–phenol–chloroform method and then mRNA was extracted using oligo(dT)-Latex beads according to the protocol provided by the manufacturer (Takara Shuzo, Otsu, Shiga, Japan). Northern blotting was performed using Northern Max-Gly™ Northern Blotting kit (Ambion, Austin, TX) as recommended by the manufacturer. The total RNA-Human Normal Colon was purchased from BioChain Institute (Hayward, CA). Each lane contained 3 μg of total RNA. Human multiple tissue northern blots (Clontech) were also used. Northern blot analysis was performed as described previously ( 29 ). The hybridization probes used were the 0.4 kb fragment for the intracellular domain of Protocadherin LKC and the 0.3 kb fragment for the intracellular domain of human E-cadherin .

RT–PCR analysis

Total cellular RNA was extracted as described above. cDNA was synthesized using 0.8 and 0.2 μg of mRNA for cell lines and tumor samples, respectively, incubated in a 20 μl reaction volume containing 100 mM Tris–HCl pH 8.3, 40 mM KCl, 10 mM MgCl 2 , 0.5 mM Spermidine, 5 mM dNTPs and 5 U AMV reverse transcriptase (Invitrogen) for 1 h at 42°C. The RT–PCR designed to evaluate the level of the protocadherin LKC mRNA expression was carried out by using LKC-TD-S (5′-GAAGCTTCAAGCTATGAAGG-3′) and LKC-TD-AS (5′-CCTTGATTTCCTGACTGTTC-3′) primers. The PCR was performed over 29 cycles, consisting of denaturation at 95°C for 30 s, annealing at 52°C for 30 s and extension at 72°C for 30 s. The amplified products were electrophoresed onto 2% agarose gels and detected by ethidium bromide fluorescence. To ensure the successful completion of cDNA synthesis for each sample, G3PDH cDNA was amplified with the following primers; sense, 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and antisense, 5′-CATGTGGGCCATGAGGTCCACCAC-3′, for 25 cycles.

Construction of Protocadherin LKC and hMAST205 expression plasmids

pEGFP- Protocadherin LKC was constructed by subcloning the full-length Protocadherin LKC into the Nhe I– Kpn I site of pEGFP-N3 vector (Clontech). The full-length Protocadherin LKC was derived from the Spe I– Hin dIII fragment of pCR2.1-LKC and the Hin dIII– Kpn I fragments synthesized by PCR using 5′-RACE-S (5′- GCCTCCGTGGGAAGGGGACACAGGT-3′) and N3-AS (5′-GGCCCGGTACCCAGGTCCGTGGTGTCCAGGC-3′) primers. pBTM122-LKC [wild-type (WT)], pGEX4T1-LKC and pME18S-HA-LKC were constructed by subcloning the intracellular domain of Protocadherin LKC (amino acids 1177–1310) into the Eco RI– Not I site of pBTM122, pGEX-4T-1 (Pharmacia Biotech, Piscataway, NJ) and pME18S-HA vectors ( 30 ), respectively. The pLexA DNA-binding domain vector, pBTM122 was a kind gift from T.Oda. pBTM122-LKC [mutant (MU)] was constructed by subcloning the LKC mutant cDNA bearing an amino acid substitution (amino acid 1310 Leu to Arg) synthesized by PCR into the Eco RI– Not I site of pBTM122 vector. The pCEP-LKC was constructed by subcloning the fragment of full-length Protocadherin LKC into the Kpn I– Xho I site of the pCEP4 vector (Invitrogen). pME18S carrying a HA-tagged C-terminal half of hMAST205 and pME18S carrying a FLAG-tagged C-terminal half of hMAST205 were constructed by subcloning a fragment of hMAST205 (amino acid 862–1798) into the Xho I site of pME18S-HA vector and Xho I site of pME18S-FLAG vector, a version of the SRα expression vector to express fusion proteins with FLAG tag at the N-terminus. pME18S carrying a HA-tagged full-length hMAST205 was constructed by subcloning the full-length hMAST205 fragment into the Xho I site of pME18S-HA vector.

Production of anti-Protocadherin LKC polyclonal antibody

The antibody against Protocadherin LKC, termed PepI, was generated by immunizing rabbits with a synthetic peptide CSGQLEGPSYTNAGLDTTDL (LKC amino acids 1292–1310), which was coupled to KLH. The antibody was affinity-purified on a Sepharose column coupled with the same peptide.

Immunohistochemical and immunocytochemical analyses

Tissue array slides, formalin-fixed paraffin-embedded tissue sections, were purchased from SuperBioChips Laboratories (Yuksam-dong, Seoul, Korea). The paraffin sections were dewaxed in xylene and rehydrated in alcohol. Rehydrated slides were placed in jars filled with 10 mM Tri–sodium citrate solution pH 6.0 and incubated three times for 5 min at 700 W in a microwave oven. After microwave heating, slides were allowed to cool to room temperature for 15 min, then washed in phosphate-buffered saline (PBS, pH 7.4), and quenched with endogenous peroxidase in 3% H 2 O 2 solution for 6 min. After blocking with normal goat serum, the slides were treated with PepI antibody (1:500) in PBS for 1 h, and washed with PBS, and incubated with biotinylated anti-rabbit antibody for 10 min and avidin–biotin-conjugated horseradish peroxidase for 5 min, with rinsing in PBS between each step. All reagents were parts of the VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA). Staining was visualized using a Vector DAB/Ni substrate kit (Vector Laboratories) and H 2 O 2 . The sections were lightly counterstained with hematoxylin.

