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Specific sequences in p120ctn determine subcellular distribution of its multiple isoforms involved in cellular adhesion of normal and malignant epithelial cells

Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

View larger version (105K): [in a new window]   Fig. 1. Differential expression of p120ctn isoforms in epidermal keratinocytes and melanocytes. Horizontal bars along the schematic structure of p120ctn (A) show the domains recognized by MAb 6H11 and MAb pp120. Cryosections of adult human epidermis (B,C) showed prominent immunofluorescence with MAb pp120 at the cell-cell adhesion junctions (C). Melanocytes residing within the basal cell layer, and their dendrites extending up to the upper spinous layer, were positive for MAb 6H11 (B, arrow). The prominent staining of the cornified layer with MAb 6H11 was considered non-specific and was not characterized further. Keratinocytes in primary cultures, when grown in KGM containing 60 µM Ca2+ were negative for MAb 6H11 signal (D), whereas MAb pp120 stained intracellular structures and gave a weak signal at the cell-cell adhesion junctions (E). Keratinocyte cultures grown in the presence of 1 mM Ca2+ (F-I) were also negative for MAb 6H11 (F), whereas pp120 brightly stained keratinocyte cell-cell adhesion junctions (G). Melanocytes (arrows in E and G) were difficult to distinguish with MAb pp120 owing to the intense staining of keratinocytes. Bright staining of melanocytes with MAb 6H11 (D,F) revealed that 1A is the major p120ctn isoform in melanocytes. Phase contrast images (H,I) of panels (F) and (G) demonstrate the pigmented melanocytes against the keratinocyte background.

View larger version (48K): [in a new window]   Fig. 2. Western blot analysis of p120ctn isoforms in normal and malignant epithelial cells. Cell lysates of neonatal human keratinocytes (NHK), HaCaT cells, squamous cell carcinomas (A431, A253, FADU, SCC12, DET562) (A) and normal and malignant melanoma cells (FM516SV3/3, WM793, 1205-LU, WM164, 451-LU) (B) were resolved on 6% PAGE and proteins detected with the MAb pp120 (A, and lower panel in B) and with MAb 6H11 (B, upper panel). The position of the bands corresponding to p120ctn isoforms 1 to 4 are shown on the right side of the blot together with the positions of molecular weight markers. Note that the isoform 1 is specific for melanocytic cells, whereas normal keratinocytes express predominantly isoform 3. A slower migrating band either corresponding to isoform 2 or to the phosphorylated form of isoform 3 is prominent, especially in squamous carcinoma cells. Isoform 4 is present in normal keratinocytes but is largely lost from the tumor cells.

View larger version (29K): [in a new window]   Fig. 3. RT-PCR analysis demonstrating the tissue-specific expression of p120ctn transcripts containing sequences encoding the C-terminal exon B. The tumor tissue cDNA panel and Human Multiple Tissue cDNA panels I and II (Clontech) were used as templates for PCR. The primers produced a 360 bp product, when exon B was included, and a 273 bp fragment when exon B was not present in the transcripts encoding p120ctn. While present in normal pancreas, prostate and colon, exon B is lost from the adenocarcinomas originating from the corresponding tissues.

View larger version (32K): [in a new window]   Fig. 4. Expression constructs encoding p120ctn isoforms, mutants and domains. Organization of p120ctn (A). Four alternative translation initiation sites within the N-terminal globular domain are shown as M1 to M4. The central part of p120ctn contains 10 Armadillo repeats. Two alternatively spliced exons, A and B, reside within the C-terminal domain. (B) Expression constructs encoding the p120ctn isoforms, deletions and domains, attached to the N-terminal FLAG-tag, were cloned into pcDNA3-vector. Proteins expressed by 1205-Lu cells, transiently transfected with each expression construct, were separated on a 9% PAGE, and the MAb M2 was used to demonstrate the FLAG-tagged proteins on the western blot (C). MW, molecular mass markers.

View larger version (38K): [in a new window]   Fig. 5. The phenotype induced by p120ctn isoforms transiently transfected into 1205-Lu melanoma cells. The expression of the FLAG-tagged polypeptides was demonstrated with MAb M2 16 hours after transfection (red signal), and nuclei were visualized with DAPI staining (blue signal). Isoforms 1A (A, B), 2A (C) and 3A (D) gave a strong signal throughout the cell, including the nucleus, and induced prominent branching phenotype (thick arrows). In some cells the signal was excluded from the nucleus (A,B,E,F; thin arrows), and these cells did not show the branching phenotype. The intermediate phenotype showed nuclear localization and lamellipodia (arrowheads in panels A,B). The isoform 4A was cytoplasmic and clearly excluded from the nucleus and did not induce the branching phenotype (E, F). Bar, 50 µm.

