Overexpressed Gα13 activates serum response factor through stoichiometric imbalance with Gßγ and mislocalization to the cytoplasm.

Gα13, a heterotrimeric G protein α subunit of the G12/13 subfamily, is an oncogenic driver in multiple cancer types. Unlike other G protein subfamilies that contribute to cancer progression via amino acid substitutions that abolish their deactivating, intrinsic GTPase activity, Gα13 rarely harbors such mutations in tumors and instead appears to stimulate aberrant cell growth via overexpression as a wildtype form. It is not known why this effect is exclusive to the G12/13 subfamily, nor has a mechanism been elucidated for overexpressed Gα13 promoting tumor progression. Using a reporter gene assay for serum response factor (SRF)-mediated transcription in HEK293 cells, we found that transiently expressed, wildtype Gα13 generates a robust SRF signal, approximately half the amplitude observed for GTPase-defective Gα13. When epitope-tagged, wildtype Gα13 was titrated upward in cells, a sharp increase in SRF stimulation was observed coincident with a "spillover" of Gα13 from membrane-associated to a soluble fraction. Overexpressing G protein ß and γ subunits caused both a decrease in this signal and a shift of wildtype Gα13 back to the membranous fraction, suggesting that stoichiometric imbalance in the αßγ heterotrimer results in aberrant subcellular localization and signalling by overexpressed Gα13. We also examined the acylation requirements of wildtype Gα13 for signalling to SRF. Similar to GTPase-defective Gα13, S-palmitoylation of the wildtype α subunit was necessary for SRF activation but could be replaced functionally by an engineered site for N-terminal myristoylation. However, a key difference was observed between wildtype and GTPase-defective Gα13: whereas the latter protein lacking palmitoylation sites was rescued in its SRF signalling by either an engineered polybasic sequence or a C-terminal isoprenylation site, these motifs failed to restore signalling by wildtype, non-palmitoylated Gα13. These findings illuminate several components of the mechanism in which overexpressed, wildtype Gα13 contributes to growth and tumorigenic signalling, and reveal greater stringency in its requirements for post-translational modification in comparison to GTPase-defective Gα13.

Divergent C-terminal motifs in Gα12 and Gα13 provide distinct mechanisms of effector binding and SRF activation.

The G12/13 subfamily of heterotrimeric guanine nucleotide binding proteins comprises the α subunits Gα12 and Gα13, which transduce signals for cell growth, cytoskeletal rearrangements, and oncogenic transformation. In an increasing range of cancers, overexpressed Gα12 or Gα13 are implicated in aberrant cell proliferation and/or metastatic invasion. Although Gα12 and Gα13 bind non-redundant sets of effector proteins and participate in unique signalling pathways, the structural features responsible for functional differences between these α subunits are largely unknown. Invertebrates encode a single G12/13 homolog that participates in cytoskeletal changes yet appears to lack signalling to SRF (serum response factor), a transcriptional activator stimulated by mammalian Gα12 and Gα13 to promote growth and tumorigenesis. Our previous studies identified an evolutionarily divergent region in Gα12 for which replacement by homologous sequence from Drosophila melanogaster abolished SRF signalling, whereas the same invertebrate substitution was fully tolerated in Gα13 [Montgomery et al. (2014) Mol. Pharmacol. 85: 586]. These findings prompted our current approach of evolution-guided mutagenesis to identify fine structural features of Gα12 and Gα13 that underlie their respective SRF activation mechanisms. Our results identified two motifs flanking the α4 helix that play a key role in Gα12 signalling to SRF. We found the region encompassing these motifs to provide an interacting surface for multiple Gα12-specific target proteins that fail to bind Gα13. Adjacent to this divergent region, a highly-conserved domain was vital for SRF activation by both Gα12 and Gα13. However, dissection of this domain using invertebrate substitutions revealed different signalling mechanisms in these α subunits and identified Gα13-specific determinants of binding Rho-specific guanine nucleotide exchange factors. Furthermore, invertebrate substitutions in the C-terminal, α5 helical region were selectively disruptive to Gα12 signalling. Taken together, our results identify key structural features near the C-terminus that evolved after the divergence of Gα12 and Gα13, and should aid the development of agents to selectively manipulate signalling by individual α subunits of the G12/13 subfamily.