The pCEP-LKC and/or pME18S carrying HA-tagged full-length hMAST205 were/was microinjected into MDCK cells growing on Cellocate (Eppendolf-Netheler-Hinz, Hamburg, Germany). Cells were fixed in 4% paraformaldehyde in PBS, permealized by 0.1% Triton-X-100, blocked by 2% normal goat serum, and incubated with rabbit anti-LKC polyclonal antibody (PepI) (1:500) and/or anti-HA monoclonal antibody (12CA5) (Boehringer Mannheim, Indianapolis, IN) (1:500). The HCT116 and HCT116-B5 cells were fixed with 1.75% formaldehyde, permealized with 1%TritonX-100 and incubated with mouse anti-human E-cadherin monoclonal antibody (HECD-1) (Takara Shuzo) (1:1250). The cells were incubated with Alexa™ 594-conjugated goat anti-rabbit (Molecular Probes, Eugene, OR) (1:200) and/or Alexa™ 488-conjugated goat anti-mouse antibodies (Molecular Probes) (1:200). For staining of actin, cells were incubated with Alexa™ 488 Phalloidin (Molecular Probes) after staining with the secondary antibody. Cells were mounted in VECTASHIELD™ Mounting Medium (Vector Laboratories) and analyzed by confocal microscopy (LSM510 Version1.5/Curl Zeiss Jena, Jena, Germany).

Cell cultures and establishment of Protocadherin LKC-expressing cell lines

MDCK cells, HEK293 cells, HCT116 cells and derivative clones were cultured in DMEM (Dulbecco's modified Eagle's minimum essential medium) supplemented with 10% (v/v) heat-inactivated fetal bovine serum in a humidified CO 2 incubator at 37°C. To establish cell lines that express Protocadherin LKC , pEGFP- Protocadherin LKC plasmid was transfected into HCT116 colon cancer cells by the liposome-mediated gene transfer method. The cells were switched, 48 h later, to a selective medium containing 800 μg/ml geneticin (Gibco BRL, Grand Island, NY). After 14 days of culture in the selective medium, >10 representative geneticin-resistant clones were isolated and expanded. The saturation density was calculated as follows: cells (2×10 5 ) were plated onto 3.5 cm dishes and grown for 12 days in the presence of serum. Cells were harvested by trypsinization and counted using a hemocytometer.

In vivo tumor growth

HCT 116 and derivative clones (3×10 6 cells) were inoculated subcutaneously into athymic BALB/cA Jcl-nu/nu mice (6–7 weeks old, male; CLEA Japan, Tokyo). The tumor was measured with calipers in a blinded fashion once a week and its size calculated according to the formula: length (mm)×width (mm)×height (mm)×0.5236 and reported in cm 3 .

Yeast two-hybrid screening and yeast two-hybrid assay

pBTM122-LKC (WT) plasmid, containing the yeast LEU2 gene and the kanamycin-resistance gene, was used as bait. A human testis MATCHMAKER LexA cDNA library (Clontech) constructed in the B42 activating domain carrying vector pB42AD (Clontech) was screened according to the protocol provided by the manufacturer. The plasmids were transformed into a L40 yeast strain. The positive clones were selected on quintet dropout media plates (SD/Gal/Raf/-His/-Leu/-Lys/-Trp/-Ura + 10 mM 3AT), and then assayed for β-galactosidase activity on other plates (SD/Gal/Raf/-His/-Leu/-Lys/-Trp/-Ura + 10 mM 3AT + X-gal). The inserts of positive clones were sequenced as described previously ( 29 ). For the yeast two-hybrid assay, pBTM122 plasmids carrying the WT and MU cDNA fragments of Protocadherin LKC were used as bait. pB42AD plasmids carrying several deletion cDNAs of hMAST205 were used as prey. The co-transformants were selected on quartet dropout media plates (SD/-Leu/-Lys/-Trp/-Ura), and then assayed for β-galactosidase activity on other plates (SD/Gal/Raf/-His/-Leu/-Lys/-Trp/-Ura + 10 mM 3AT + X-gal).

Immunoprecipitation and western blot analysis

HEK293 cells (1.5×10 6 cells) were co-transfected with 5 μg of pCEP-LKC and 5 μg of pME18S carrying FLAG-tagged C-terminal half of hMAST205 or transfected with only 10 μg of pCEP-LKC using Lipofectamine (Gibco BRL) and Plus Reagent (Gibco BRL). After 72 h incubation, HEK293 cells were lyzed with 2 ml of NP-40 buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM MgCl 2 and 5 mM CaCl 2 ) containing 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, 2 μg/ml pepstatin A and 0.2 mM phenylmethylsulfonyl fluoride. The HEK293 cell lysates (0.5 ml) were incubated with 1 μl of anti-FLAG antibody M2 (Eastman Kodak Company, New Haven, CT) and Protein G plus/Protein A agarose (Oncogene Research Products, Boston, MA) overnight at 4°C. Immune complexes were collected by centrifugation, and washed five times in the NP-40 buffer. After washing, bound proteins were eluted by boiling for 5 min in 2× Laemmeli sample buffer. Samples were separated by 7.5% SDS–PAGE, and transferred onto Immun-Blot PVDF membrane (Bio-Rad Laboratories, Hercules, CA). Blots were blocked with TPBS (PBS, 0.05% Tween 20) containing 5% skimmed milk for 1 h and then incubated with an appropriate dilution of each antibody (1:5000 for PepI, 1:1000 for anti-FLAG) in TPBS for 1 h. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Amersham Pharmacia Biotech, Uppsala, Sweden) (1:10 000) for 1 h, and specific proteins were detected using an enhanced chemiluminescence system (NEN Life Science Products, Boston, MA).

Results

Isolation of cDNA coding for a new Protocadherin that has a C-terminal peptide motif to bind the PDZ domain

In an effort to isolate a new human cadherin gene, we searched Hunt (Human Novel Transcripts) Human Full Length cDNA database ( http://helix-www.hri.co.jp:443/HRIDB ) using a contents search classified based on prosite motifs ( 31 ). The contents search of the Hunt database categorized cadherin extracellular repeated domain signature revealed that several helix clones contained this domain. One of these clones (identical to the hypothetical protein FLJ20383 in the GenBank database: accession no. AK000390) had moderate similarity ( E value = 6e-67) to fat protein which has been identified as a tumor suppressor in Drosophila melanogaster . As the open reading frame (ORF) of this cDNA clone did not contain a signal peptide for membranous expression of cadherins, we performed 5′ RACE and obtained a cDNA that contained a predicted signal peptide. The entire ORF encoded a polypeptide of 1310 amino acids and contained seven cadherin repeats. Both the human protocadherin and Drosophila fat containing the possible PDZ recognized peptide motif at the C-terminus, we identified as shown in Figure 1a ( 21 ).

