The heterotrimeric G protein G(12) has been implicated in such cellular regulatory processes as cytoskeletal rearrangement, cell-cell adhesion, and oncogenic transformation. Although the activated alpha-subunit of G(12) has been shown to interact directly with a number of protein effectors, the roles of many of these protein-protein interactions in G(12)-mediated cell physiology are poorly understood. To begin dissecting the specific cellular pathways engaged upon G(12) activation, we produced a series of substitution mutants in the regions of Galpha(12) predicted to play a role in effector binding. Here we report the identification and characterization of an altered form of Galpha(12) that is functionally uncoupled from signaling through the monomeric G protein Rho, a protein known to propagate several Galpha(12)-mediated signals. This mutant of Galpha(12) fails to bind the Rho-specific guanine nucleotide exchange factors p115RhoGEF and LARG (leukemia-associated RhoGEF), fails to stimulate Rho-dependent transcriptional activation, and fails to trigger activation of RhoA and the Rho-mediated cellular responses of cell rounding and c-jun N-terminal kinase activation. Importantly, this mutant of Galpha(12) retains coupling to the effector protein E-cadherin, as evidenced by its ability both to bind E-cadherin in vitro and to disrupt E-cadherin-mediated cell-cell adhesion. Furthermore, this mutant retains the ability to trigger beta-catenin release from the cytoplasmic domain of cadherin. This identification of a variant of Galpha(12) that is selectively uncoupled from one signaling pathway while retaining signaling capacity through a separate pathway will facilitate investigations into the mechanisms through which G(12) proteins mediate diverse biological responses.

beta-Catenin is a multifunctional protein that is known to participate in two well defined cellular processes, cell-cell adhesion and Wnt-stimulated transcriptional activation. Here we report that beta-catenin participates in a third cellular process, the establishment of a bipolar mitotic spindle. During mitosis, beta-catenin relocalizes to mitotic spindle poles and to the midbody. Furthermore, biochemical fractionation demonstrates the presence of beta-catenin in purified centrosome preparations. Reduction of cellular beta-catenin by RNA interference leads to the failure of centrosomes to fully separate, resulting in a marked increase in the frequency of monoastral mitotic spindles. Our results define a new and important function for beta-catenin in mitosis and demonstrate that beta-catenin is involved in vital biological processes beyond cell adhesion and Wnt signaling.

E-cadherin has been termed an "invasion suppressor," yet the mechanism of this suppression is not known. In contrast, several reports indicate N-cadherin does not suppress but, rather, promotes cell motility and invasion. Here, by characterizing a series of chimeric cadherins we defined a previously uncharacterized region consisting of the transmembrane domain and an adjacent portion of the cytoplasmic segment that is responsible for the difference in ability of E- and N-cadherin to suppress movement of mammary carcinoma cells, as quantified from time-lapse video recordings. A mutation in this region enabled N-cadherin to suppress motility, indicating that both E- and N-cadherin can suppress, but the activity of N-cadherin is latent, presumably repressed by binding of a specific inhibitor. To define regions common to E- and N-cadherin that are required for suppression, we analyzed a series of deletion mutants. We found that suppression of movement requires E-cadherin amino acids 699-710. Strikingly, beta-catenin binding is not sufficient for and p120ctn is not involved in suppression of these mammary carcinoma cells. Furthermore, the comparable region of N-cadherin can substitute for this required region in E-cadherin and is required for suppression by the mutant form of N-cadherin that is capable of suppressing. Variations in expression of factors that bind to the two regions we have identified may explain previously observed differences in response of tumor cells to cadherins.

Cadherins function to promote adhesion between adjacent cells and play critical roles in such cellular processes as development, tissue maintenance, and tumor suppression. We previously demonstrated that heterotrimeric G proteins of the G(12) subfamily comprised of Galpha(12) and Galpha(13) interact with the cytoplasmic domain of cadherins and cause the release of the transcriptional activator beta-catenin (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 519-524). Because of the importance of beta-catenin in cadherin-mediated cell-cell adhesion, we examined whether G(12) subfamily proteins could also regulate cadherin function. The introduction of mutationally activated G(12) proteins into K562 cells expressing E-cadherin blocked cadherin-mediated cell adhesion in steady-state assays. Also, in breast cancer cells, the introduction of activated G(12) proteins blocked E-cadherin function in a fast aggregation assay. Aggregation mediated by a mutant cadherin that lacks G(12) binding ability was not affected by activated G(12) proteins, indicating a requirement for direct G(12)-cadherin interaction. Furthermore, in wound-filling assays in which ectopic expression of E-cadherin inhibits cell migration, the expression of activated G(12) proteins reversed the inhibition via a mechanism that was independent of G(12)-mediated Rho activation. These results validate the G(12)-cadherin interaction as a potentially important event in cell biology and suggest novel roles for G(12) proteins in the regulation of cadherin-mediated developmental events and in the loss of cadherin function that is characteristic of metastatic tumor progression.

