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Semaphorin3A (Sema3A) exerts a wide variety of biological functions by regulating reorganization of actin and tubulin cytoskeletal proteins through signaling pathways including sequential phosphorylation of collapsin response mediator protein 1 (CRMP1) and CRMP2 by cyclin-dependent kinase-5 and glycogen synthase kinase-3β (GSK3β). To delineate how GSK3β mediates Sema3A signaling, we here determined the substrates of GSK3β involved. Introduction of either GSK3β mutants, GSK3β-R96A, L128A, or K85M into chick dorsal root ganglion (DRG) neurons suppressed Sema3A-induced growth cone collapse, thereby suggesting that unprimed as well as primed substrates are involved in Sema3A signaling. Axin-1, a key player in Wnt signaling, is an unprimed substrate of GSK3β. The phosphorylation of Axin-1 by GSK3β accelerates the association of Axin-1 with β-catenin. Immunocytochemical studies revealed that Sema3A induced an increase in the intensity levels of β-catenin in the DRG growth cones. Axin-1 siRNA knockdown suppressed Sema3A-induced growth cone collapse. The reintroduction of RNAi-resistant Axin-1 (rAxin-l)-wt rescued the responsiveness to Sema3A, while that of nonphosphorylated mutants, rAxin S322A/S326A/S330A and T485A/S490A/S497A, did not. Sema3A also enhanced the colocal-ization of GSK3β, Axin-1, and β-catenin in the growth cones. The increase of β-catenin in the growth cones was suppressed by the siRNA knockdown of Axin-1. Furthermore, either Axin-1 or β-catenin RNAi knockdown suppressed the internalization of Sema3A. These results suggest that Sema3A induces the formation of GSK3β/Axin-l/β-catenin complex, which regulates signaling cascade of Sema3A via an endocytotic mechanism. This finding should provide clue for understanding of mechanisms of a wide variety of biological functions of Sema3A [ABSTRACT FROM AUTHOR]

Wnt signalling is known to promote G1/S progression through the stimulation of gene expression, but whether this signalling regulates mitotic progression is not clear. Here, the function of dishevelled 2 (Dvl2), which transmits the Wnt signal, in mitosis was examined. Dvl2 localized to the spindles and spindle poles during mitosis. When cells were treated with nocodazole, Dvl2 was observed at the kinetochores (KTs). Dvl2 bound to and was phosphorylated at Thr206 by a mitotic kinase, Polo-like kinase 1 (Plk1), and this phosphorylation was required for spindle orientation and stable microtubule (MT)-KT attachment. Dvl2 was also found to be involved in the activation of a spindle assembly checkpoint (SAC) kinase, Mps1, and the recruitment of other SAC components, Bub1 and BubR1, to the KTs. However, the phosphorylation of Dvl2 by Plk1 was dispensable for SAC. Furthermore, Wnt receptors were involved in spindle orientation, but not in MT-KT attachment or SAC. These results suggested that Dvl2 is involved in mitotic progression by regulating the dynamics of MT plus-ends and the SAC in Plk1-dependent and -independent manners. [ABSTRACT FROM AUTHOR]

Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the β-catenin-independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin-mediated route in response to Wnt5a, and inhibition of clathrin-dependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a-dependent lipoprotein receptor-related protein 6 (LRP6) phosphorylation and β-catenin accumulation. Wnt3a-dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a-dependent accumulation of β-catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the β-catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin-dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalization-dependent and -independent mechanisms. [ABSTRACT FROM AUTHOR]

The mechanism of cross talk between the Wnt signaling and cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) pathways was studied. Prostaglandin E1 (PGE1), isoproterenol, and dibutyryl cAMP (Bt2cAMP), all of which activate PKA, increased the cytoplasmic and nuclear β-catenin protein level, and these actions were suppressed by a PKA inhibitor and RNA interference for PKA. PGE1 and Bt2cAMP also increased T-cell factor (Tcf)-dependent transcription through β-catenin. Bt2cAMP suppressed degradation of β-catenin at the protein level. Although PKA did not affect the formation of a complex between glycogen synthase kinase 3β (GSK-3β), β-catenin, and Axin, phosphorylation of β-catenin by PKA inhibited ubiquitination of β-catenin in intact cells and in vitro. Ser675 was found to be a site for phosphorylation by PKA, and substitution of this serine residue with alanine in β-catenin attenuated inhibition of the ubiquitination of β-catenin by PKA, PKA-induced stabilization of β-catenin, and PKA-dependent activation of Tcf. These results indicate that PKA inhibits the ubiquitination of β-catenin by phosphorylating β-catenin, thereby causing β-catenin to accumulate and the Wnt signaling pathway to be activated. [ABSTRACT FROM AUTHOR]