Northern blot analysis of various adult human tissues using this cDNA as a probe showed that a transcript of ∼5 kb in length, corresponding to the full-length cDNA obtained in our experiment, was highly expressed in the liver, kidney and colon and moderately expressed in the small intestine (Figure 1b ). Because of the specific distribution pattern of the 5 kb transcript, we termed the gene Protocadherin LKC (Protocadherin l iver, k idney and c olon).

Down regulation of Protocadherin LKC transcript in liver and colon cancers

It is well known that the expression of E-cadherin is down regulated in some epithelial tumor cells at the transcriptional level ( 32 ). Therefore, we analyzed the expression of Protocadherin LKC in human colon cancer cell lines by northern blot analysis. In most cell lines, Protocadherin LKC mRNA was markedly down regulated (Figure 2a , upper panel). The down regulation of this mRNA was assumed associating with down regulated levels of E-cadherin (Figure 2a , middle panel). We next examined the expression level of Protocadherin LKC mRNA in clinical samples: 14 matched pairs of colon and liver human cancers and surrounding normal tissues. For the application of small amounts of biopsied samples, we performed RT-PCR analysis. In this PCR condition, we reproduced the results of northern blot analysis on colon cancer cell lines by RT–PCR analysis (Figure 2b ). Five out of eight colon cancer tissues and four out of six liver cancer tissues showed low levels of expression of Protocadherin LKC mRNA compared with the surrounding normal tissues (Figure 2c ).

Down regulation of Protocadherin LKC protein in liver, kidney and colon tumors

To examine the expression of endogenous Protocadherin LKC protein, we raised and purified a polyclonal antibody termed PepI against a synthetic peptide derived from the C-terminal region of Protocadherin LKC protein. The specificity of this antibody was evaluated by western blot analysis of HEK293 cells transiently transfected with the Protocadherin LKC expression plasmid (pCEP-LKC) and with a mock plasmid. An ∼180 kDa band corresponding to the Protocadherin LKC was specifically detected from the lysate of pCEP-LKC transfected HEK293 cells with PepI antibody, but not with preimmune serum (Figure 3a ).

To determine the expression level of Protocadherin LKC protein, we performed immunohistochemical analysis of formalin-fixed paraffin-embedded tissue sections. As the expression of Protocadherin LKC mRNA was specifically decreased or absent in liver, kidney and colon cancers, we performed immunohistochemical analysis for the matched pairs of cancer and surrounding normal tissue sections of these organs using PepI antibody. Protocadherin LKC was detected in cell-to-cell junctions of hepatocytes, the apical surface of renal tubular epithelial cells and absorptive epithelial cells, and at the apical and lateral surfaces of goblet cells, but it was hardly detected in the tumor tissues (Figure 3b ).

Subcellular localization of Protocadherin LKC and its relationship with actin cytoskeleton

To examine the precise subcellular localization of Protocadherin LKC in epithelial cell cultures, the Protocadherin LKC -expression plasmid was microinjected into Madin–Darby canine kidney (MDCK) cells, and examined using confocal microscopy. In cells expressing low level of Protocadherin LKC, the staining was predominantly observed at the cell-to-cell junctions both in horizontal and vertical views. In cells expressing high level of the protein, the staining was not only at the cell-to-cell junctions but also on the apical surface of the cells (Figure 4a ). At higher magnifications, in cells expressing low levels of Protocadherin LKC, staining was detected between the cortical actin rim of adjacent cells. In cells expressing high levels of the protein, Protocadherin LKC and actin were colocalized at the protruding microvilli-like structures from the apical surface of the cells (Figure 4b ).

To examine the role of actin cytoskeleton for the subcellular localization of Protocadherin LKC, we examined cells expressing EGFP-fusion Protocadherin LKC protein (HCT116-B5, see next section) after treatment with several reagents known to alter cytoskeletal reorganization. Protocadherin LKC was observed at cell-to-cell junctions in untreated cells (Figure 4c , control), whereas it was redistributed after treatment with the actin-depolymerizing reagent cytochalasin D (Figure 4c , +cytochalasin D). In contrast, treatment with the microtubule-destabilizing reagent, nocodazole, did not affect the subcellular localization of Protocadherin LKC, even when the reagent was used at a concentration high enough to induce morphological changes (Figure 4c , +nocodazole). These results could not clarify whether the link between Protocadherin LKC and actin cytoskeleton is direct or indirect, but indicated that Protocadherin LKC is certainly associated with the actin cytoskeleton.

Overexpression of Protocadherin LKC induces morphological change and affects cell saturation density

Down regulation of Protocadherin LKC protein in liver, kidney and colon cancers led us to hypothesize that Protocadherin LKC acts as a tumor suppressor in those tissues. To examine this possibility, we established the Protocadherin LKC -expressing clones (HCT116-B5, HCT116-B6) and investigated the effect of Protocadherin LKC expression on growth of a parental colon cancer cell line HCT116, which does not express Protocadherin LKC protein (Figure 5a ).

Expression of EGFP- Protocadherin LKC was associated with a marked change in cell morphology. HCT116-B5 and HCT116-B6 clones (also other clones established in our study; data not shown) grew as islands with strong cell-to-cell contacts, and in stationary phase exhibited the typical paving stone appearance of normal epithelial cells (Figure 5c , right panels). In contrast, HCT116 and HCT116-EGFP cells were round in shape, showed loose cell-to-cell contacts and grew over neighboring cells (Figure 5c , right panels). As the Protocadherin LKC -expressing clones (HCT116-B5, HCT116-B6) showed a marked change in cell morphology, we co-cultured these cells with the parental cells at low density and observed the border area between each clone and parental cells. Protocadherin LKC -expressing clones showed clear borders with the parental cells, while control HCT116-EGFP integrated with parental cells (Figure 5b , left panels). Further cultivation showed that parental cells grew in a vertical direction along the border with the EGFP- Protocadherin LKC -expressing cells (Figure 5b , right panels).

