Dianqing Wu

Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.

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.

Axin and Frat1 interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1.

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.

Dishevelled Proteins Lead to Two Signaling Pathways REGULATION OF LEF-1 AND c-Jun N-TERMINAL KINASE IN MAMMALIAN CELLS

Dishevelled (Dsh/Dvl) proteins are known to mediate Wnt signaling by up-regulating β-catenin levels and stimulating T cell factor (TCF)/LEF-1-dependent transcription. We have identified a new Dvl-mediated signaling pathway in that mouse Dvl proteins, when expressed in COS-7 cells, stimulate c-Jun-dependent transcription activity and the kinase activity of the c-Jun N-terminal kinase (JNK). The DEP domain of Dvl1 is essential for JNK activation. By contrast, all three conserved domains of Dvl, including DIX, PDZ, and DEP, are required for up-regulation of β-catenin and for stimulation of LEF-1-mediated transcription in mammalian cells. Thus, Dvl can lead to two different signaling pathways. Furthermore, the small G proteins of Cdc42 or Rac1, which are involved in JNK activation by many stimuli, do not appear to play a major role in Dvl-mediated JNK activation, because the dominant negative mutants of Cdc42 and Rac1 could not inhibit Dvl-induced JNK activation. This suggests that Dvl may activate JNK via novel pathways.

Specific Involvement of G Proteins in Regulation of Serum Response Factor-mediated Gene Transcription by Different Receptors

Regulation of serum response factor (SRF)-mediated gene transcription by G protein subunits and G protein-coupled receptors was investigated in transfected NIH3T3 cells and in a cell line that was derived from mice lacking Gαq and Gα11. We found that the constitutively active forms of the α subunits of the Gqand G12 class of G proteins, including Gαq, Gα11, Gα14, Gα16, Gα12, and Gα13, can activate SRF in NIH3T3 cells. We also found that the type 1 muscarinic receptor (m1R) and α1-adrenergic receptor (AR)-mediated SRF activation is exclusively dependent on Gαq/11, while the receptors for thrombin, lysophosphatidic acid (LPA), thromboxane A2, and endothelin can activate SRF in the absence of Gαq/11. Moreover, RGS12 but not RGS2, RGS4, or Axin was able to inhibit Gα12 and Gα13-mediated SRF activation. And RGS12, but not other RGS proteins, blocked thrombin- and LPA-mediated SRF activation in the Gαq/11-deficient cells. Therefore, the thrombin, LPA, thromboxane A2, and endothelin receptors may be able to couple to Gα12/13. On the contrary, receptors including β2- and α2-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 Gαq/11, suggesting that these receptors cannot couple to endogenous G proteins of the G12 or Gqclasses.

The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd.

ABSTRACT The mechanism by which the high-bone-mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates canonical Wnt signaling was investigated. The mutation was previously shown to reduce DKK1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for DKK-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperone protein for LRP5/6 that is required for transport of the coreceptors to cell surfaces, resulting in fewer LRP5 molecules on the cell surface. Although the reduction in the number of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical Wnt, Wnt7b, and that osteocytes produce paracrine DKK1, we think that the G171V mutation may cause an increase in Wnt activity in osteoblasts by reducing the number of targets for paracrine DKK1 to antagonize without affecting the activity of autocrine Wnt.

Regulation of Gli1 Transcriptional Activity in the Nucleus by Dyrk1

To investigate the cellular role of dual specificity Yak1-related kinase (Dyrk) 1, a nuclear localized dual specificity protein kinase, we examined its effect on transcriptional regulation using reporter gene assays. We found that Dyrk1 can substantially enhance Gli1-dependent, but not LEF-1-, c-Jun-, or Elk-dependent, gene transcription. In part, Dyrk1 does this through retaining Gli1 in the nucleus. However, we also demonstrate that Dyrk1 can enhance the transcriptional activity of Gli1-AHA, a nuclear export mutant, suggesting that Dyrk1 may be more directly involved in regulating the transcriptional activity of Gli1. In addition, Dyrk1 acted synergistically with Sonic hedgehog (Shh) to induce gene transcription and differentiation in mouse C3H10T1/2 cells. The failure of Shh to stimulate Dyrk1 kinase activity suggests that Dyrk1 may not be directly regulated by the Shh signaling pathway but functionally interacts with it. Thus, Gli1 transcriptional activity may be subjected to further regulation in the cell nucleus by a pathway distinct from Shh signaling, one mediated by Dyrk1.

Pertussis Toxin-sensitive Activation of Phospholipase C by the C5a and fMet-Leu-Phe Receptors

Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC β2 and PLC β3 through the Gβγ subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC β2 and PLC β3 were shown to be activated by the βγ subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC β3 or recombinant PLC β3 in transfected COS-7 cells. However, both receptors can clearly activate PLC β2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC β2 probably through the Gβγ subunits. These findings were further corroborated by the results that PLC β3 could only be slightly activated by Gβ1γ1 or Gβ1γ5 in the cotransfection assay, whereas the Gβγ subunits strongly activated PLC β2 under the same conditions. PLC β3 can be activated by Gαq, Gα11, and Gα16 in the cotransfection assay. In addition, the Gγ2 and Gγ3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gβγ-mediated activation of PLC β2, were able to inhibit C5a or fMLP-mediated activation of PLC β2. These Gγ mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gβγ subunits are involved in PLC β2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC β3 by Gβγ may not be the primary pathway for the receptors.

Suppression of glycogen synthase kinase activity is not sufficient for leukemia enhancer factor-1 activation.

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 β-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 β-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.

Prostate-Specific Membrane Antigen Regulates Angiogenesis by Modulating Integrin Signal Transduction

ABSTRACT The transmembrane peptidase prostate-specific membrane antigen (PSMA) is universally upregulated in the vasculature of solid tumors, but its functional role in tumor angiogenesis has not been investigated. Here we show that angiogenesis is severely impaired in PSMA-null animals and that this angiogenic defect occurs at the level of endothelial cell invasion through the extracellular matrix barrier. Because proteolytic degradation of the extracellular matrix is a critical component of endothelial invasion in angiogenesis, it is logical to assume that PSMA participates in matrix degradation. However, we demonstrate a novel and more complex role for PSMA in angiogenesis, where it is a principal component of a regulatory loop that is tightly modulating laminin-specific integrin signaling and GTPase-dependent, p21-activated kinase 1 (PAK-1) activity. We show that PSMA inhibition, knockdown, or deficiency decreases endothelial cell invasion in vitro via integrin and PAK, thus abrogating angiogenesis. Interestingly, the neutralization of β1 or the inactivation of PAK increases PSMA activity, suggesting that they negatively regulate PSMA. This negative regulation is mediated by the cytoskeleton as the disruption of interactions between the PSMA cytoplasmic tail and the anchor protein filamin A decreases PSMA activity, integrin function, and PAK activation. Finally, the inhibition of PAK activation enhances the PSMA/filamin A interaction and, thus, boosts PSMA activity. These data imply that PSMA participates in an autoregulatory loop, wherein active PSMA facilitates integrin signaling and PAK activation, leading to both productive invasion and downregulation of integrin β1 signaling via reduced PSMA activity. Therefore, we have identified a novel role for PSMA as a true molecular interface, integrating both extracellular and intracellular signals during angiogenesis.

Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Gα12/13

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 thrombins 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, Rho‐mediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho‐independent 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.