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First published online 2 September 2003
doi: 10.1242/jcs.00724

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Regulation of p120-catenin nucleocytoplasmic shuttling activity

Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

View larger version (30K): [in a new window]   Fig. 2. Nuclear localization of p120-3A is not dependent on its classical nuclear localization sequences. (A) p120 structure. Multiple isoforms of p120 exist due to the use of alternative ATG start sites and alternative splicing of exons A, B, and C (CC, coiled-coil domain; PD, phosphorylation domain). p120 has two consensus NLS sequences, one localized in the phosphorylation domain and the other in the Arm repeat 6. (B) Deletion mutants were expressed by retroviral transduction in cadherin-deficient A431D cells and their distribution examined by immunofluorescence. p120-3A localizes in the nucleus (i), but deletion of each NLS separately (ii and iii) or in combination (iv) does not affect nuclear localization of p120-3A.

View larger version (56K): [in a new window]   Fig. 1. Endogenous p120 shuttles between the cytoplasm and nucleus in cells lacking E-cadherin expression. Endogenous p120 was localized by immunofluorescence in a panel of E-cadherin-deficient cell lines before (panels A-D) and after (panels E-H) LMB treatment. Treatment with LMB significantly increased nuclear levels of p120, indicating that it is subject to nucleocytoplasmic shuttling.

View larger version (52K): [in a new window]   Fig. 3. The N-terminus regulates the subcellular distribution of p120. (A) Naturally occurring isoforms of p120 that differ only in the length of their N-termini. (B) p120 isoforms 1A, 3A and 4A were retrovirally transduced into E-cadherin-positive (A431) and E-cadherin-negative (A431D) cells. The cells in each figure are representative of the localization seen in the majority of cells. The isoforms show identical distribution in A431 cells (i-iii) but differ in their localization in A431D cells (iv-vi). Incubation with LMB enhanced nuclear levels of p120 1A (vii) and 3A (viii) but had little effect on 4A (ix). (C) The distribution of p120 isoforms in A431D cells before and after LMB treatment was scored by counting random fields of cells (200 cells per slide), and the results are indicated by the bar graphs (n=3). NC, nuclear and cytoplasmic; PC, predominantly cytoplasmic; PN, predominantly nuclear.

View larger version (83K): [in a new window]   Fig. 4. The Arm repeats are necessary for the nucleocytoplasmic shuttling of p120. (A) Individual Arm repeats were deleted in p120-3A. The mutant constructs were transduced into A431D cells and their distribution examined by immunofluorescence. Pictures shown are representative of the localization seen in the majority of cells. Deletion of Arm repeats 3 and 5 abolished nuclear localization of p120 and could not be rescued with LMB incubation. However, deletion of Arm 8 enhanced nuclear localization of p120-3A with little further effect of LMB. (B) The distribution of p120-3A deletion mutants before and after LMB treatment was scored by counting random fields of cells (200 cells per slide), and the results are indicated by the bar graphs (n=1). NC, nuclear and cytoplasmic; PC, predominantly cytoplasmic; PN, predominantly nuclear.

View larger version (65K): [in a new window]   Fig. 5. E-cadherin inhibits nucleocytoplasmic shuttling of p120. (A) E-cadherin-expressing cells (A431 or HT29) were incubated with LMB and the localization of endogenous p120 was detected by immunofluorescence. Incubation with LMB does not result in significant nuclear accumulation of p120 when E-cadherin is present (i-iv). For B, C and D, SKBr3 cells were retrovirally transduced with either vector alone (neo), full-length E-cadherin (E-cadherin) or p120-uncoupled E-cadherin (E-cadherin764), and the shuttling activity of endogenous p120 was examined by immunofluorescence in the absence (CTR) and presence (LMB) of LMB. (B) In the absence of E-cadherin p120 shuttles between the nucleus and the cytoplasm. (C) Re-expression of E-cadherin strongly inhibits the nucleocytoplasmic shuttling of p120. (D) Restoration of adherens junctions with the p120-uncoupled E-cadherin rescues epithelial morphology but does not inhibit p120 shuttling to the nucleus.

View larger version (49K): [in a new window]   Fig. 6. p120 Arm repeats necessary for E-cadherin binding overlap with those necessary for nuclear import. (A) p120 deletion mutants are compared with respect to E-cadherin binding and nuclear localization. + indicates that the deletion mutant can bind E-cadherin and/or localize in the nucleus; ± indicates diminished steady-state levels in the nucleus; and - indicates lack of binding to E-cadherin and/or localization in the nucleus. The data show that Arm repeats 3, 4, 5 and 7 are necessary for both E-cadherin binding and nuclear localization. (B) E-cadherin-positive A431 cells were transduced with Arm 3 and Arm 5 deletion mutants of p120-3A and their distribution compared by immunofluorescence to that of E-cadherin. Both Arm 3 and Arm 5 p120 deletion mutants are found in the cytoplasm; they do not colocalize with E-cadherin, nor do they traffic into the nucleus.

View larger version (82K): [in a new window]   Fig. 7. Increased nucleocytoplasmic shuttling of p120 after deletion of Arm repeat 10. E-cadherin-positive A431 cells were transduced with p120-3A, p120-3AArm8 or p120-3AArm10 and their distribution examined by immunofluorescence p120-3A localizes to the adherens junctions of A431 cell before and after LMB treatment (A and B). The arm 8 deletion mutant localizes to the adherens junctions, the cytoplasm and the nucleus both before and after LMB treatment (C and D). The Arm 10 deletion mutant localizes to the adherens junctions before LMB treatment (E) and prominently to the nucleus after LMB treatment (F).

View larger version (63K): [in a new window]   Fig. 8. p120 nucleocytoplasmic shuttling is modulated by interactions with the microtubule system. (A) A431D cells transduced with p120-3A were double labeled with p120 (i) and -tubulin (ii) antibodies and visualized by conventional immunofluorescent microscopy. p120 colocalized with the microtubules, particularly at the nuclear periphery. (B) A431D cells transduced with p120-1A were double labeled with p120 and -tubulin antibodies before (i, ii) and after (iii, iv) LMB treatment, and visualized at the plane of the nucleus by deconvolution microscopy. Incubation with LMB shifted p120 staining from the microtubules to the nucleus, whereas tubulin staining was unaffected by LMB. The incidence of perinuclear staining of p120 was quantified by counting random fields of cells before and after LMB treatment (v). (C) A431D cells transduced with p120-1A were double labeled with p120 and -tubulin antibodies before (i, ii) and after (iii, iv) nocodazole treatment. Disruption of the microtubule network with nocodazole caused nuclear accumulation of p120-1A (iii) but not tubulin (iv). (D) p120 and microtubules were localized in p120-3A-infected A431D cells before (i, ii) and after (iii, iv) treatment with the microtubule-stabilizing drug taxol. P120-3A is strongly nuclear before taxol treatment but leaves the nucleus to associate with microtubules after stabilization of these structures by taxol.