Because the expression of Protocadherin LKC seemed to suppress the piling up of malignant cells, we compared the growth rates and saturation density between the stable clones and parental cells. No significant differences in the growth rates were observed during the exponential growth phase (Figure 5c , left panel). In contrast, the saturation density of EGFP- Protocadherin LKC -expressing clones was reduced to approximately half that of parental cells (Figure 5c , middle and right panels). Furthermore, we investigated whether E-cadherin contribute to the growth property of Protocadherin LKC -expressing cells. No significant difference of the E-cadherin expression was observed between the parental and Protocadherin LKC -expressing cells in spite of the cell density (Figure 5c ). Even though E-cadherin accumulated at the cell-to-cell junctions in parental HCT116 cells, the cells were able to overcome the contact inhibition. These results suggest that Protocadherin LKC is involved in cell-to-cell adhesion and acts as a trigger in the contact inhibition of epithelial cells in liver, kidney and colon tissues.

In vivo effect of Protocadherin LKC expression

We showed that expression of Protocadherin LKC decreased the saturation density of monolayer cell cultures and suppressed piling up of the colon cancer cells. To extend these findings to three-dimensional growth of the cells, we compared in vivo growth of HCT116 colon cancer cell lines and its derivative Protocadherin LKC-expressing clones in nude mice. Protocadherin LKC-expressing clones and the control clones were inoculated subcutaneously into the same mice. As Protocadherin LKC is expressed as EGFP-fusion protein, we also used HCT116-EGFP clone that expresses EGFP alone as control. Both parental HCT116 and HCT116-EGFP cells resulted in marked tumor formation, but Protocadherin LKC-expressing HCT116-B5 and HCT116-B6 clones completely inhibited tumor growth (Table I ).

Interaction of Protocadherin LKC with a protein kinase containing a PDZ domain

To elucidate the signal transduction, which involves Protocadherin LKC, we identified the molecules that interact with the intracellular domain of Protocadherin LKC. We screened ∼8 million clones of a human testis cDNA library in the yeast two-hybrid system using the intracellular domain of Protocadherin LKC including the C-terminal PDZ-recognized peptide motif as bait. The most frequently obtained clone (24 independent cDNAs) encoded a human homologue of mouse MAST205 (microtubule-associated in the spermatid manchette serine/threonine kinase-205 kDa) ( 33 ), a member of the STP family (Ser/Thr kinase and PDZ domains) ( 34 ). The human MAST205 (hMAST205) is strictly homologous (95% identical) to mouse MAST205 and consists of a kinase domain and a PDZ domain (Figure 6a ).

The yeast two-hybrid assay using several deletion constructs revealed that the region from amino acid 1032 to 1294 of hMAST205 including the PDZ domain was sufficient for this binding (Fig. 7a ). Conversely, a Protocadherin LKC mutant bearing an amino acid substitution (Leu to Arg) at the putative C-terminal PDZ-recognized peptide motif did not interact with hMAST205 [Fig. 7a , pBTM-LKC(MU)]. These results indicate that Protocadherin LKC interacts with the PDZ domain of hMAST205 via its C-terminal tail region.

The above interaction was also confirmed by immunoprecipitation in mammalian cells. The full-length Protocadherin LKC and the FLAG-tagged C-terminal half of hMAST205 including the PDZ domain were co-expressed in HEK293 cells. The lysate was immunoprecipitated with anti-FLAG antibody, and Protocadherin LKC was detected in the FLAG-tagged hMAST205 immune complex. Protocadherin LKC was not immunoprecipitated with anti-FLAG antibody in cells that expressed Protocadherin LKC alone (Figure 7b ).

We then examined whether the Protocadherin LKC and hMAST205 co-localized at the subcellular level. Protocadherin LKC and/or HA-tagged full-length hMAST205 expression plasmids were microinjected into MDCK cells. hMAST205 distributed throughout the cytoplasm in the cells microinjected with hMAST205 expression plasmid alone. Co-expression of Protocadherin LKC resulted in hMAST205 being recruited from the cytoplasm to the submembranous area (Figure 7c ). These findings were not specific to MDCK cells; similar results were obtained in SW480 cells (data not shown).

Discussion

In this study, we identified and characterized a new protocadherin containing seven cadherin repeats, designated Protocadherin LKC based on its specific tissue expression pattern. Recent studies have implicated some cadherins in tumorigenesis ( 11,12,17 ), and Protocadherin LKC shows moderate similarity to one such cadherin, Drosophila fat ( 22 ). One of the characteristics of tumor cells is inability of contact inhibition of growth, and E-cadherin as well as other cadherins appears to contribute to the contact inhibition ( 35,36 ).

Our results showed that Protocadherin LKC in the tumor is down regulated at both transcriptional and protein levels. Overexpression of Protocadherin LKC protein restored the contact inhibition of cell proliferation in colon cancer cell line HCT116 cells, which are known to have a profound defect in contact inhibition ( 37 ). Expressed Protocadherin LKC protein accumulated at cell-to-cell adhesion sites, and suppressed HCT116 cells to grow three-dimensionally in a nude mouse model. These results suggest that Protocadherin LKC is important for contact inhibition on the lateral surface of epithelial cells, and absence or repression of Protocadherin LKC induces tumor growth and expansion, more concretely, Protocadherin LKC acts as a tumor suppressor protein, at least in HCT116 colon cancer cell line.

Although recent studies of E-cadherin have suggested that the E-cadherin functions as contact inhibition ( 36 ), it is evident that Protocadherin LKC did it in our experimental system. One concern was that quantitative change of E-cadherin expression might occur in the Protocadherin LKC -expressing clones (HCT116-B5, HCT116-B6). However, overexpression of Protocadherin LKC did not affect expression of E-cadherin. This result suggests that suppression of the abnormal growth in HCT116 cells is due primarily to the expression of Protocadherin LKC rather than the expression of E-cadherin.

Expression of the E-cadherin is down regulated in human colon cancers ( 38–41 ). Furthermore, our recent observation revealed that both of the Protocadherin LKC and E-cadherin expressions are dominantly decreased in high-grade hepatomas (H.Ota et al ., manuscript in preparation). The molecular mechanism of the link between Protocadherin LKC and E-cadherin expressions is still obscure. However, these observations suggest that expression level of Protocadherin LKC is a potential marker for the diagnosis of colon and liver cancers.