View larger version (93K): [in a new window]   Fig. 6. Demonstration of functional nuclear localization signal within the N-terminus of p120ctn. Both expression constructs, N1 and N3, localized in the nucleus (A-D), whereas the C-terminally deleted N1A (see Fig. 4) demonstrated diffuse staining throughout the cell (E,F). Transiently transfected 1205-Lu cells were detected with MAb M2 (A,C,E) and the nuclei demonstrated with DAPI (B,D,F). Bar, 50 µm.

View larger version (40K): [in a new window]   Fig. 7. Effects of deletion mutants and modifications of p120ctn on their subcellular distribution and on the morphology of 1205-Lu cells. The branching phenotype induced by isoforms 2A and 3A was not affected by the deletion of the N-terminal nuclear localization signal (A,B). The deletion of amino acids 63-232 from isoform 2A (2ANS) did not completely abolish the branching phenotype (C), but the deletion construct of isoform 3A (3AAK), with amino acids 205-316 deleted, showed predominantly cytoplasmic localization and only a few lamellipodial protrusions of transiently transfected 1205-Lu cells (D). The nuclear localization of isoform 4A, induced either through the SV40 nuclear localization signal (E,G) or ubinuclein nuclear localization domain (F,H) joined to the amino terminus of 4A, did not induce the branching phenotype in transiently transfected 1205-Lu cells. When the alternatively spliced exon B was included in the transcript (1AB), it abolished both the nuclear localization and the prominent branching phenotype induced by isoform 1A (I). However, the site-directed mutagenesis of the nuclear export signal within exon B (1ABm) was not sufficient to restore the phenotype induced by isoform 1A (J). Red signal, MAb M2. Blue signal, DAPI. Bar, 50 µm.

View larger version (51K): [in a new window]   Fig. 8. The spectrum of cellular phenotypes induced by p120ctn isoforms. P120ctn isoform 1A, expressed in 1205-Lu cells, fills up the entire cell volume, but less dense staining is evident in the cell nucleus (A) and in the lamellipodial extensions (a in panels A,B). Some cells demonstrate flattening and extensive lamellipodial protrusions (B,C). Thin filopodia (b in panels B,C) emerge from the cell body. The extending filopodia form branches (c in panel C). In the fully developed branching phenotype (D), dendritic extensions send out secondary branches (c in panel D). 1205-Lu cells grown in the chamber slides were transiently transfected with p120ctn isoforms and deletion constructs in parallel. Slides were processed for indirect immunofluorescence 16 hours later. Cells positive for the FLAG-tag-specific antibody M2 in each 0.7 cm2 well were counted and divided into four categories (A-D) on the basis of their appearance. The percentage of cells in each category is shown (E). The total number of cells assessed in each well varied from 150 to 350. Bar, 50 µm.

View larger version (132K): [in a new window]   Fig. 9. The phenotype induced by p120ctn isoforms and deletion constructs transiently transfected into HaCaT cells. 16 hours after the transfection, a serum-free medium containing 1 mM Ca2+ was added and cells were incubated for 8 hours before preparing for the immunofluorescence staining. The expression of the FLAG-tagged polypeptides was demonstrated with FLAG-tag-specific rabbit antibody (FITC, green signal), endogenous E-cadherin is shown as red signal (Texas Red), and nuclei were visualized with DAPI staining (blue signal). Isoforms 1A (A,B,E,F), 2A (I,J,L,M) and 3A (O,P,S,T), as well as the deletion construct 2ANS (K,N), colocalized with E-cadherin at the cell-cell junctions, but the overexpression of each transgene resulted in the branching phenotype and the simultaneous disappearance of the E-cadherin staining both from the cell-cell adhesion borders as well as from the cytoplasmic pool (arrowheads in K and N). The isoforms 4A (R, V) and 1AB (C,G), as well as the deletion construct 3AAK (Q,U), displayed cytoplasmic localization and did not interfere with the endogenous E-cadherin signal. The C-terminally truncated isoform 1, 1X, (D,H) localized to the nucleus but did not affect the E-cadherin signal. Bar, 50 µm. The bar in (A) applies to panels E,I,K,L,N,O,S, and the bar in (B) applies to panels C,D,F,G,H,J,M,P,Q,R,T,U,V.

View larger version (77K): [in a new window]   Fig. 10. The nuclear localization of p120ctn isoforms correlates with their capacity to induce the branching phenotype. The isoforms 1A (A), 2A (D) and 3A (F) as well as the deletion construct 2ANS (E) localized equally in the cytoplasm and in the cell nucleus. In addition to the prominent signal at the cell-cell adhesion junctions, filopodial structures are starting to emerge. Isoforms 1AB (B), 4A (H) and the deletion construct 3AAK (G) were excluded from the nucleus but localized to the cytoplasm. The C-terminally truncated 1A, 1X, showed nuclear localization but was not sufficient for the induction of branching cellular phenotype (C). Bar, 50 µm.