Inhibitor 2 (I-2) is a ubiquitous regulator of type 1 protein phosphatase (PP1). Previous in vitro studies suggested that its inhibitory activity towards PP1 is regulated by phosphorylation at Thr72 by glycogen synthase kinase-3β (GSK-3β), and at Ser86, Ser120, and Ser121 by casein kinase 2 (CK2). Here we report that GSK-3β expressed in COS-7 cells phosphorylates wild-type I-2 but not an I-2 mutant carrying a T to A substitution at residue 72, showing that GSK-3β phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3β and I-2-FLAG co-exist in a same complex in the intact cells, but they do not bind directly. It is noteworthy that co-expression of Myc-PP1C significantly increased co-precipitation of HA-GSK-3β with I-2-FLAG, showing a complex formation of HA-GSK-3β/Myc-PP1C / I-2-FLAG in vivo. Further studies using a GSK-3β kinase–dead mutant and LiCl, an inhibitor of GSK-3β, showed that the enzyme activity of GSK-3β is required for co-precipitation. IP-Western study using several I-2 mutants substituted at phosphorylation sites (T72, S86, S120, and S121) suggested that phosphorylation of I-2 by CK2 is also involved in enhancement of association between GSK-3β and I-2 in vivo. This study is the first demonstration that GSK-3β associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells. [ABSTRACT FROM PUBLISHER]

Axin forms a complex with adenomatous polyposis coli gene product (APC), glycogen synthase kinase-3β (GSK-3β), and β-catenin through different binding sites and downregulates β-catenin. GSK-3β-dependent phosphorylation of APC-(1211-2075) which has the Axin-binding site was facilitated by Axin, but that of APC-(959-1338) which lacks the Axin-binding site was not. Axin-(298-506) or Axin-(298-832), which has the GSK-3β- and β-catenin- but not APC-binding sites, did not enhance GSK-3β-dependent phosphorylation of either APC-(1211-2075) or APC-(959-1338). Furthermore, β-catenin stimulated the phosphorylation of APC-(959-1338) and APC-(1211-2075) by GSK-3β in the presence of Axin. Consistent with these in vitro observations, expression of β-catenin or Axin in COS cells promoted an SDS gel band shift of APC. These results indicate that APC complexed with Axin is effectively phosphorylated by GSK-3β and that β-catenin may modulate this phosphorylation. In addition, the heterodimeric form of protein phosphatase 2A (PP2A) directly bound to Axin, and PP2A complexed with Axin dephosphorylated APC phosphorylated by GSK-3β. Taken together, these results suggest that GSK-3β-dependent phosphorylation of APC can be modulated by β-catenin and PP2A complexed with Axin. Oncogene (2000) 19, 537–545. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. We have isolated here a GSK-3β-interacting protein from a rat brain cDNA library using a yeast twohybrid method. This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and dishevelled (Dsh) homologous domains in its N- and C-terminal regions, respectively. The predicted amino acid sequence of this GSK-3β-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, we termed this protein rAxin (rat Axin). rAxin interacted directly with, and was phosphorylated by, GSK-3β. rAxin also interacted directly with the armadillo repeats of β-catenin. The binding site of rAxin for GSK-3β was distinct from the β-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promoted GSK-3β-dependent phosphorylation of β-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3β and β-catenin and mediating the signal from GSK-3β to β-catenin. [ABSTRACT FROM AUTHOR]

Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of β-catenin by glycogen synthase kinase 3β. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P61 or P65, with unit-cell parameters a = b = 91.49, c = 84.92 Å. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 Å. [ABSTRACT FROM AUTHOR]

β-catenin-mediated Wnt signaling is crucial in animal development and tumor progression. The phosphorylation of low-density lipoprotein receptor-related protein 6 (LRP6), a single-span transmembrane Wnt receptor, plays a vital role in this signaling. Dickkopf1 (Dkk1) has been shown to inhibit the Wnt— β-catenin pathway, but the mechanism is not yet clear. Here, evidence is presented that Wnt3a-dependent phosphorylation of LRP6 occurs in the lipid raft and that Dkk1 inhibits the formation of a complex between LRP6 and casein kinase 1 γ (CK1γ) by removing LRP6 from the lipid raft. Dkk1 internalized LRP6 in a Rab5-dependent mechanism to prevent phosphorylation mediated by CK1γ. The internalized LRP6 was recycled back in a Rab11-dependent mechanism to the cell-surface membrane, and the recycled LRP6 again responded to Wnt3a and Dkk1. Internalized Dkk1 was trafficked in a Rab7-mediated route and degraded in the lysosome. These results suggest that Dkk1 induces the internalization of LRP6 to suppress its phosphorylation in the lipid raft and allows subsequent recycling of LRP6 so that it can be reused for signaling. [ABSTRACT FROM AUTHOR]