The Protocadherin LKC gene was mapped to chromosome 5q35, as indicated in the genomic information of NCBI. Major genetic alterations including large deletions and translocations of this region have not been reported in the liver, kidney or colon cancers ( 42 ), although minor alterations of the gene might be present in those cancers.

Another functional aspect of Protocadherin LKC is the formation of cytoplasmic protrusions. Immunohistochemical staining for Protocadherin LKC protein was intense at the apical surface of renal tubular epithelial cells and absorptive epithelial cells, and goblet cells. These cells commonly have microvilli and face the lumen in bile capillaries, urinary tubules and the digestive tract. We also showed overexpression of Protocadherin LKC in protruding microvilli-like structures on the apical surface of the MDCK cells. Recent studies reported that certain protocadherins are located on the apical surface of cells and are involved in various functions, in addition to cell-to-cell adhesion ( 43–46 ). Protocadherin Chd23, also known as otocadherin, is a critical component of hair bundles within the cochlea. Mutations of Chd23 are known to cause disorganization of the inner stereocilia and deafness in the Waltzer mouse ( 47 ), and Usher syndrome type 1D in humans ( 48 ). Our findings, together with the evidence for Chd23, allow us to postulate that specific protocadherins are required for the proper formation of cytoplasmic protrusions such as stereocilia in every organ.

Intracellular signal transduction of classical cadherins has been extensively studied and characterized. Their intracellular domains interact with β-catenin or plakoglobin and consequently with α-catenin and the actin cytoskeleton ( 3 ). Classical cadherins indirectly interact with PDZ proteins such as ZO1 and ZO2 through α-catenin ( 49 ). PDZ proteins are thought to play important roles in cadherin signal transduction as well as cytoskeletal organization induced by extracellular signals like cell-to-cell adhesion. On the other hand, little is known about the intracellular signal transduction of protocadherins except for CNR (cadherin-related neuronal receptor) ( 2 ). CNR interacts with Fyn, a Src family tyrosine kinase, modulates its enzymatic activity for DAB1 adaptor protein, and subsequently activates downstream signals such as CDK5 ( 50–53 ). Based on the structure variability of the cytoplasmic domains of protocadherins, they should connect protocadherins with their different and specific cytoplasmic partners. The adhesion activity (and probably other activities) of protocadherins appears to require yet unknown intracellular molecules, other than catenins ( 7 ).

To clarify Protocadherin LKC-signal transduction, we identified the PDZ domain containing protein kinase hMAST205 as a protocadherin LKC-interacting protein. MAST205 was originally identified as a molecule that interacts with and potentially modulates the function of microtubules during spermatogenesis ( 33 ). Recently, it was reported that MAST205 also interacts with cortical actin filaments through the formation of a β 2 -syntrophin-dystrophin/utrophin complex ( 34 ). As PDZ proteins act as scaffolding to organize other proteins into large supramolecular complexes ( 28 ), it is fair to speculate that such a protein complex links MAST205 together with Protocadherin LKC to the actin cytoskeleton. Our observation of the redistribution of Protocadherin LKC following cytochalasin D treatment supports this conclusion. However, it is possible that other LKC-interacting molecules link the actin cytoskeleton to its intracellular domain. Further studies are required to identify the components of the Protocadherin LKC-MAST205 protein complex with actin-binding activity.

The PDZ domains recognize a specific peptide sequence motif located at the extreme C-termini of their target proteins with the consensus sequence -(S/T)-X-(V/I/L)-COOH (where X is any residue) ( 54 ). We noticed that not only Protocadherin LKC but also the following human cadherins have a PDZ-recognized peptide motif in the C-terminal tail: Fat, Cadherin-11, μ-cadherin and Cadherin-23 ( 47,48,55–58 ). In addition, we identified a new protocadherin cDNA clone that had a PDZ-recognized peptide motif in the NCBI gene database (cDNA FLJ20377 fis, clone KIAA0462). This is the first report of a PDZ protein that interacts directly with a cadherin. It is still unclear whether the intracellular domains of these cadherins have common functions, but we propose a new category for cadherins that directly interact with PDZ proteins.

In summary, we have demonstrated in the present study a significant reduction of Protocadherin LKC expression in liver, kidney, and colon cancers and that overexpression of Protocadherin LKC prevented HCT116 cells from overcoming contact inhibition and formation of tumors in a nude mouse model. These results suggest that Protocadherin LKC is a tumor suppressor for epithelial cells in liver, kidney and colon tissues. Furthermore, we demonstrated that Protocadherin LKC has a PDZ-recognized peptide motif in its C-terminal tail and associates with hMAST205 protein. As PDZ proteins act as scaffolding to organize other proteins into large supramolecular complexes ( 24 ), we speculate that such protein complexes link Protocadherin LKC to the actin cytoskeleton thereby forming the molecular switch for contact inhibition.

Accession numbers

The human Protocadherin LKC (accession no. AB047004) cDNA sequence and the human MAST205 (accession no. AB047005) have been deposited in the DDBJ/EMBL/ GeneBank databases.

Table I.