A Gα12-specific Binding Domain in AKAP-Lbc and p114RhoGEF.

AKAP-Lbc is a Rho-activating guanine nucleotide exchange factor (RhoGEF) important in heart development and pro-fibrotic signaling in cardiomyocytes. Heterotrimeric G proteins of the G12/13 subfamily, comprising Gα12 and Gα13, are well characterized as stimulating a specialized group of RhoGEFs through interaction with their RGS-homology (RH) domain. Despite lacking an RH domain, AKAP-Lbc is bound by Gα12 through an unknown mechanism to activate Rho signaling. We identified a Gα12-binding region near the C-terminus of AKAP-Lbc, closely homologous to a region of p114RhoGEF that we also discovered to interact with Gα12. This binding mechanism is distinct from the well-studied interface between RH-RhoGEFs and G12/13 α subunits, as demonstrated by Gα12 mutants selectively impaired in binding either this AKAP-Lbc/p114RhoGEF region or RH-RhoGEFs. AKAP-Lbc and p114RhoGEF showed high specificity for binding Gα12 in comparison to Gα13, and experiments using chimeric G12/13 α subunits mapped determinants of this selectivity to the N-terminal region of Gα12. In cultured cells expressing constitutively GDP-bound Gα12 or Gα13, the Gα12 construct was more potent in exerting a dominant-negative effect on serum-mediated signaling to p114RhoGEF, demonstrating coupling of these signaling proteins in a cellular pathway. In addition, charge-reversal of conserved residues in AKAP-Lbc and p114RhoGEF disrupted Gα12 binding for both proteins, suggesting they harbor a common structural mechanism for interaction with this α subunit. Our results provide the first evidence of p114RhoGEF as a Gα12 signaling effector, and define a novel region conserved between AKAP-Lbc and p114RhoGEF that allows Gα12 signaling input to these non-RH RhoGEFs.

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 Gα12 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.

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.

Gα(12) binds to the N-terminal regulatory domain of p120(ctn), and downregulates p120(ctn) 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.

Isolation of centrosomes from cultured Mammalian cells.

INTRODUCTIONThe centrosome is a cell's 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.

Biologic functions of the G12 subfamily of heterotrimeric g proteins: growth, migration, and metastasis.

The G12 subfamily of heterotrimeric G proteins has been the subject of intense scientific interest for more than 15 years. During this period, studies have revealed more than 20 potential G12-interacting proteins and numerous signaling axes emanating from the G12 proteins, Galpha12 and Galpha13. In addition, more recent studies have begun to illuminate the various and sundry functions that the G12 subfamily plays in biology. In this review, we summarize the diverse range of proteins that have been identified as Galpha12 and/or Galpha13 interactors and describe ongoing studies designed to dissect the biological roles of specific Galpha-effector protein interactions. Further, we describe and discuss the expanding role of G12 proteins in the biology of cells, focusing on the distinct properties of this subfamily in regulating cell proliferation, cell migration, and metastatic invasion.

Domains necessary for Galpha12 binding and stimulation of protein phosphatase-2A (PP2A): Is Galpha12 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 Galpha(12). PP2A activity is altered in carcinogenesis and in some neurodegenerative diseases. We have identified binding of Galpha(12) with the Aalpha subunit of PP2A, a trimeric enzyme composed of A (scaffolding), B (regulatory), and C (catalytic) subunits and demonstrated that Galpha(12) stimulated phosphatase activity (J Biol Chem 279: 54983-54986, 2004). We now show in substrate-velocity analysis using purified PP2A that V(max) was stimulated 3- to 4-fold by glutathione transferase (GST)-Galpha(12) with little effect on K(m) values. To identify the binding domains mediating the Aalpha-Galpha(12) interaction, an extensive mutational analysis was performed. Well-characterized mutations of Aalpha were expressed in vitro and tested for binding to GST-Galpha(12) in pull-down assays. Galpha(12) binds to Aalpha along repeats 7 to 10, and PP2A B subunits are not necessary for binding. To identify where Aalpha binds to Galpha(12), a series of 61 Galpha(12) mutants were engineered to contain the sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS) in place of 6 consecutive amino acids. Mutant Galpha(12) proteins were individually expressed in human embryonic kidney cells and analyzed for interaction with GST or GST-Aalpha in pull-down assays. The Aalpha binding sites were localized to regions near the N and C termini of Galpha(12). The expression of constitutively activated Galpha(12) (QLalpha(12)) 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 Galpha(12) and PP2A Aalpha, a model describing the binding surfaces and potential mechanisms of Galpha(12)-mediated PP2A activation is presented.

