Panos Z. Anastasiadis

A novel role for p120 catenin in E-cadherin function.

Îndirect evidence suggests that p120-catenin (p120) can both positively and negatively affect cadherin adhesiveness. Here we show that the p120 gene is mutated in SW48 cells, and that the cadherin adhesion system is impaired as a direct consequence of p120 insufficiency. Restoring normal levels of p120 caused a striking reversion from poorly differentiated to cobblestone-like epithelial morphology, indicating a crucial role for p120 in reactivation of E-cadherin function. The rescue efficiency was enhanced by increased levels of p120, and reduced by the presence of the phosphorylation domain, a region previously postulated to confer negative regulation. Surprisingly, the rescue was associated with substantially increased levels of E-cadherin. E-cadherin mRNA levels were unaffected by p120 expression, but E-cadherin half-life was more than doubled. Direct p120–E-cadherin interaction was crucial, as p120 deletion analysis revealed a perfect correlation between E-cadherin binding and rescue of epithelial morphology. Interestingly, the epithelial morphology could also be rescued by forced expression of either WT E-cadherin or a p120-uncoupled mutant. Thus, the effects of uncoupling p120 from E-cadherin can be at least partially overcome by artificially maintaining high levels of cadherin expression. These data reveal a cooperative interaction between p120 and E-cadherin and a novel role for p120 that is likely indispensable in normal cells.

Inhibition of RhoA by p120 catenin

RhoA organizes actin stress fibres and is necessary for cell transformation by oncogenes such as src and ras. Moreover, RhoA is implicated in cadherin clustering during the formation of adherens junctions. The catenin p120 has also been implicated in cadherin clustering through an unknown mechanism. Here we show that p120 selectively inhibits RhoA activity in vitro and in vivo. RhoA inhibition and the interaction of p120 with cadherins are mutually exclusive, suggesting a mechanism for regulating the recruitment and exchange of RhoA at nascent cell–cell contacts. By affecting RhoA activation, p120 could modulate cadherin functions, including suppression of invasion, neurite extension and junction formation.

Diverse compounds mimic Alzheimer disease–causing mutations by augmenting Aβ42 production

Increased Aβ42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Aβ42. Among the more potent Aβ42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2–selective NSAID. Many COX-2–selective NSAIDs tested raised Aβ42, including multiple COX-2–selective derivatives of two Aβ42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Aβ42. These compounds seem to target the γ-secretase complex, increasing γ-secretase–catalyzed production of Aβ42 in vitro. Short-term in vivo studies show that two Aβ42-raising compounds increase Aβ42 levels in the brains of mice. The elevations in Aβ42 by these compounds are comparable to the increases in Aβ42 induced by Alzheimer disease–causing mutations in the genes encoding amyloid β protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Aβ42 production in humans.

Regulation of Rho GTPases by p120-catenin.

Three recent reports indicate that p120-catenin can modulate the activities of RhoA, Rac and Cdc42, suggesting an elegant and previously unexpected mechanism for regulating the balance between adhesive and motile cellular phenotypes. The observations in these reports provide important new clues toward p120s mechanism of action and provide a potential explanation for the metastatic phenotype exhibited in carcinoma cells that have lost E cadherin expression.

Protein kinase Cι is required for Ras transformation and colon carcinogenesis in vivo

Protein kinase C ι (PKCι) has been implicated in Ras signaling, however, a role for PKCι in oncogenic Ras-mediated transformation has not been established. Here, we show that PKCι is a critical downstream effector of oncogenic Ras in the colonic epithelium. Transgenic mice expressing constitutively active PKCι in the colon are highly susceptible to carcinogen-induced colon carcinogenesis, whereas mice expressing kinase-deficient PKCι (kdPKCι) are resistant to both carcinogen- and oncogenic Ras-mediated carcinogenesis. Expression of kdPKCι in Ras-transformed rat intestinal epithelial cells blocks oncogenic Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Constitutively active Rac1 (RacV12) restores invasiveness and anchorage-independent growth in Ras-transformed rat intestinal epithelial cells expressing kdPKCι. Our data demonstrate that PKCι is required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis in vivo and define a procarcinogenic signaling axis consisting of Ras, PKCι, and Rac1.

Protein Kinase C (PKC) βII Induces Cell Invasion through a Ras/Mek-, PKCι/Rac 1-dependent Signaling Pathway

Protein kinase C βII (PKCβII) promotes colon carcinogenesis. Expression of PKCβII in the colon of transgenic mice induces hyperproliferation and increased susceptibility to colon cancer. To determine molecular mechanisms by which PKCβII promotes colon cancer, we established rat intestinal epithelial (RIE) cells stably expressing PKCβII. Here we show that RIE/PKCβII cells acquire an invasive phenotype that is blocked by the PKCβ inhibitor LY379196. Invasion is not observed in RIE cells expressing a kinase-deficient PKCβII, indicating that PKCβII activity is required for the invasive phenotype. PKCβII induces activation of K-Ras and the Ras effector, Rac1, in RIE/PKCβII cells. PKCβII-mediated invasion is blocked by the Mek inhibitor, U0126, and by expression of either dominant negative Rac1 or kinase-deficient atypical PKCι. Expression of constitutively active Rac1 induces Mek activation and invasion in RIE cells, indicating that Rac1 is the critical downstream effector of PKCβII-mediated invasion. Taken together, our results define a novel PKCβII → Ras → PKCι /Rac1 → Mek signaling pathway that induces invasion in intestinal epithelial cells. This pathway provides a plausible mechanism by which PKCβII promotes colon carcinogenesis.