Effect of Protocadherin LKC expression on tumor formation of HCT116 in nude mice

 Volume of tumors 
  After 2 weeks (mm 3 )   After 4 weeks (mm 3 )  
 Left flank Right flank Left flank Right flank 
 (–LKC) (+LKC) (–LKC) (+LKC) 
Experiment 1. Control cells HCT116 were injected into the left flank and Protocadherin LKC -expressing cells HCT116-B5 were injected into the right flank.  
Experiment 2. Control cells HCT116 were injected into the left flank and Protocadherin LKC -expressing cells HCT116-B6 were injected into the right flank.  
Experiment 3. Control cells GFP-expressing HCT116 were injected into the left flank and Protocadherin LKC -expressing cells HCT116-B5 were injected into the right flank.  
Experiment 4. Control cells GFP-expressing HCT116 were injected into the left flank and Protocadherin LKC -expressing cells HCT116-B6 were injected into the right flank.  
Experiment 1     
Mouse 1  20 1456 
Mouse 2 320 
Mouse 3  4.5 
Experiment 2     
Mouse 4  1.5 320 
Mouse 5  4.5 135 
Mouse 6 
Experiment 3     
Mouse 7 180 5187 
Mouse 8 408 0.6 2912 0.6 
Mouse 9 202 0.6 2100 0.6 
Experiment 4     
Mouse 10  30 1584 
Mouse 11  18 910 
Mouse 12 72 
 Volume of tumors 
  After 2 weeks (mm 3 )   After 4 weeks (mm 3 )  
 Left flank Right flank Left flank Right flank 
 (–LKC) (+LKC) (–LKC) (+LKC) 
Experiment 1. Control cells HCT116 were injected into the left flank and Protocadherin LKC -expressing cells HCT116-B5 were injected into the right flank.  
Experiment 2. Control cells HCT116 were injected into the left flank and Protocadherin LKC -expressing cells HCT116-B6 were injected into the right flank.  
Experiment 3. Control cells GFP-expressing HCT116 were injected into the left flank and Protocadherin LKC -expressing cells HCT116-B5 were injected into the right flank.  
Experiment 4. Control cells GFP-expressing HCT116 were injected into the left flank and Protocadherin LKC -expressing cells HCT116-B6 were injected into the right flank.  
Experiment 1     
Mouse 1  20 1456 
Mouse 2 320 
Mouse 3  4.5 
Experiment 2     
Mouse 4  1.5 320 
Mouse 5  4.5 135 
Mouse 6 
Experiment 3     
Mouse 7 180 5187 
Mouse 8 408 0.6 2912 0.6 
Mouse 9 202 0.6 2100 0.6 
Experiment 4     
Mouse 10  30 1584 
Mouse 11  18 910 
Mouse 12 72 
Fig. 1.

A novel human Protocadherin LKC is predominantly expressed in liver, kidney and colon tissues. ( a ) The deduced amino acid sequence of Protocadherin LKC and its schematic representation. Protocadherin LKC has a signal peptide sequence (thin underline; 1–17), seven cadherin repeats (white boxes; 31–107, 129–226, 246–340, 485–577, 590–686, 700–797, 934–1043), a transmembrane domain (bold underline; 1152–1176) and a PDZ recognition sequence (double-bold underline; 1307–1310). ( b ) Northern blot analysis of Protocadherin LKC in several human tissues. Human multiple tissue northern blots (Clontech) were probed with Protocadherin LKC probe. Each lane contains 2 μg of poly(A)+ RNA. The source of each tissue sample appears on the top of the panel. The 5 kb Protocadherin LKC transcript is indicated by an arrow.

Fig. 1.

A novel human Protocadherin LKC is predominantly expressed in liver, kidney and colon tissues. ( a ) The deduced amino acid sequence of Protocadherin LKC and its schematic representation. Protocadherin LKC has a signal peptide sequence (thin underline; 1–17), seven cadherin repeats (white boxes; 31–107, 129–226, 246–340, 485–577, 590–686, 700–797, 934–1043), a transmembrane domain (bold underline; 1152–1176) and a PDZ recognition sequence (double-bold underline; 1307–1310). ( b ) Northern blot analysis of Protocadherin LKC in several human tissues. Human multiple tissue northern blots (Clontech) were probed with Protocadherin LKC probe. Each lane contains 2 μg of poly(A)+ RNA. The source of each tissue sample appears on the top of the panel. The 5 kb Protocadherin LKC transcript is indicated by an arrow.

Fig. 2.

Down regulation of Protocadherin LKC transcripts in colon and liver cancers. ( a ) Expression of Protocadherin LKC and E-cadherin in colon cancer cell lines was determined by northern blot analysis. Each lane contains ∼3 μg of total RNA prepared from human colon cancer cell lines and normal colon tissue. The source of each tissue sample appears on the top of the panel. As a control, ethidium bromide (EtBr) staining was performed. ( b ) Expression of Protocadherin LKC in human colon cancer cell lines was determined by RT–PCR analysis. The name of each cell line appears on the top of the panel. As a control, G3PDH (glycerol 3-phosphate dehydrogenase) was amplified. ( c ) The expression of Protocadherin LKC in 14 matched pairs of colon and liver cancers (T) and surrounding normal (N) tissues was determined by RT–PCR analysis.

Fig. 2.

Down regulation of Protocadherin LKC transcripts in colon and liver cancers. ( a ) Expression of Protocadherin LKC and E-cadherin in colon cancer cell lines was determined by northern blot analysis. Each lane contains ∼3 μg of total RNA prepared from human colon cancer cell lines and normal colon tissue. The source of each tissue sample appears on the top of the panel. As a control, ethidium bromide (EtBr) staining was performed. ( b ) Expression of Protocadherin LKC in human colon cancer cell lines was determined by RT–PCR analysis. The name of each cell line appears on the top of the panel. As a control, G3PDH (glycerol 3-phosphate dehydrogenase) was amplified. ( c ) The expression of Protocadherin LKC in 14 matched pairs of colon and liver cancers (T) and surrounding normal (N) tissues was determined by RT–PCR analysis.

Fig. 3.

Detection of Protocadherin LKC protein in liver, kidney and colon tissues and cancers. ( a ) Lysates from HEK293 cells transfected with Protocadherin LKC -expression (pCEP-LKC) and mock (pCEP) plasmids were resolved by 10% SDS–PAGE, and analyzed by western blot using anti-Protocadherin LKC antibody (PepI) and pre-immune serum. Approximately 180 kDa band, corresponding to Protocadherin LKC, is indicated by the arrow. ( b ) Immunohistochemical staining for Protocadherin LKC protein in four matched pairs of liver, kidney and colon cancer and surrounding normal tissues. Left panels: normal tissues, right panels: tumor tissues. Sections were stained with PepI antibody. Subsequently, 3,3′-diaminobenzidine tetrahydrochloride (DAB) was used for color development (brown) and sections were lightly counterstained with hematoxylin. Histopathologically, the tumors are hepatocellular carcinoma, clear cell type renal cell carcinoma, and moderately differentiated colon adenocarcinoma.

Fig. 3.

