Thomas E. Meigs

Interaction of Gα12 and Gα13 with the cytoplasmic domain of cadherin provides a mechanism for β-catenin release

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.

Gα12 Directly Interacts with PP2A EVIDENCE FOR Gα12-STIMULATED PP2A PHOSPHATASE ACTIVITY AND DEPHOSPHORYLATION OF MICROTUBULE-ASSOCIATED PROTEIN, Tau

The Gα12/13 family of heterotrimeric G proteins modulate multiple cellular processes including regulation of the actin cytoskeleton. Gα12/13 interact with several cytoskeletal/scaffolding proteins, and in a yeast two-hybrid screen with Gα12, we detected an interaction with the scaffolding subunit (Aα) of the Ser/Thr phosphatase, protein phosphatase 2A (PP2A). PP2A dephosphorylates multiple substrates including tau, a microtubule-associated protein that is hyperphosphorylated in neurofibrillary tangles. The interaction of Aα and Gα12 was confirmed by coimmunoprecipitation studies in transfected COS cells and by glutathione S-transferase (GST)-Gα12 pull-downs from cell lysates of primary neurons. The interaction was specific for Aα and Gα12 and was independent of Gα12 conformation. Endogenous Aα and Gα12 colocalized by immunofluorescent microscopy in Caco-2 cells and in neurons. In vitro reconstitution of GST-Gα12 or recombinant Gα12 with PP2A core enzyme resulted in ∼300% stimulation of PP2A activity that was not detected with other Gα subunits and was similar with GTPγS- and GDP-liganded Gα12. When tau and active kinase (Cdk5 and p25) were cotransfected in to COS cells, there was robust tau phosphorylation. Co-expression of wild type or QLα12 with tau and the active kinase resulted in 60 ± 15% reductions in tau phosphorylation. In primary cortical neurons stimulated with lysophosphatitic acid, a 50% decrease in tau phosphorylation was observed. The Gα12 effect on tau phosphorylation was inhibited by the PP2A inhibitor, okadaic acid (50 nm), in COS cells and neurons. Taken together, these findings reveal novel, direct regulation of PP2A activity by Gα12 and potential in vivo modulation of PP2A target proteins including tau.

Gα12 Interaction with αSNAP Induces VE-cadherin Localization at Endothelial Junctions and Regulates Barrier Function

The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified αSNAP as an interactive partner of Gα12 using yeast two-hybrid screening. glutathione S-transferase pull-down assays showed the selective interaction of αSNAP with Gα12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of Gα12 (1–37 amino acids) was necessary and sufficient for its interaction with αSNAP. Gα13 with its N-terminal extension replaced by that of Gα12 acquired the ability to bind to αSNAP, whereas Gα12 with its N terminus replaced by that of Gα13 lost this ability. Using four point mutants of αSNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of αSNAP was involved in its interaction with Gα12. Co-transfection of human umbilical vein endothelial cells with Gα12 and αSNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of αSNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with Gα12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the α subunit of G12 and αSNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function.

Domains Necessary for Gα12 Binding and Stimulation of Protein Phosphatase-2A (PP2A): Is Gα12 a Novel Regulatory Subunit of PP2A?

Many cellular signaling pathways share regulation by protein phosphatase-2A (PP2A), a widely expressed serine/threonine phosphatase, and the heterotrimeric G protein Gα12. PP2A activity is altered in carcinogenesis and in some neurodegenerative diseases. We have identified binding of Gα12 with the Aα subunit of PP2A, a trimeric enzyme composed of A (scaffolding), B (regulatory), and C (catalytic) subunits and demonstrated that Gα12 stimulated phosphatase activity (J Biol Chem 279: 54983–54986, 2004). We now show in substrate-velocity analysis using purified PP2A that Vmax was stimulated 3- to 4-fold by glutathione transferase (GST)-Gα12 with little effect on Km values. To identify the binding domains mediating the Aα-Gα12 interaction, an extensive mutational analysis was performed. Well-characterized mutations of Aα were expressed in vitro and tested for binding to GST-Gα12 in pull-down assays. Gα12 binds to Aα along repeats 7 to 10, and PP2A B subunits are not necessary for binding. To identify where Aα binds to Gα12, a series of 61 Gα12 mutants were engineered to contain the sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS) in place of 6 consecutive amino acids. Mutant Gα12 proteins were individually expressed in human embryonic kidney cells and analyzed for interaction with GST or GST-Aα in pull-down assays. The Aα binding sites were localized to regions near the N and C termini of Gα12. The expression of constitutively activated Gα12 (QLα12) in Madin Darby canine kidney cells stimulated PP2A activity as determined by decreased phosphorylation of tyrosine 307 on the catalytic subunit. Based on crystal structures of Gα12 and PP2A Aα, a model describing the binding surfaces and potential mechanisms of Gα12-mediated PP2A activation is presented.