During dehydration, protein kinase A phosphorylates aquaporin 2 (AQP2) at serine 256 and this is essential for apical membrane sorting of AQP2 in the collecting ducts. A-kinase anchoring proteins (AKAPs) bind protein kinase A and protein phosphatases conferring substrate specificity to these enzymes and localize them to the appropriate intracellular compartment. We found that AKAP220 bound to AQP2 in a yeast two-hybrid screen. Further, it was highly localized to the papilla compared to other regions of the kidney. Using double immunofluorescence and immunoelectron microscopy we found that AKAP220 co-localized with AQP2 in the cytosol of the inner medullary collecting ducts. Forskolin-mediated phosphorylation of AQP2, transiently expressed in COS cells, was increased by AKAP220 co-expression. Our results suggest that AKAP220 may be involved in the phosphorylation of AQP2 by recruiting protein kinase A.Kidney International (2008) 74, 1429–1433; doi:10.1038/ki.2008.402; published online 13 August 2008 [ABSTRACT FROM AUTHOR]

Summary: Wnt and Dickkopf (Dkk) regulate the stabilization of β-catenin antagonistically in the Wnt signaling pathway; however, the molecular mechanism is not clear. In this study, we found that Wnt3a acts in parallel to induce the caveolin-dependent internalization of low-density-lipoprotein receptor-related protein 6 (LRP6), as well as the phosphorylation of LRP6 and the recruitment of Axin to LRP6 on the cell surface membrane. The phosphorylation and internalization of LRP6 occurred independently of one another, and both were necessary for the accumulation of β-catenin. In contrast, Dkk1, which inhibits Wnt3a-dependent stabilization of β-catenin, induced the internalization of LRP6 with clathrin. Knockdown of clathrin suppressed the Dkk1-dependent inhibition of the Wnt3a response. Furthermore, Dkk1 reduced the distribution of LRP6 in the lipid raft fraction where caveolin is associated. These results indicate that Wnt3a and Dkk1 shunt LRP6 to distinct internalization pathways in order to activate and inhibit the β-catenin signaling, respectively. [Copyright &y& Elsevier]

Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. We previously showed that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate, possibly by promoting neu rite elongation via microtubule assembly. Here, we showed that glycogen synthase kinase -3β (GSK-3β) phosphorylated CRMP-2 at Thr-514 and inactivated it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3β induced the formation of multiple axon-like neurites in hippocampal neurons. The expression of constitutively active GSK-3β impaired neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracted the inhibitory effects of GSK-3β, indicating that GSK-3β regulates neuronal polarity through the phosphorylation of CRMP-2. Treatment of hippocampal neurons with neurotrophin-3 (NT-3) induced inactivation of GSK-3β and dephosphorylation of CRMP-2. Knockdown of CRMP-2 inhibited NT-3-induced axon outgrowth. These results suggest that NT-3 decreases phosphorylated CRMP-2 and increases nonphosphorylated active CRMP-2, thereby promoting axon outgrowth. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase-3β (GSK-3β) activity is suppressed when it becomes phosphorylated on serine 9 by protein kinase B (Akt). To determine how GSK-3β activity opposes Akt function we used various methods to alleviate GSK-3β suppression in prostate carcinoma cells. In some experiments, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (a kinase involved in activating Akt) and tumor necrosis factor-α (TNF-α) were used to activate GSK-3β. In other experiments mutant forms of GSK-3β, GSK-3β[sup Δ9] (a constitutively active deletion mutant of GSK-3β) and GSK-3β[sup Y216F] (an inactive point mutant of GSK-3β) were used to alter GSK-3β activity. LY294002, TNF-α, and overexpression of wild-type GSK-3β or of GSK-3β[sup Δ9], but not GSK-3β[sup Y216F], alleviated the suppression of GSK-3β activity in prostate carcinoma cells and enhanced the turnover of β-catenin. Forced expression of wild-type GSK-3β or of GSK-3β[sup Δ9], but not GSK3β[sup Y216F], suppressed cell growth and showed that the phosphorylation status of GSK-3β can affect its intracellular distribution. When transcription factors activator protein-1 and cyclic AMP-response element (CRE).binding protein were analyzed as targets of GSK-3β activity, overexpression of wild-type GSK-3β suppressed AP1-mediated transcription and activated CRE-mediated transcription. Overexpression of GSK-3β[sup Δ9] caused an (80-fold) increase in CRE-mediated transcription, which was further amplifled (up to 130-fold) by combining GSK-3β[sup Δ9] overexpression with the suppression of Jun activity. This study also demonstrated for the first time that expression of constitutively active GSK-3β[sup Δ9] results in the phosphorylation of CRE-binding protein on serine 129 and enhancement of CRE-mediated transcription in intact cell nuclei. [ABSTRACT FROM AUTHOR]