Regulation of E-cadherin/Catenin association by tyrosine phosphorylation

Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120- and beta-catenins. We have examined the role of this modification in these proteins in the control of beta-catenin/E-cadherin binding using in vitro assays with recombinant proteins. Recombinant pp60(c-src) efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for beta-catenin and p120-catenin, respectively. pp60(c-src) phosphorylation had opposing effects on the affinities of beta-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of beta-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of beta-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) beta-catenin for E-cadherin. The phosphorylated Tyr residues were identified as Tyr-86 and Tyr-654. Experiments using point mutants in these two residues indicated that, although Tyr-86 was a better substrate for pp60(c-src), only modification of Tyr-654 was relevant for the interaction with E-cadherin. Transient transfections of different mutants demonstrated that Tyr-654 is phosphorylated in conditions in which adherens junctions are disrupted and evidenced that binding of beta-catenin to E-cadherin in vivo is controlled by phosphorylation of beta-catenin Tyr-654.     

Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells.

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.     

Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia

Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-cell and cell-substrate interactions. Here we have identified changes in the adhesions and cytoskeletal interactions of transformed epithelial cells that contribute to their altered morphology. Using MCF-10A human breast epithelial cells as a model system, we have found that transformation by an activated form of ras is characterized by less developed adherens- type junctions between cells but increased focal adhesions. Contributing to the modified adherens junctions of the transformed cells are decreased interactions among beta-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including beta-catenin and p120 Cas. Whereas in the normal cells beta-catenin is found in association with E- cadherin, p120 Cas is not. In the ras-transformed cells, the situation is reversed; tyrosine-phosphorylated p120 Cas, but not tyrosine- phosphorylated beta-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated beta-catenin also shows increased detergent solubility, suggesting a decreased association with the actin cytoskeleton. p120 Cas, whether tyrosine phosphorylated or not, partitions into the detergent soluble fraction, suggesting that it is not tightly bound to the actin cytoskeleton in either the normal or ras- transformed cells. Inhibitors of tyrosine kinases decrease the level of tyrosine phosphorylation and restore a normal epithelial morphology to the ras-transformed cells. In particular, decreased tyrosine phosphorylation of beta-catenin is accompanied by increased interaction with both E-cadherin and the detergent insoluble cytoskeletal fraction. These results suggest that elevated tyrosine phosphorylation of proteins such as beta-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia.     

CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

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 in vitro. 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.     

Tyrosine phosphorylations in vivo associated with v-fms transformation.

The role of tyrosine-specific phosphorylation in v-fms-mediated transformation was examined by immunoblotting techniques together with a high-affinity antibody that is specific for phosphotyrosine. This antiphosphotyrosine antibody detected phosphorylated tyrosine residues on the gp140v-fms molecule, but not gP180v-fms or gp120v-fms, in v-fms-transformed cells. This antibody also identified a number of cellular proteins that were either newly phosphorylated on tyrosine residues or showed enhanced phosphorylation on tyrosine residues as a result of v-fms transformation. However, the substrates of the v-fms-induced tyrosine kinase activity were not the characterized pp60v-src substrates. The phosphorylation of some of these cellular proteins and of the gp140fms molecule was found to correlate with the ability of v-fms/c-fms hybrids to transform cells. In addition, immunoblotting with the phosphotyrosine antibody allowed a comparison to be made of the substrates phosphorylated on tyrosine residues in various transformed cell lines. This study indicates that the pattern of tyrosine phosphorylation in v-fms-transformed cells is strikingly similar to that in v-sis-transformed cells.     

The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane

In contrast to growth factor-stimulated tyrosine phosphorylation of p120, its relatively constitutive serine/threonine phosphorylation is not well understood. Here we examined the role of serine/threonine phosphorylation of p120 in cadherin function. Expression of cadherins in cadherin-null cells converted them to an epithelial phenotype, induced p120 phosphorylation and localized it to sites of cell contact. Detergent solubility and immunofluorescence confirmed that phosphorylated p120 was at the plasma membrane. E-cadherin constructs incapable of traveling to the plasma membrane did not induce serine/threonine phosphorylation of p120, nor did cadherins constructs incapable of binding p120. However, an E-cadherin cytoplasmic domain construct artificially targeted to the plasma membrane did induce serine/threonine phosphorylation of p120, suggesting phosphorylation occurs independently of signals from cadherin dimerization and trafficking through the ER/Golgi. Solubility assays following calcium switch showed that p120 isoform 3A was more effective at stabilizing E-cadherin at the plasma membrane relative to isoform 4A. Since the major phosphorylation domain of p120 is included in isoform 3A but not 4A, we tested p120 mutated in the known phosphorylation sites in this domain and found that it was even less effective at stabilizing E-cadherin. These data suggest that serine/threonine phosphorylation of p120 influences the dynamics of E-cadherin in junctions.     

Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation

p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.     

Phosphorylation of focal adhesion kinase at tyrosine 861 is crucial for Ras transformation of fibroblasts

Although elevated expression and increased tyrosine phosphorylation of focal adhesion kinase (FAK) are crucial for tumor progression, the mechanism by which FAK promotes oncogenic transformation is unclear. We have therefore determined the role of FAK phosphorylation at tyrosine 861 in the oncogenic transformation of NIH3T3 fibroblasts. FAK phosphorylation at tyrosine 861 was increased in both constitutively H-Ras-transformed and H-Ras-inducible NIH3T3 cells, in parallel with cell transformation. However, H-Ras-inducible cells transfected with the nonphosphorylatable mutant FAK Y861F showed decreased migration/invasion, focus forming activity and anchorage-independent growth, compared with either wild-type or kinase-defective FAK. In contrast to unaltered FAK/Src activity, the association of FAK and p130(CAS) was decreased in FAK Y861F-transfected cells, and FAK phosphorylation at tyrosine 861 enhanced this association in vitro. Consistently, FAK Y861F-transfected cells were defective in activation of c-Jun NH(2)-terminal kinase and in expression of matrix metalloproteinase-9 during transformation. Taken together, these results strongly suggest that FAK phosphorylation at tyrosine 861 is crucial for H-Ras-induced transformation through regulation of the association of FAK with p130(CAS).     

Association of p120, a tyrosine kinase substrate, with E- cadherin/catenin complexes

p120 was originally identified as a substrate of pp60src and several receptor tyrosine kinases, but its function is not known. Recent studies revealed that this protein shows homology to a group of proteins, beta-catenin/Armadillo and plakoglobin (gamma-catenin), which are associated with the cell adhesion molecules cadherins. In this study, we examined whether p120 is associated with E-cadherin using the human carcinoma cell line HT29, as well as other cell lines, which express both of these proteins. When proteins that copurified with E- cadherin were analyzed, not only alpha-catenin, beta-catenin, and plakoglobin but also p120 were detected. Conversely, immunoprecipitates of p120 contained E-cadherin and all the catenins, although a large subpopulation of p120 was not associated with E-cadherin. Analysis of these immunoprecipitates suggests that 20% or less of the extractable E- cadherin is associated with p120. When p120 immunoprecipitation was performed with cell lysates depleted of E-cadherin, beta-catenin was no longer coprecipitated, and the amount of plakoglobin copurified was greatly reduced. This finding suggests that there are various forms of p120 complexes, including p120/E-cadherin/beta-catenin and p120/E- cadherin/plakoglobin complexes; this association profile contrasts with the mutually exclusive association of beta-catenin and plakoglobin with cadherins. When the COOH-terminal catenin binding site was truncated from E-cadherin, not only beta-catenin but also p120 did not coprecipitate with this mutated E-cadherin. Immunocytological studies showed that p120 colocalized with E-cadherin at cell-cell contact sites, even after non-ionic detergent extraction. Treatment of cells with hepatocyte growth factor/scatter factor altered the level of tyrosine phosphorylation of p120 as well as of beta-catenin and plakoglobin. These results suggest that p120 associates with E-cadherin at its COOH-terminal region, but the mechanism for this association differs from that for the association of beta-catenin and plakoglobin with E-cadherin, and thus, that p120, whose function could be modulated by growth factors, may play a unique role in regulation of the cadherin- catenin adhesion system.     

Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene.

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.     

Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen.

We characterized the tyrosine phosphorylation sites of free pp60c-src and of pp60c-src associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60c-src were different, both in vitro and in vivo. Free pp60c-src was phosphorylated in vitro at a single site, tyrosine 416. pp60c-src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60c-src in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60c-src associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60c-src associated with mT in transformed cells are identical to those of pp60v-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60c-src associated with mT may play a role in the enhancement of the pp60c-src protein kinase activity and in cell transformation by polyomavirus.     