Continuous association of cadherin with β-catenin requires the non-receptor tyrosine-kinase Fer

The function of Type 1, classic cadherins depends on their association with the actin cytoskeleton, a connection mediated by α- and β-catenin. The phosphorylation state of β-catenin is crucial for its association with cadherin and thus the association of cadherin with the cytoskeleton. We now show that the phosphorylation of β-catenin is regulated by the combined activities of the tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer phosphorylates PTP1B at tyrosine 152, regulating its binding to cadherin and the continuous dephosphorylation of β-catenin at tyrosine 654. Fer interacts with cadherin indirectly, through p120ctn. We have mapped the interaction domains of Fer and p120ctn and peptides corresponding to these sequences release Fer from p120ctn in vitro and in live cells, resulting in loss of cadherin-associated PTP1B, an increase in the pool of tyrosine phosphorylated β-catenin and loss of cadherin adhesion function. The effect of the peptides is lost when a β-catenin mutant with a substitution at tyrosine 654 is introduced into cells. Thus, Fer phosphorylates PTP1B at tyrosine 152 enabling it to bind to the cytoplasmic domain of cadherin, where it maintains β-catenin in a dephosphorylated state. Cultured fibroblasts from mouse embryos targeted with a kinase-inactivating ferD743R mutation have lost cadherin-associated PTP1B and β-catenin, as well as localization of cadherin and β-catenin in areas of cell-cell contacts. Expression of wild-type Fer or culture in epidermal growth factor restores the cadherin complex and localization at cell-cell contacts.

A Novel Interaction between Kinesin and p120 Modulates p120 Localization and Function

p120-catenin exists in a membrane-associated cadherin-bound pool, a cytosolic pool that affects Rho GTPases, and a nuclear pool that is thought to associate with the methylation-relevant transcriptional repressor Kaiso. We show here that cytoplasmic p120 can also associate both directly and indirectly with the microtubule network, and that p120 traffics along microtubules toward their plus ends. The direct binding required most of the armadillo repeats and was mutually exclusive for interaction with E-cadherin. Perturbing the p120-microtubule interaction with nocodazole or taxol markedly affected both the tubulin interaction and the balance between cytoplasmic and nuclear p120. The indirect binding occurred via a novel interaction between a segment of the p120 N-terminal domain and conventional kinesin heavy chains. Selective uncoupling of the p120-kinesin interaction by overexpression of the respective p120 and kinesin-binding fragments promoted nuclear p120 accumulation. In addition, expression of full-length kinesin reduced the nuclear accumulation of p120 and blocked the branching phenotype associated with p120 overexpression. Taken together, the data suggest that kinesin affects both the targeting and activity of p120 at several cellular locations.

A p120 Catenin Isoform Switch Affects Rho Activity, Induces Tumor Cell Invasion, and Predicts Metastatic Disease

p120 catenin is a cadherin-associated protein that regulates Rho GTPases and promotes the invasiveness of E-cadherin-deficient cancer cells. Multiple p120 isoforms are expressed in cells via alternative splicing, and all of them are essential for HGF signaling to Rac1. However, only full-length p120 (isoform 1) promotes invasiveness. This selective ability of p120 isoform 1 is mediated by reduced RhoA activity, both under basal conditions and following HGF treatment. All p120 isoforms can bind RhoA in vitro, via a central RhoA binding site. However, only the cooperative binding of RhoA to the central p120 domain and to the alternatively spliced p120 N terminus stabilizes RhoA binding and inhibits RhoA activity. Consistent with this, increased expression of p120 isoform 1, when compared with other p120 isoforms, is predictive of renal tumor micrometastasis and systemic progression, following nephrectomy. Furthermore, ectopic expression of the RhoA-binding, N-terminal domain of p120 is sufficient to block the ability of p120 isoform 1 to inhibit RhoA and to promote invasiveness. The data indicate that the increased expression of p120 isoform 1 during tumor progression contributes to the invasive phenotype of cadherin-deficient carcinomas and that the N-terminal domain of p120 is a valid therapeutic target.

p120 catenin is essential for mesenchymal cadherin-mediated regulation of cell motility and invasiveness.

During epithelial tumor progression, the loss of E-cadherin expression and inappropriate expression of mesenchymal cadherins coincide with increased invasiveness. Reexpression experiments have established E-cadherin as an invasion suppressor. However, the mechanism by which E-cadherin suppresses invasiveness and the role of mesenchymal cadherins are poorly understood. We show that both p120 catenin and mesenchymal cadherins are required for the invasiveness of E-cadherin–deficient cells. p120 binding promotes the up-regulation of mesenchymal cadherins and the activation of Rac1, which are essential for cell migration and invasiveness. p120 also promotes invasiveness by inhibiting RhoA activity, independently of cadherin association. Furthermore, association of endogenous p120 with E-cadherin is required for E-cadherin–mediated suppression of invasiveness and is accompanied by a reduction in mesenchymal cadherin levels. The data indicate that p120 acts as a rheostat, promoting a sessile cellular phenotype when associated with E-cadherin or a motile phenotype when associated with mesenchymal cadherins.