Two Regions of Cadherin Cytoplasmic Domains Are Involved in Suppressing Motility of a Mammary Carcinoma Cell Line

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, β-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.

Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis.

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations in PKD1, the gene for polycystin-1 (PC1), a transmembrane protein with a cytoplasmic C-terminus that interacts with numerous signaling molecules, including Gα12. The functions of PC1 and the mechanisms of cyst development leading to renal failure are complex. Recently, we reported that PC1 expression levels modulate activity of Gα12-stimulated apoptosis (Yu et al., J. Biol. Chem. 2010 285(14):10243-51). Herein, a mutational analysis of Gα12 and PC1 was undertaken to identify regions required for their interaction and ability to modulate apoptosis. A set of Gα12 mutations with systematic replacement of six amino acids with NAAIRS was tested for binding to the PC1 C-terminus in GST pulldowns. Additionally, a series of deletions within the PC1 C-terminus was examined for binding to Gα12. We identified 3 NAAIRS substitutions in Gα12 that completely abrogated binding, and identified a previously described 74 amino acid Gαi/o binding domain in the PC1 C-terminus as necessary for Gα12 interaction. The functional consequences of uncoupling PC1/Gα12 binding were studied in apoptosis assays utilizing HEK293 cells with inducible PC1 overexpression. Gα12 mutants deficient in PC1 binding were refractory to PC1 inhibition of Gα12-stimulated apoptosis. Likewise, deletion of the Gα12-interacting sequence from the PC1 cytoplasmic domain abrogated its inhibition of Gα12-stimulated apoptosis. Based on the crystal structure of Gα12, the PC1 interaction sites are likely to reside on exposed regions within the G protein helical domain. These structural details should facilitate the design of reagents to uncouple PC1/Gα12 signaling in ADPKD.

Isolation of Centrosomes from Cultured Mammalian Cells

INTRODUCTIONThe centrosome is a cells primary microtubule-organizing center. In most mammalian cells, the centrosome is composed of a pair of centrioles and surrounding pericentriolar material. The centrosome is duplicated exactly once per cell cycle such that at the onset of mitosis, a cell has two centrosomes, which serve as poles of the mitotic spindle. During cytokinesis, one centrosome is segregated to each daughter cell. This protocol describes the isolation of centrosomes from asynchronous cells, and thus the purified material will consist primarily of interphase centrosomes. Isolated centrosomes can be used in a variety of assays, including studies of microtubule function and the identification of centrosome-associated proteins and their interactions.

Gα12 Structural Determinants of Hsp90 Interaction Are Necessary for Serum Response Element–Mediated Transcriptional Activation

