To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, we identified Axin as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, showed no effect on the canonical Wnt signaling pathway by itself, but acted synergistically with Wnt. In contrast, LRP-5 mutants lacking the extracellular domain functioned as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt caused the translocation of Axin to the membrane and enhanced the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, we conclude that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway.

contributed equally to this work Wnt proteins transduce their signals through dishevelled (Dvl) proteins to inhibit glycogen synthase kinase 3β (GSK), leading to the accumulation of cytosolic β-catenin and activation of TCF/LEF-1 transcription factors. To understand the mechanism by which Dvl acts through GSK to regulate LEF-1, we investigated the roles of Axin and Frat1 in Wnt-mediated activation of LEF-1 in mammalian cells. We found that Dvl interacts with Axin and with Frat1, both of which interact with GSK. Similarly, the Frat1 homolog GBP binds Xenopus Dishevelled in an interaction that requires GSK. We also found that Dvl, Axin and GSK can form a ternary complex bridged by Axin, and that Frat1 can be recruited into this complex probably by Dvl. The observation that the Dvl-binding domain of either Frat1 or Axin was able to inhibit Wnt-1-induced LEF-1 activation suggests that the interactions between Dvl and Axin and between Dvl and Frat may be important for this signaling pathway. Furthermore, Wnt-1 appeared to promote the disintegration of the Frat1-Dvl-GSK-Axin complex, resulting in the dissociation of GSK from Axin. Thus, formation of the quaternary complex may be an important step in Wnt signaling, by which Dvl recruits Frat1, leading to Frat1-mediated dissociation of GSK from Axin.

Regulation of serum response factor (SRF)-mediated gene transcription by G protein subunits and G proteincoupled receptors was investigated in transfected NIH3T3 cells and in a cell line that was derived from mice lacking G␣ q and G␣ 11 . We found that the constitutively active forms of the ␣ subunits of the G q and G 12 class of G proteins, including G␣ q , G␣ 11 , G␣ 14 , G␣ 16 , G␣ 12 , and G␣ 13 , can activate SRF in NIH3T3 cells. We also found that the type 1 muscarinic receptor (m1R) and ␣ 1 -adrenergic receptor (AR)-mediated SRF activation is exclusively dependent on G␣ q/11 , while the receptors for thrombin, lysophosphatidic acid (LPA), thromboxane A2, and endothelin can activate SRF in the absence of G␣ q/11 . Moreover, RGS12 but not RGS2, RGS4, or Axin was able to inhibit G␣ 12 and G␣ 13 -mediated SRF activation. And RGS12, but not other RGS proteins, blocked thrombin-and LPA-mediated SRF activation in the G␣ q/11 -deficient cells. Therefore, the thrombin, LPA, thromboxane A2, and endothelin receptors may be able to couple to G␣ 12/13 . On the contrary, receptors including ␤ 2 -and ␣ 2 -ARs, m2R, the dopamine receptors type 1 and 2, angiotensin receptors types 1 and 2, and interleukin-8 receptor could not activate SRF in the presence or absence of G␣ q/11 , suggesting that these receptors cannot couple to endogenous G proteins of the G 12 or G q classes.Hormones, neurotransmitters, and many other biologically active molecules, such as lysophosphatidic acid (LPA), 1 thrombin, catecholamines, endothelin, etc., transduce their signals through heterotrimeric G proteins (1, 2). Molecular cloning has revealed at least four classes of G protein ␣ subunits: G␣ s , G␣ i , G␣ q , and G␣ 12 (3). The G␣ s subunits and G␣ i subunits regulate adenylyl cyclase activities, while the G␣ q subunits regulate phospholipase C activities. However, the function of the G␣ 12 class of G proteins, which includes G␣ 12 and G␣ 13 , remains to be elucidated. Activated forms of G␣ 12 and G␣ 13 , when transfected into fibroblast cells, were shown to induce transformation phenotypes (4 -6), suggesting that this class of G proteins may be involved in cell growth regulation. Moreover, G␣ 12 and G␣ 13 were shown to induce formation of stress fibers in fibroblast cells through small G protein RhoA (7). This observation was supported by the report that G␣ 12 activated serum response factor (SRF) through RhoA (8). The in vivo function of G␣ 13 was also investigated using the gene-targeting technique in mice. Mice lacking G␣ 13 are embryonic lethal apparently due to the failure to develop vasculature structures, indicating that G␣ 13 may be involved in the function of endothelial cells (9). In the same study, thrombin-mediated chemotaxis of fibroblasts lacking G␣ 13 was blocked, indicating that the thrombin receptor couples to G␣ 13 . This is consistent with the observation that thrombin as well as a thromboxane A2 receptor agonist could stimulate the binding of a photo-affinity GTP analog to G␣ 13 (10). However, there were contradictory r...