G alpha12 interaction with alphaSNAP 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 alphaSNAP as an interactive partner of G alpha12 using yeast two-hybrid screening. Glutathione S-transferase pull-down assays showed the selective interaction of alphaSNAP with G alpha12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of G alpha12 (1-37 amino acids) was necessary and sufficient for its interaction with alphaSNAP. G alpha13 with its N-terminal extension replaced by that of G alpha12 acquired the ability to bind to alphaSNAP, whereas G alpha12 with its N terminus replaced by that of G alpha13 lost this ability. Using four point mutants of alphaSNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of alphaSNAP was involved in its interaction with G alpha12. Co-transfection of human umbilical vein endothelial cells with G alpha12 and alphaSNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of alphaSNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with G alpha12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the alpha subunit of G12 and alphaSNAP, 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.

Selective uncoupling of G alpha 12 from Rho-mediated signaling.

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.

Galpha12 directly interacts with PP2A: evidence FOR Galpha12-stimulated PP2A phosphatase activity and dephosphorylation of microtubule-associated protein, tau.

The Galpha(12/13) family of heterotrimeric G proteins modulate multiple cellular processes including regulation of the actin cytoskeleton. Galpha(12/13) interact with several cytoskeletal/scaffolding proteins, and in a yeast two-hybrid screen with Galpha(12), we detected an interaction with the scaffolding subunit (Aalpha) 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 Aalpha and Galpha(12) was confirmed by coimmunoprecipitation studies in transfected COS cells and by glutathione S-transferase (GST)-Galpha(12) pull-downs from cell lysates of primary neurons. The interaction was specific for Aalpha and Galpha(12) and was independent of Galpha(12) conformation. Endogenous Aalpha and Galpha(12) colocalized by immunofluorescent microscopy in Caco-2 cells and in neurons. In vitro reconstitution of GST-Galpha(12) or recombinant Galpha(12) with PP2A core enzyme resulted in approximately 300% stimulation of PP2A activity that was not detected with other Galpha subunits and was similar with GTPgammaS- and GDP-liganded Galpha(12). 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 QLalpha(12) 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 Galpha(12) 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 Galpha(12) and potential in vivo modulation of PP2A target proteins including tau.

Identification of a role for beta-catenin in the establishment of a bipolar mitotic spindle.

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.

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, 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.

Galpha12 and Galpha13 negatively regulate the adhesive functions of cadherin.

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 G12 subfamily comprised of Galpha12 and Galpha13 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 G12 subfamily proteins could also regulate cadherin function. The introduction of mutationally activated G12 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 G12 proteins blocked E-cadherin function in a fast aggregation assay. Aggregation mediated by a mutant cadherin that lacks G12 binding ability was not affected by activated G12 proteins, indicating a requirement for direct G12-cadherin interaction. Furthermore, in wound-filling assays in which ectopic expression of E-cadherin inhibits cell migration, the expression of activated G12 proteins reversed the inhibition via a mechanism that was independent of G12-mediated Rho activation. These results validate the G12-cadherin interaction as a potentially important event in cell biology and suggest novel roles for G12 proteins in the regulation of cadherin-mediated developmental events and in the loss of cadherin function that is characteristic of metastatic tumor progression.