Heterotrimeric G proteins of the G(12) subfamily mediate cellular signals leading to events such as cytoskeletal rearrangements, cell proliferation, and oncogenic transformation. Several recent studies have revealed direct effector proteins through which G(12) subfamily members may transmit signals leading to various cellular responses. Our laboratory recently demonstrated that G alpha (12) and G alpha (13) specifically interact with the cytoplasmic domains of several members of the cadherin family of cell adhesion molecules (Meigs, T. E., Fields, T. A., McKee, D. D., and Casey, P. J. (2001) Proc. Natl. Acad. Sei. U. S. A. 98, 519-524). This interaction causes beta -catenin to release from cadherin and relocalize to the cytoplasm and nucleus, where it participates in transcriptional activation. Here we report that two distinct regions of the epithelial cadherin (E-cadherin) tail are required for interaction with beta -catenin and G alpha (12), respectively. Deletion of an acidic, 19-amino acid region of E-cadherin abolishes its ability to bind beta -catenin in vitro, to inhibit beta -catenin-mediated transactivation, or to stabilize beta -catenin; causes subcellular mislocalization of beta -catenin; and disrupts cadherin-mediated cell adhesion. On the other hand, deletion of a distinct 11-amino acid region of E-cadherin dramatically attenuates interaction with G alpha (12); furthermore, G alpha (12) is ineffective in stimulating beta -catenin release from an E-cadherin cytoplasmic domain lacking this putative G alpha (12)-binding region. These findings indicate that G alpha (12) and beta -catenin do not compete for the same binding site on cadherin and provide molecular targets for selectively disrupting the interaction of these proteins with cadherin.

The G12 subfamily of heterotrimeric G proteins, comprised of the alpha-subunits Galpha12 and Galpha13, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to cell growth and oncogenesis. In an attempt to elucidate specific roles of this subfamily in cell regulation, we sought to identify molecular targets of Galpha12. Here we show a specific interaction between the G12 subfamily and the cytoplasmic tails of several members of the cadherin family of cell-surface adhesion proteins. Galpha12 or Galpha13 binding causes dissociation of the transcriptional activator beta-catenin from cadherins. Furthermore, in cells lacking the adenomatous polyposis coli protein required for beta-catenin degradation, expression of mutationally activated Galpha12 or Galpha13 causes an increase in beta-catenin-mediated transcriptional activation. These findings provide a potential molecular mechanism for the previously reported cellular transforming ability of the G12 subfamily and reveal a link between heterotrimeric G proteins and cellular processes controlling growth and differentiation.

Regulators of G protein signaling (RGS) are a family of proteins that attenuate the activity of the trimeric G proteins, RGS proteins act as GTPase-activating proteins (GAPs) for the alpha subunits of several trimeric G proteins, much like the GAPs that regulate the activity of monomeric G proteins such as Ras. RGS proteins have been cloned from many eukaryotes, and those whose biochemical activity has been characterized regulate the members of the G(i) family of G proteins; some forms can also act on G(q) proteins. In an ongoing effort to elucidate the role of G(z)alpha in cell signaling, the yeast two-hybrid system was employed to identify proteins that could interact with a mutationally activated form of G(z)alpha. A novel RGS, termed RGSZ1, was identified that is most closely related to two existing RGS proteins termed RetRGS1 and GAIP. Northern blot analysis revealed that expression of RGSZ1 was Limited to brain, and expression was particularly high in the caudate nucleus. Biochemical characterization of recombinant RGSZ1 protein revealed that RGSZ1 was indeed a GAP and, most significantly, showed a marked preference for G(z)alpha over other members of the G(i)alpha family. Phosphorylation of G(z)alpha by protein kinase C, an event known to occur in cells and that was previously shown to influence alpha-beta gamma interactions of G(z), rendered the G protein much less susceptible to RGSZ1 action.