Tyrosine Phosphorylation and Src Family Kinases Control Keratinocyte Cell–Cell Adhesion

In their progression from the basal to upper differentiated layers of the epidermis, keratinocytes undergo significant structural changes, including establishment of close intercellular contacts. An important but so far unexplored question is how these early structural events are related to the biochemical pathways that trigger differentiation. We show here that β-catenin, γ-catenin/plakoglobin, and p120-Cas are all significantly tyrosine phosphorylated in primary mouse keratinocytes induced to differentiate by calcium, with a time course similar to that of cell junction formation. Together with these changes, there is an increased association of α-catenin and p120-Cas with E-cadherin, which is prevented by tyrosine kinase inhibition. Treatment of E-cadherin complexes with tyrosine-specific phosphatase reveals that the strength of α-catenin association is directly dependent on tyrosine phosphorylation. In parallel with the biochemical effects, tyrosine kinase inhibition suppresses formation of cell adhesive structures, and causes a significant reduction in adhesive strength of differentiating keratinocytes. The Fyn tyrosine kinase colocalizes with E-cadherin at the cell membrane in calcium-treated keratinocytes. Consistent with an involvement of this kinase, fyn-deficient keratinocytes have strongly decreased tyrosine phosphorylation levels of β- and γ-catenins and p120-Cas, and structural and functional abnormalities in cell adhesion similar to those caused by tyrosine kinase inhibitors. Whereas skin of fyn−/− mice appears normal, skin of mice with a disruption in both the fyn and src genes shows intrinsically reduced tyrosine phosphorylation of β-catenin, strongly decreased p120-Cas levels, and important structural changes consistent with impaired keratinocyte cell adhesion. Thus, unlike what has been proposed for oncogene-transformed or mitogenically stimulated cells, in differentiating keratinocytes tyrosine phosphorylation plays a positive role in control of cell adhesion, and this regulatory function appears to be important both in vitro and in vivo.     

Functional role of GTPase-activating protein in cell transformation by pp60v-src.

Morphological transformation of NIH 3T3 cells was observed following coexpression of a portion of the ras GTPase-activating protein (GAP) comprising the amino terminus (GAP-N) and a mutant of v-src (MDSRC) lacking the membrane-localizing sequence. Cells expressing either of these genes alone remained nontransformed. Coexpression of GAP-N with MDSRC did not alter the subcellular localization, kinase activity, or pattern of cellular substrates phosphorylated by the MDSRC product. In contrast to SHC, phospholipase C-gamma 1, and the p85 alpha phosphatidylinositol 3'-kinase subunit, the endogenous GAP product (p120GAP) was highly tyrosine-phosphorylated only in cells transformed by wild-type v-src. Furthermore, for transformation induced by wild-type v-src as well as by coexpression of MDSRC and GAP-N, a strict correlation was observed between cell transformation, elevated tyrosine phosphorylation of p62, p190, and a novel protein of 150 kDa, and complex formation between these proteins and p120GAP. As with cells transformed by wild-type v-src, the MDSRC plus GAP-N transformants remained dependent on endogenous Ras. The results suggest that tyrosine phosphorylation and complex formation involving p120GAP represent critical elements of cell transformation by v-src and that complementation of the cytosolic v-src mutant by GAP-N results, at least in part, from the formation of these complexes.     

Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes.

p120cas is a tyrosine kinase substrate implicated in ligand-induced receptor signaling through the epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor receptors and in cell transformation by Src. Here we report that p120 associates with a complex containing E-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Furthermore, p120 precisely colocalizes with E-cadherin and catenins in vivo in both normal and Src-transformed MDCK cells. Unlike beta-catenin and plakoglobin, p120 has at least four isoforms which are differentially expressed in a variety of cell types, suggesting novel means of modulating cadherin activities in cells. In Src-transformed MDCK cells, p120, beta-catenin, and plakoglobin were heavily phosphorylated on tyrosine, but the physical associations between these proteins were not disrupted. Association of p120 with the cadherin machinery indicates that both Src and receptor tyrosine kinases cross talk with proteins important for cadherin-mediated cell adhesion. These results also strongly suggest a role for p120 in cell adhesion.     

The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110.

Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine-containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both.     

p120 Catenin-associated Fer and Fyn tyrosine kinases regulate beta-catenin Tyr-142 phosphorylation and beta-catenin-alpha-catenin Interaction

beta-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and alpha-catenin. We show here that interaction of beta-catenin with alpha-catenin is regulated by the phosphorylation of beta-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupted the association between both catenins. We have also examined whether these kinases are involved in the regulation of this interaction by K-ras. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little alpha-catenin-beta-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of beta-catenin-alpha-catenin interaction.     

Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts

Rat 3Y1 cells acquire metastatic potential when transformed with v-src, and this potential is enhanced by double transformation with v-src and v-fos (Taniguchi, S., T. Kawano, T. Mitsudomi, G. Kimura, and T. Baba. 1986. Jpn. J. Cancer Res. 77:1193-1197). We compared the activity of cadherin cell adhesion molecules of normal 3Y1 cells with that of v-src transformed (SR3Y1) and v-src and v-fos double transformed (fosSR3Y1) 3Y1 cells. These cells expressed similar amounts of P-cadherin, and showed similar rates of cadherin-mediated aggregation under suspended conditions. However, the aggregates or colonies of these cells were morphologically distinct. Normal 3Y1 cells formed compacted aggregates in which cells are firmly connected with each other, whereas the transformed cells were more loosely associated, and could freely migrate out of the colonies. Overexpression of exogenous E-cadherin in these transformed cells had no significant effect on their adhesive properties. We then found that herbimycin A, a tyrosine kinase inhibitor, induced tighter cell-cell associations in the aggregates of the transformed cells. In contrast, vanadate, a tyrosine phosphatase inhibitor, inhibited the cadherin-mediated aggregation of SR3Y1 and fosSR3Y1 cells but had little effect on that of normal 3Y1 cells. These results suggest that v-src-mediated tyrosine phosphorylation perturbs cadherin function directly or indirectly, and the inhibition of tyrosine phosphorylation restores cadherin action to the normal state. We next studied tyrosine phosphorylation on cadherins and the cadherin-associated proteins, catenins. While similar amounts of catenins were expressed in all of these cells, the 98-kD catenin was strongly tyrosine phosphorylated only in SR3Y1 and fosSR3Y1 cells. Cadherins were also weakly tyrosine phosphorylated only in the transformed cells. The tyrosine phosphorylation of these proteins was enhanced by vanadate, and inhibited by herbimycin A. Thus, the tyrosine phosphorylation of the cadherin-catenin system itself might affect its function, causing instable cell-cell adhesion.     

Adhesion-associated and PKC-modulated changes in serine/threonine phosphorylation of p120-catenin

p120-catenin (p120) was originally identified as a tyrosine kinase substrate, and subsequently shown to regulate cadherin-mediated cell-cell adhesion. Binding of the p120 Arm domain to E-cadherin appears to be necessary to maintain adequate cadherin levels for strong adhesion. In contrast, the sequence amino-terminal to the Arm domain confers a negative regulatory function that is likely to be modulated by phosphorylation. Several agents that induce rapid changes in cell-cell adhesion, including PDBu, histamine, thrombin, and LPA, result in significant changes in p120 S/T phosphorylation. In some cases, these changes are PKC-dependent, but the relationship among adhesion, PKC activation, and p120 phosphorylation is unclear, in part because the relevant p120 phosphorylation sites are unknown. As a crucial step toward directly identifying the function of these modifications in adhesion, we have used two-dimensional tryptic mapping and site-directed mutagenesis to pinpoint the constitutive and PKC-modulated sites of p120 S/T phosphorylation. Of eight sites that have been identified, two were selectively phosphorylated in vitro by GSK3 beta, but in vivo treatment of cells with GSK3 beta inhibitors did not eliminate these sites. PKC stimulation in vivo induced potent dephosphorylation at S268, and partial dephosphorylation of several additional sites. Surprisingly, PKC also strongly induced phosphorylation at S873. These data directly link PKC activation to specific changes in p120 phosphorylation, and identify the target sites associated with the mechanism of PKC-dependent adhesive changes induced by agents such as histamine and PDBu.     

Specific phosphorylation of p120-catenin regulatory domain differently modulates its binding to RhoA

p120-catenin is an adherens junction-associated protein that controls E-cadherin function and stability. p120-catenin also binds intracellular proteins, such as the small GTPase RhoA. In this paper, we identify the p120-catenin N-terminal regulatory domain as the docking site for RhoA. Moreover, we demonstrate that the binding of RhoA to p120-catenin is tightly controlled by the Src family-dependent phosphorylation of p120-catenin on tyrosine residues. The phosphorylation induced by Src and Fyn tyrosine kinases on p120-catenin induces opposite effects on RhoA binding. Fyn, by phosphorylating a residue located in the regulatory domain of p120-catenin (Tyr112), inhibits the interaction of this protein with RhoA. By contrast, the phosphorylation of Tyr217 and Tyr228 by Src promotes a better affinity of p120-catenin towards RhoA. In agreement with these biochemical data, results obtained in cell lines support the important role of these phosphorylation sites in the regulation of RhoA activity by p120-catenin. Taken together, these observations uncover a new regulatory mechanism acting on p120-catenin that contributes to the fine-tuned regulation of the RhoA pathways during specific signaling events.     

Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases

p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.