CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

<div><p>CD148 is a transmembrane tyrosine phosphatase that is expressed at cell junctions. Recent studies have shown that CD148 associates with the cadherin/catenin complex and p120 catenin (p120) may serve as a substrate. However, the role of CD148 in cadherin cell-cell adhesion remains unknown. Therefore, here we addressed this issue using a series of stable cells and cell-based assays. Wild-type (WT) and catalytically inactive (CS) CD148 were introduced to A431D (lacking classical cadherins), A431D/E-cadherin WT (expressing wild-type E-cadherin), and A431D/E-cadherin 764AAA (expressing p120-uncoupled E-cadherin mutant) cells. The effects of CD148 in cadherin adhesion were assessed by Ca²⁺ switch and cell aggregation assays. Phosphorylation of E-cadherin/catenin complex and Rho family GTPase activities were also examined. Although CD148 introduction did not alter the expression levels and complex formation of E-cadherin, p120, and β-catenin, CD148 WT, but not CS, promoted cadherin contacts and strengthened cell-cell adhesion in A431D/E-cadherin WT cells. This effect was accompanied by an increase in Rac1, but not RhoA and Cdc42, activity and largely diminished by Rac1 inhibition. Further, we demonstrate that CD148 reduces the tyrosine phosphorylation of p120 and β-catenin; causes the dephosphorylation of Y529 suppressive tyrosine residue in Src, a well-known CD148 site, increasing Src activity and enhancing the phosphorylation of Y228 (a Src kinase site) in p120, in E-cadherin contacts. Consistent with these findings, CD148 dephosphorylated both p120 and β-catenin <i>in vitro</i>. The shRNA-mediated CD148 knockdown in A431 cells showed opposite effects. CD148 showed no effects in A431D and A431D/E-cadherin 764AAA cells. In aggregate, these findings provide the first evidence that CD148 promotes E-cadherin adhesion by regulating Rac1 activity concomitant with modulation of p120, β-catenin, and Src tyrosine phosphorylation. This effect requires E-cadherin and p120 association.</p></div

CD148 regulates the tyrosine phosphorylation of p120, β-catenin, and Src upon E-cadherin engagement.

<p>Effects of CD148 in the cadherin adhesion-associated tyrosine phosphorylation of p120, β-catenin, and Src were assessed by a calcium-switch assay and immunoblot analysis using A431D/E-caherin WT (left panels) and A431D/E-cadherin 764AAA (right panels) cells. For p120 and β-catenin, tyrosine phosphorylation of p120 and β-catenin that were co-immunoprecipitated with E-cadherin was assessed by immunoblotting. In A431D/E-cadherin 764 AAA cells, p120 was immunoprecipitated. The membranes were reprobed with p120, β-catenin and Src antibodies and a ratio of phosphorylated to total protein was quantified by densitometry. Data are representative of five independent experiments. CD148 WT, but not CS, reduces the tyrosine phosphorylation of p120, β-catenin, and Src (Y529) upon E-cadherin engagement in A431D/E-cadherin WT cells, while it increases the phosphorylation of Y228 (a Src site) in p120. These effects are not observed in A431D/E-cadherin 764 AAA cells.</p

CD148 knockdown reduces cell-cell adhesion and E-cadherin contacts accompanied by a decrease in Rac1 activity in A431 cells.

<p><b>A)</b> A431 cells was infected with a lentivirus encoding CD148-targeting or scrambled shRNA. Cells were harvested at 72 h after infection and the expression of CD148, E-cadherin, p120, and β- catenin was assessed by immunoblot analysis. Equal loading was confirmed by reblotting the membrane for β-actin. The targeting shRNA reduces CD148 expression (∼65%) without altering the E-cadherin, p120, and β-catenin expression. <b>B)</b> CD148 knock-down cells and the control cells treated with scrambled shRNA were subjected to a handing drop assay. Data are representative of five independent experiments. CD148 knockdown reduces cell aggregation in A431 cells. <b>C and D)</b> CD148 knock-down and control cells were subjected to a calcium switch assay and were immunostained for E-cadherin (panel C). Rac1 activity in these cells was also assessed (panel D). Representative results of four independent experiments are shown. The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148-targeting cells. CD148 knockdown reduces the E-cadherin contact formation accompanied by a decrease in Rac1 activity.</p

A Hypothetical model depicting CD148 regulation of E-cadherin cell-cell adhesion.

<p>CD148 dephosphorylates p120 and β-catenin in E-cadherin contacts. It also dephosphorylates the suppressive tyrosine residue (Y529) in Src, increasing Src activity and possibly enhancing the phosphorylation of Y228 in p120. These signaling events increase Rac1 activity and promote the expansion of contact zones and the stabilization of cadherin complexes, resulting in stronger E-cadherin cell-cell adhesion.</p

Introduction of CD148 forms to A431D and A431D/E-cadherin cells and its effects for E-cadherin and catenin expression and complex formation.