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Glycogen synthase kinase-3 (GSK) can be regulated by different signaling pathways including those mediated by protein kinase Akt and Wnt proteins. Wnt proteins are believed to activate a transcription factor leukemia enhancer factor-1 (LEF-1) by inhibiting GSK, and Akt was shown to phosphorylate GSK and inhibit its kinase activity. We investigated the effect of an activated Akt on the accumulation of cytosolic ␤-catenin and LEF-1-dependent transcription. Although the activated Akt, mAkt, clearly inhibited the kinase activity of GSK, mAkt alone did not induce accumulation of cytosolic ␤-catenin or activate LEF-1-dependent transcription. On the contrary, coexpressed Wnt-1 and Frat activated LEF-1 but did not show significant inhibition of GSK-mediated phosphorylation of a peptide substrate. However, mAkt could act synergistically with Wnt-1 or Frat to activate LEF-1. In addition, the interaction of GSK for Axin appeared to decrease in the presence of mAkt, whereas the interaction for Frat remained unchanged. Consistently, a GSK mutant with substitution of a Phe residue for residue Tyr-216, which showed one-fifth of kinase activity of the wild-type GSK, exhibited a reduced association for Axin than the wild-type GSK. These results suggest that inhibition of GSK kinase activity is not sufficient for activation of LEF-1 but may facilitate the activation by reducing the interaction of GSK for Axin. The additional mechanism for LEF-1 activation may require dissociation of GSK from Axin as Frat facilitates the dissociation of GSK from Axin.The Wnt family of secretory glycoproteins is one of the major families of developmentally important signaling molecules and plays important roles in embryonic induction, generation of cell polarity, and specification of cell fate. Wnt pathways are also closely linked to tumorigenesis. A large amount of knowledge about Wnt-mediated signal transduction comes from genetic studies in Drosophila. A genetic order of these signal transducers has been established, in which Wg appears to negatively regulate Zeste-White 3 (Zw3) 1 through Dishevelled (Dsh), thus relieving the suppression of Armadillo by Zw3 with a net result of up-regulation of Armadillo. Armadillo interacts with Pangolin-regulating gene transcription. Recent evidence suggests that the Frizzled proteins may function as receptors for Wnt (for review, see Refs. 1-6).Wnt-linked pathways are apparently conserved in higher organisms including mammals. More than 20 Wnt and 8 Frizzled homologs have been cloned and sequenced in mammals (5,7,8). Molecular cloning also revealed mammalian homologs to Dsh (9 -11), Zw3, and Armadillo; they are Dvl, GSK-3␤ and ␤-catenin, respectively (5). Fly zw3 mutants and frog embryos expressing dominant negative mutants of GSK-3␤ showed phenotypes consistent with constitutive activation of the ␤-catenin by the Wnt pathway (5). This led to a model suggesting that Wnt inhibits GSK activity. Additionally, soluble Wg was shown to inhibit GSK activity in mouse fibroblasts (12).Although the molecular mechanism ...

A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. We found that Tec and Bmx activate serum response factor (SRF), in synergy with constitutively active α subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Gα13-and Gα12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Gαq/11, where thrombin's effect is mediated by G12/ 13 proteins. Moreover, expression of Gα12 and Gα13 stimulates autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Gα12/13-induced, Rhomediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rhoindependent pathways in 3T3 cells, as shown by the fact that C3 did not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed.

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein ␣ subunits were characterized in transfection systems. G␣q, G␣12, and G␣13, but not G␣i, activate SRF through RhoA. When G␣q, ␣12, or ␣13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, G␣13, but not G␣q or G␣12, showed synergistic activation of SRF with GEF115. The synergy between G␣13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between G␣13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited G␣13-and G␣12-induced, but not GEF115 itself-or G␣q-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin-and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of G␣12͞13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both G␣12 and G␣13. Thus, the inhibition of G␣12͞13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between G␣13 and GEF115 indicates that GEF115 mediates G␣13-induced activation of Rho and SRF.Four classes of G protein ␣ subunits-G␣s, G␣i, G␣q, and G␣12 (1)-are involved in signal transduction of various hormones, neurotransmitters, and many other biologically active molecules such as lysophosphatidic acid (LPA) and thrombin (2-4). The G␣s subunits and G␣i subunits regulate adenyl cyclase activities, and the G␣q subunits regulate phospholipase C activities (5, 6). However, the direct effectors for the G␣12 class of G proteins, which includes G␣12 and G␣13 (7), remains to be elucidated. Activated forms of G␣12 and G␣13 were shown to induce transformation phenotypes when transfected into fibroblasts, suggesting that they are involved in regulation of cell growth (8-10). In addition, G␣12 and G␣13 were shown to induce formation of stress fibers in fibroblast cells and apoptosis through the small G protein RhoA (11,12). This observation was supported by the report that G␣12 activated serum response factor (SRF) through RhoA (13). Moreover, a study using mice that lack G␣13 indicates that G␣13 is involved in the function of endothelial cells because mice lacking G␣13 are embryonic lethal apparently due to the failure to develop vasculature (14). In this study, thrombinmediated chemotaxis of fibroblasts lacking G␣13 was blocked, indicating that the thrombin receptor couples to G␣13. This is consistent with the observation that thrombin could stimulate the binding of a photoaffinity GTP analog to G␣13 (15).In this report, we describe the involvement of a Rho-specific guanine nucleotide exchange factor, GEF115 (16), in G␣13-but not G␣12-or G␣q-mediated SRF activation. We found that the N-terminal portion of GEF115, which contains a region homologous to the regulator of G protein signaling (RGS) domain of the RGS12 protein, is required for mediatin...

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