Detection of Protocadherin LKC protein in liver, kidney and colon tissues and cancers. ( a ) Lysates from HEK293 cells transfected with Protocadherin LKC -expression (pCEP-LKC) and mock (pCEP) plasmids were resolved by 10% SDS–PAGE, and analyzed by western blot using anti-Protocadherin LKC antibody (PepI) and pre-immune serum. Approximately 180 kDa band, corresponding to Protocadherin LKC, is indicated by the arrow. ( b ) Immunohistochemical staining for Protocadherin LKC protein in four matched pairs of liver, kidney and colon cancer and surrounding normal tissues. Left panels: normal tissues, right panels: tumor tissues. Sections were stained with PepI antibody. Subsequently, 3,3′-diaminobenzidine tetrahydrochloride (DAB) was used for color development (brown) and sections were lightly counterstained with hematoxylin. Histopathologically, the tumors are hepatocellular carcinoma, clear cell type renal cell carcinoma, and moderately differentiated colon adenocarcinoma.

Fig. 4.

Protocadherin LKC localizes at cell-to-cell junctions and the apical surface microvilli-like structures related to actin cytoskeleton. ( a ) pCEP-LKC plasmid was microinjected into MDCK cells, and after 48 h the cells were stained with anti-Protocadherin LKC antibody (red fluorescence). Microscopic images were obtained by laser scanning confocal microscopy (LSM510 Version 1.5/Curl Zeiss) and show the horizontal planes at different depths and in vertical sections (bar, 5 μm). ( b ) The microinjected cells were double-stained with anti-Protocadherin LKC antibody and Alexa™ 488 Phalloidin. The red fluorescence (LKC) and green fluorescence (phalloidin) indicate Protocadherin LKC protein and actin cytoskeleton, respectively. Merged images are also shown (merge). The cell-to-cell junction of microinjected and non-injected cells (left upper panel; bar, 20 μm white rectangles) was magnified to examine the relationship between Protocadherin LKC and cortical actin rim (left lower panels; bar, 10 μm). The protruding microvilli-like structures from the apical surface of the cells were also observed at high magnification (right panel; bar, 4 μm) ( c ) We established HCT116 clones stably expressing EGFP-fusion Protocadherin LKC (HCT116-B5) and treated them with cytochalasin D (0.5 μg/ml, 6 h), or nocodazole (0.5 μg/ml, 30 h). EGFP-fusion Protocadherin LKC protein (green) in living cells was observed using fluorescence microscopy.

Fig. 4.

Protocadherin LKC localizes at cell-to-cell junctions and the apical surface microvilli-like structures related to actin cytoskeleton. ( a ) pCEP-LKC plasmid was microinjected into MDCK cells, and after 48 h the cells were stained with anti-Protocadherin LKC antibody (red fluorescence). Microscopic images were obtained by laser scanning confocal microscopy (LSM510 Version 1.5/Curl Zeiss) and show the horizontal planes at different depths and in vertical sections (bar, 5 μm). ( b ) The microinjected cells were double-stained with anti-Protocadherin LKC antibody and Alexa™ 488 Phalloidin. The red fluorescence (LKC) and green fluorescence (phalloidin) indicate Protocadherin LKC protein and actin cytoskeleton, respectively. Merged images are also shown (merge). The cell-to-cell junction of microinjected and non-injected cells (left upper panel; bar, 20 μm white rectangles) was magnified to examine the relationship between Protocadherin LKC and cortical actin rim (left lower panels; bar, 10 μm). The protruding microvilli-like structures from the apical surface of the cells were also observed at high magnification (right panel; bar, 4 μm) ( c ) We established HCT116 clones stably expressing EGFP-fusion Protocadherin LKC (HCT116-B5) and treated them with cytochalasin D (0.5 μg/ml, 6 h), or nocodazole (0.5 μg/ml, 30 h). EGFP-fusion Protocadherin LKC protein (green) in living cells was observed using fluorescence microscopy.

Fig. 5.

Overexpression of Protocadherin LKC induces morphological changes and affects cell saturation density. ( a ) Lysates from HCT116 clones stably expressing EGFP-fusion Protocadherin LKC (HCT116-B5) and parental cells (HCT116) were analyzed by western blot using PepI antibody. An ∼180 kDa band corresponding to Protocadherin LKC is indicated by arrow. ( b ) HCT116 clones stably expressing EGFP (HCT116-EGFP) or EGFP-fusion Protocadherin LKC (HCT116-B5, HCT116-B6) were co-cultured with parental cells (HCT116). A mixture of parental cells and clone (1×10 3 cells) were seeded on 10 cm diameter dishes. The border of parental cells and each clone was examined at day 14 (left panels) and 21 (right panels) by fluorescence phase contrast microscopy. Upper panels indicate phase contrast microscopic images and lower panels fluorescence microscopic images. ( c ) 2×10 5 cells of HCT116-B5, HCT116-B6, HCT116-EGFP and parental HCT116 line were plated on 3.5-cm diameter dishes and cultured in the presence of serum. Triplicate dishes were counted at the indicated time points of the growth curves, and each determination represents the average value (left panel). Cell saturation density was estimated at day 12 (middle panel). Data represent the mean ± SD. At the same time point, cell morphology was observed by phase contrast microscopy (right panels). ( d ) Parental HCT116 and HCT116-B5 cells were stained with anti-E-cadherin antibody.

Fig. 5.

Overexpression of Protocadherin LKC induces morphological changes and affects cell saturation density. ( a ) Lysates from HCT116 clones stably expressing EGFP-fusion Protocadherin LKC (HCT116-B5) and parental cells (HCT116) were analyzed by western blot using PepI antibody. An ∼180 kDa band corresponding to Protocadherin LKC is indicated by arrow. ( b ) HCT116 clones stably expressing EGFP (HCT116-EGFP) or EGFP-fusion Protocadherin LKC (HCT116-B5, HCT116-B6) were co-cultured with parental cells (HCT116). A mixture of parental cells and clone (1×10 3 cells) were seeded on 10 cm diameter dishes. The border of parental cells and each clone was examined at day 14 (left panels) and 21 (right panels) by fluorescence phase contrast microscopy. Upper panels indicate phase contrast microscopic images and lower panels fluorescence microscopic images. ( c ) 2×10 5 cells of HCT116-B5, HCT116-B6, HCT116-EGFP and parental HCT116 line were plated on 3.5-cm diameter dishes and cultured in the presence of serum. Triplicate dishes were counted at the indicated time points of the growth curves, and each determination represents the average value (left panel). Cell saturation density was estimated at day 12 (middle panel). Data represent the mean ± SD. At the same time point, cell morphology was observed by phase contrast microscopy (right panels). ( d ) Parental HCT116 and HCT116-B5 cells were stained with anti-E-cadherin antibody.