<p><b>A)</b> Wild-type (WT) or catalytically inactive (CS) CD148 was stably introduced to A431D cells lacking classical cadherins [E-cad (−)] or expressing wild-type (WT) or p120-uncoupled mutant (764AAA) E-cadherin. The expression levels of CD148 in these cells were examined by immunoblotting (upper panel) and flow cytometry (lower panel). The loading was assessed by reblotting the membrane for β-actin. <b>B)</b> The levels of E-cadherin, p120, and β-catenin in nearly confluent CD148 stable cells were assessed by immunoblotting, comparing with CD148-negative cells (upper panels). The formation of E-cadherin/catenin complex was assessed by co-immunoprecipitation with E-cadherin (lower panels). Note: The association of E-cadherin with p120 is not observed in A431D/E-cadherin 764 AAA cells.</p

CD148 increases Rac1 activity in the condition of a hanging drop assay.

<p><b>A and B)</b> CD148-introduced or CD148-negative A431D/E-cadherin WT (panel A) and A431D/E-cadherin 764 AAA (panel B) cells were subjected to a hanging drop assay. Rac1, Cdc42, and RhoA activities were assessed at the indicated time points. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by pull-down assays and/or immunoblot analysis (left panels). The relative levels of active versus total Rac1, Cdc42, and RhoA were quantified by densitometric analysis (right panels). The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148 (−) cells. CD148 WT, but not CS, increases Rac1 activity in A431D/E-cadherin WT cells, while this effect is not observed in A431D/E-cadherin 764 AAA cells. <b>C)</b> Effects of CD148 WT in cell-cell adhesion were assessed by a hanging drop assay in the presence or absence of Rac1 inhibitor NSC 23766 (75 µM). CD148 increase of cell-cell adhesion is largely diminished by Rac1 inhibition.</p

CD148 strengthens cell-cell adhesion in A431D/E-cadherin WT, but not A431D or A431D/E-cadherin 764AAA, cells.

<p>Effects of CD148 in cell-cell adhesion were assessed by a hanging drop assay. Images show representative data of ten independent experiments. CD148 WT, but not CS, remarkably increases the cell-cell adhesion in A431D/E-cadherin WT cells, while it shows no effects in A431D or A431D/E-cadherin 764 AAA cells.</p

Effects of CD148 in E-cadherin distribution.

<p>Immunofluorescence localization of CD148 (green), E-cadherin (red), and p120 (purple) were examined in CD148 WT or CS-introduced A431D/E-cadherin WT (left panels) and A431D/E-cadherin 764AAA (right panels) cells and compared with CD148-negative cells. Lower panels show a higher magnification of E-cadherin immunofluorescence. Wild-type E-cadherin is more broadly distributed at cell junctions in CD148 WT-introduced cells (arrowheads in left panels), while the distribution of p120-uncoupled E-cadherin is unaltered by CD148 WT introduction (right panels).</p

CD148 dephosphorylates p120 and β-catenin <i>in vitro</i>.

<p><b>A)</b> A431D/E-cadherin WT and A431D cells were treated with (+) or without (−) pervandadate (PV) and cell lysates were incubated with GST or GST-CD148 (WT, DA) proteins. GST-protein complex were pulled-down using glutathione beads and the protein interactions were examined by immunoblotting (left panels). Vandadate (VO4) competition was also assessed (right panels). Substrate-trapping (DA), but not WT, form of CD148 binds to p120 and β-catenin in a phosphorylation dependent manner and these interactions are blocked by vanadate (VO4). <b>B)</b> CD148 dephosphorylation of E-cadherin, p120, and β-catenin was assessed <i>in vitro</i>. E-cadherin, p120, and β-catenin were immunoprecipitated from the pervanadate (PV)-treated or untreated A431D/E-cadherin WT cells. The immunoprecipitates were incubated with GST or GST-CD148 proteins and its effects were assessed by immunoblotting with a pY20 phosphotyrosine antibody (pY) (left panels). The amount of protein was assessed by reprobing the membranes with specific antibodies. Vanadate (VO4) competition was also assessed (right panels). CD148 WT, but not CS, dephosphorylates p120 and β-catenin in a dose dependent manner, while its effects for E-cadherin are limited. CD148 dephosphorylation of p120 and β-catenin is blocked by vanadate (VO₄).</p

VE-PTP promoter activity in adult mouse kidney.

<p><b>(A)</b> Whole mount X-gal staining of adult VE-PTP^tlacZ/+ mouse kidney. Evident β-galactosidase activity is observed in renal cortex (C), inner stripe (IS) of outer medulla, inner medulla (IM), and papilla (P). Right panel shows vascular distribution of β-galactosidase activity in renal cortex. C, cortex; OS, outer stripe; IS, inner stripe; IM, inner medulla; P, papilla. <b>(B)</b> β-galactosidase activity in adult VE-PTP^tlacZ/+ mouse kidney sections. In medulla, β-galactosidase activity is observed in vascular bundle (VB), subpopulations of medullary tubules (red arrows in panels c and d), and papillary cells (panel e). VB, vascular bundle. <b>(C)</b> β-galactosidase activity in cortical renal vasculature. RA, renal artery: RV, renal vein; AA, arcuate artery; AV, arcuate vein; IA, interlobular artery; IV, interlobular vein; Art, arteriole; G, glomerulus; af, afferent arteriole; ef, efferent arteriole. Note: VE-PTP promoter activity is limited in efferent arterioles, peritubular capillaries, and venous circulations. Scale bar, 200 μm in B-a and B-b; 100 μm in B-c, B-d, B-e, and C; 50 μm in an insert of C.</p