The G12/13 class of heterotrimeric G proteins, comprising the α-subunits Gα12 and Gα13, regulates multiple aspects of cellular behavior, including proliferation and cytoskeletal rearrangements. Although guanine nucleotide exchange factors for the monomeric G protein Rho (RhoGEFs) are well characterized as effectors of this G protein class, a variety of other downstream targets has been reported. To identify Gα12 determinants that mediate specific protein interactions, we used a structural and evolutionary comparison between the G12/13, Gs, Gi, and Gq classes to identify “class-distinctive” residues in Gα12 and Gα13. Mutation of these residues in Gα12 to their deduced ancestral forms revealed a subset necessary for activation of serum response element (SRE)–mediated transcription, a G12/13-stimulated pathway implicated in cell proliferative signaling. Unexpectedly, this subset of Gα12 mutants showed impaired binding to heat-shock protein 90 (Hsp90) while retaining binding to RhoGEFs. Corresponding mutants of Gα13 exhibited robust SRE activation, suggesting a Gα12-specific mechanism, and inhibition of Hsp90 by geldanamycin or small interfering RNA–mediated lowering of Hsp90 levels resulted in greater downregulation of Gα12 than Gα13 signaling in SRE activation experiments. Furthermore, the Drosophila G12/13 homolog Concertina was unable to signal to SRE in mammalian cells, and Gα12:Concertina chimeras revealed Gα12-specific determinants of SRE activation within the switch regions and a C-terminal region. These findings identify Gα12 determinants of SRE activation, implicate Gα12:Hsp90 interaction in this signaling mechanism, and illuminate structural features that arose during evolution of Gα12 and Gα13 to allow bifurcated mechanisms of signaling to a common cell proliferative pathway.

Determinants at the N- and C-termini of Gα 12 required for activation of Rho-mediated signaling

Background Heterotrimeric guanine nucleotide binding proteins of the G12/13 subfamily, which includes the α-subunits Gα12 and Gα13, stimulate the monomeric G protein RhoA through interaction with a distinct subset of Rho-specific guanine nucleotide exchange factors (RhoGEFs). The structural features that mediate interaction between Gα13 and RhoGEFs have been examined in crystallographic studies of the purified complex, whereas a Gα12:RhoGEF complex has not been reported. Several signaling responses and effector interactions appear unique to Gα12 or Gα13, despite their similarity in amino acid sequence. Methods To comprehensively examine Gα12 for regions involved in RhoGEF interaction, we screened a panel of Gα12 cassette substitution mutants for binding to leukemia-associated RhoGEF (LARG) and for activation of serum response element mediated transcription. Results We identified several cassette substitutions that disrupt Gα12 binding to LARG and the related p115RhoGEF. These Gα12 mutants also were impaired in activating serum response element mediated signaling, a Rho-dependent response. Most of these mutants matched corresponding regions of Gα13 reported to contact p115RhoGEF, but unexpectedly, several RhoGEF-uncoupling mutations were found within the N- and C-terminal regions of Gα12. Trypsin protection assays revealed several mutants in these regions as retaining conformational activation. In addition, charge substitutions near the Gα12 N-terminus selectively disrupted binding to LARG but not p115RhoGEF. Conclusions Several structural aspects of the Gα12:RhoGEF interface differ from the reported Gα13:RhoGEF complex, particularly determinants within the C-terminal α5 helix and structurally uncharacterized N-terminus of Gα12. Furthermore, key residues at the Gα12 N-terminus may confer selectivity for LARG as a downstream effector.

Gα12 binds to the N-terminal regulatory domain of p120ctn, and downregulates p120ctn tyrosine phosphorylation induced by Src family kinases via a RhoA independent mechanism

p120 Catenin (p120(ctn)) regulates cadherin stability, and thus facilitates strong cell-cell adhesion. Previously, we demonstrated that Gα(12) interacts with p120(ctn). In the present study, we have delineated a region of p120(ctn) that binds to Gα(12). We report that the N-terminal region of p120(ctn) (amino acids 1-346) is necessary and sufficient for the interaction. While the coiled-coiled domain and a charged region, comprising a.a 102-120, were found to be dispensable, amino acids 121-323 were required for p120(ctn) binding to Gα(12). This region harbors the phosphorylation domain of p120(ctn) and has been postulated as important for RhoA regulation. Downregulation of Src family kinase-induced tyrosine phosphorylation of p120(ctn) was observed in the presence of activated Gα(12). This down-regulation was triggered by three different Gα(12) mutants uncoupled from RhoA signalling. Furthermore, a dominant active form of RhoA did not reduce Src-induced phosphoryaltion of p120(ctn). In summary, our results suggest that Gα(12) binds to p120(ctn) and modulates its phosphorylation status through a Rho-independent mechanism. Gα(12) emerges as an important regulator of p120(ctn) function, and possibly of cadherin-mediated adhesion and/or cell motility.