Fig. 6.

Protocadherin LKC-interacting protein hMAST205. The deduced amino acid sequence of hMAST205 (human microtubule-associated serine/threonine kinase-205 kDa) and its schematic representation. hMAST205 has a Ser/Thr kinase domain (512–785) and a PDZ domain (1104–1191). Northern blot analysis of hMAST205 in several human tissues. Human multiple tissue northern blots (Clontech) were probed with hMAST205 probe. Each lane contains 2 μg of poly(A)+ RNA. The names of the tissues are shown on the top of the panels. The 7 kb hMAST205 transcript is indicated by an arrow.

Fig. 6.

Protocadherin LKC-interacting protein hMAST205. The deduced amino acid sequence of hMAST205 (human microtubule-associated serine/threonine kinase-205 kDa) and its schematic representation. hMAST205 has a Ser/Thr kinase domain (512–785) and a PDZ domain (1104–1191). Northern blot analysis of hMAST205 in several human tissues. Human multiple tissue northern blots (Clontech) were probed with hMAST205 probe. Each lane contains 2 μg of poly(A)+ RNA. The names of the tissues are shown on the top of the panels. The 7 kb hMAST205 transcript is indicated by an arrow.

Fig. 7.

Protocadherin LKC interacts with the PDZ domain of hMAST205 via its C-terminal tail. ( a ) Interaction of Protocadherin LKC with hMAST205 was detected by a yeast two-hybrid assay. The pLexA DNA-binding domain vector containing wild-type (pBTM-LKC(WT)) or mutant-type (pBTM-LKC(MU); Leu to Arg substitution at the PDZ-recognized peptide motif) intracellular domain of Protocadherin LKC was used as bait. Several deletion constructs of pB42AD-hMAST205 used as prey are shown on the left. For the negative controls, empty pB42AD and pBTM122DBD (pBTM) vectors were used. (Middle panel) β-Galactosidase activity and (right panel) growth of the yeast harboring each plasmid. ( b ) Immunoprecipitation of HEK293 cells transiently transfected with full-length Protocadherin LKC and/or FLAG-tagged C-terminal half of hMAST205 expression plasmids. The lysates were immunoprecipitated with anti-FLAG or anti-Protocadherin LKC antibody. The immunoprecipitates (IP) were separated by SDS–PAGE and analyzed by western blotting using anti-FLAG or anti- Protocadherin LKC antibody. Aliquots of the lysates (total lysate) were analyzed by western blotting using the same antibody. ( c ) Protocadherin LKC and/or HA-tagged hMAST205 expression plasmids were microinjected into the MDCK cells. After 48 h, the cells were fixed and double-stained with anti-Protocadherin LKC and anti-HA antibodies. The red fluorescence (left) and the green fluorescence (center) indicate Protocadherin LKC and hMAST205 proteins, respectively. Merged images are shown on the right (bar, 10 μm).

Fig. 7.

Protocadherin LKC interacts with the PDZ domain of hMAST205 via its C-terminal tail. ( a ) Interaction of Protocadherin LKC with hMAST205 was detected by a yeast two-hybrid assay. The pLexA DNA-binding domain vector containing wild-type (pBTM-LKC(WT)) or mutant-type (pBTM-LKC(MU); Leu to Arg substitution at the PDZ-recognized peptide motif) intracellular domain of Protocadherin LKC was used as bait. Several deletion constructs of pB42AD-hMAST205 used as prey are shown on the left. For the negative controls, empty pB42AD and pBTM122DBD (pBTM) vectors were used. (Middle panel) β-Galactosidase activity and (right panel) growth of the yeast harboring each plasmid. ( b ) Immunoprecipitation of HEK293 cells transiently transfected with full-length Protocadherin LKC and/or FLAG-tagged C-terminal half of hMAST205 expression plasmids. The lysates were immunoprecipitated with anti-FLAG or anti-Protocadherin LKC antibody. The immunoprecipitates (IP) were separated by SDS–PAGE and analyzed by western blotting using anti-FLAG or anti- Protocadherin LKC antibody. Aliquots of the lysates (total lysate) were analyzed by western blotting using the same antibody. ( c ) Protocadherin LKC and/or HA-tagged hMAST205 expression plasmids were microinjected into the MDCK cells. After 48 h, the cells were fixed and double-stained with anti-Protocadherin LKC and anti-HA antibodies. The red fluorescence (left) and the green fluorescence (center) indicate Protocadherin LKC and hMAST205 proteins, respectively. Merged images are shown on the right (bar, 10 μm).

1
Present address: Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu-city, Chiba 292-0812, Japan
2
To whom correspondence should be addressed Email: hkoga@kazusa.or.jp

We thank Drs M.Omata, T.Ikenoue and G.Togo (Second Department of Medicine, University of Tokyo), and Drs A.Nakagawara and M.Ohira (Division of Biochemistry, Chiba Cancer Center Research Institute) for providing the tumor samples and cancer cell lines; Dr T.Kawasaki (Department of Urology, School of Medicine, Niigata) for providing cancer cell line. We also thank Drs O.Ohara and T.Nagase (Kazusa DNA Research Institute) for providing the human MAST205 cDNA clone (KIAA0807) and Dr S.Sugano (Department of Virology, Institute of Medical Science, the University of Tokyo) for providing the human cDNA clone (KAIA2948). We are also grateful to Drs H.Saya, Y.Kawano, N.Hanada (Department of Tumor Genetics and Biology, Kumamoto University School of Medicine) and T.Oda (Division of Genetic Diagnosis, Institute of Medical Science, University of Tokyo) for the helpful suggestions and providing cancer cell lines.

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