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Dvl is a key protein that transmits the Wnt signal to the canonical beta-catenin pathway and the noncanonical planar cell polarity (PCP) pathway. We studied the roles of Rho-associated kinase (Rho-kinase), which is activated by Dvl in the PCP pathway of mammalian cells. The expression of Dvl-1, Wnt-1, or Wnt-3a activated Rho-kinase in COS cells, and this activation was inhibited by the Rho-binding domain of Rho-kinase. The expression of Dvl-1 in PC12 cells activated Rho and inhibited nerve growth factor (NGF)-induced neurite outgrowth. This inhibition was reversed by a Rho-kinase inhibitor but not by a c-Jun N-terminal kinase inhibitor. Dvl-1 also inhibited serum starvation-dependent neurite outgrowth of N1E-115 cells, and expression of the Rho-binding domain of Rho-kinase reversed this inhibitory activity of Dvl-1. Dvl-1 mutants that did not activate Rho-kinase did not inhibit the neurite outgrowth of N1E-115 cells. Furthermore, the purified Wnt-3a protein activated Rho-kinase and inhibited the NGF-dependent neurite outgrowth of PC12 cells. Wnt-3a-dependent neurite retraction was also prevented by a Rho-kinase inhibitor and a Dvl-1 mutant that suppresses Wnt-3a-dependent activation of Rho-kinase. These results suggest that Wnt-3a and Dvl regulate neurite formation through Rho-kinase and that PC12 and N1E-115 cells are useful for analyzing the PCP pathway.

We demonstrate that Dvl-1, casein kinase I epsilon (CKI epsilon), and Frat-1 activate the Wnt signaling pathway cooperatively. The amino acid region 228-250 of Dvl-1 was necessary for its binding to Frat-1, and the interaction of Dvl-1 with Frat-1 was enhanced by CKI epsilon. Coexpression of Dvl-1 and Frat-1 caused accumulation of beta-catenin synergistically in L cells. Both proteins also activated the transcriptional activity of T-cell factor-4 (Tcf-4) synergistically in human embryonic kidney 293 cells, but coexpression of Dvl-1-(Delta 228-250), which lacks the amino acid region 228-250 from Dvl-1, and Frat-1 did not. Dvl-1, but not Dvl-1-(Delta 228-250), acted synergistically with CKI epsilon to activate Tcf-4. Depletion of CKI epsilon by double-stranded RNA interference in HeLa S3 cells led to the inhibition of Wnt-3a-induced phosphorylation of Dvl and the binding of Dvl-1 to Frat-1. Furthermore, depletion of CKI epsilon reduced the Wnt-3a-induced accumulation of beta-catenin, although it did not affect the basal level of beta-catenin. These results indicate that CKI epsilon-dependent phosphorylation of Dvl enhances the formation of a complex of Dvl-1 with Frat-1 and that this complex leads to the activation of the Wnt signaling pathway.

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-3beta (GSK-3beta), and at Ser86, Ser120, and Ser121 by casein kinase 2 (CK2). Here we report that GSK-3beta 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-3beta phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3beta 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-3beta with I-2-FLAG, showing a complex formation of HA-GSK-3beta/Myc-PP1C / I-2-FLAG in vivo. Further studies using a GSK-3beta kinase-dead mutant and LiCl, an inhibitor of GSK-3beta, showed that the enzyme activity of GSK-3beta 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-3beta and I-2 in vivo. This study is the first demonstration that GSK-3beta associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.

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]

Duplin was originally isolated as a negative regulator of β-catenin–dependent T-cell factor (Tcf) transcriptional activity in the Wnt signaling pathway. However, Duplin knockout mice exhibit embryonic lethality at 5.5-em day, suggesting that Duplin has important roles other than as a negative regulator of the Wnt signal. To identify new roles of Duplin, the Duplin-binding proteins were screened. PIAS3, which is a SUMO E3 ligase and acts as an inhibitor of signal transducer and activator of transcription (STAT3), was identified as a Duplin-binding protein. Duplin was sumoylated, but PIAS3 affected neither the sumoylation of Duplin nor its ability to inhibit Tcf-4 activity. Like PIAS3, Duplin suppressed the leukemia-inhibitory factor (LIF)–induced STAT3 transcriptional activity. Duplin did not affect the LIF-dependent tyrosine phosphorylation or nuclear localization of STAT3 but inhibited the formation of complex between STAT3 and DNA. Although STAT3 is not modified with SUMO, PIAS3 inhibited the STAT3 activity in a manner partially depending on its SUMO E3 ligase activity. Duplin suppressed the LIF-dependent STAT3 activity independently of sumoylation. These results demonstrate that Duplin inhibits not only Tcf-4 but also STAT3, suggesting that Duplin may act as a repressor for multiple transcriptional factors. [ABSTRACT FROM PUBLISHER]

We have previously shown that modification of Tcf-4, a transcription factor in the Wnt pathway, with SUMO by PIASy, a SUMO E3 ligase, enhances its transcriptional activity. Since PIASy itself was also modified with SUMO-1, we studied the role of sumoylation of PIASy in the regulation of Tcf-4. Lys35 was found to be a sumoylation site of PIASy. PIASyK35R, in which Lys35 was mutated to Arg, did not enhance sumoylation of Tcf-4, although this PIASy mutant did not lose the ligase activity of sumoylation for other proteins. Wild-type PIASy and PIASyK35R showed a distinct distribution in the nucleus, although both were colocalized with Tcf-4. Promyelocytic leukemia protein, which is involved in transcriptional regulation, was associated with PIASyK35R more frequently than wild-type PIASy in the nucleus. PIASyK35R could not stimulate the transcriptional activity of Tcf-4 under the conditions in which wild-type PIASy enhanced it. Conjugation of SUMO-1 to the amino terminus of PIASyK35R neither enhanced sumoylation of Tcf-4 nor stimulated the transcriptional activity of Tcf-4. These results suggest that sumoylation of Lys35 in PIASy determines the nuclear localization of PIASy and that it is necessary for PIASy-dependent sumoylation and transcriptional activation of Tcf-4. [ABSTRACT FROM AUTHOR]

Synaptic scaffolding molecule (S-SCAM) is a synaptic protein that consists of PDZ domains, a guanylate kinase domain, and WW domains. It interacts with N-methyl- d-aspartate receptor subunits, neuroligin, and β-catenin. Here, we identified Axin as a novel binding partner of S-SCAM. Axin was co-immunoprecipitated with S-SCAM from rat brain, detected in the post-synaptic density fraction in rat brain subcellular fractionation, and partially co-localized with S-SCAM in neurons. The guanylate kinase domain of S-SCAM directly bound to the GSK3β-binding region of Axin. S-SCAM formed a complex with β-catenin and Axin, but competed with GSK3β for Axin-binding. Thereby, S-SCAM inhibited the Axin-mediated phosphorylation of β-catenin by GSK3β. [ABSTRACT FROM AUTHOR]

Dvl is a cytoplasmic protein to regulate the stability of β-catenin in the Wnt signalling pathway. However, the molecular mechanism by which Dvl regulates the Wnt signalling pathway is not fully understood. We identified a novel protein that binds to Dvl and named it Daple. Daple consisted of 2009 amino acids with a high frequency of leucine residues and formed a homo-oligomer. The C-terminal three amino acids of Daple were necessary for binding to the region containing the PDZ domain of Dvl. Expression of Daple in mouse fibroblast L cells inhibited Wnt-3a-induced accumulation of β-catenin. Furthermore, Daple inhibited Wnt-3a-dependent activation of T-cell factor (Tcf) transcriptional activity. Expression of Daple in the dorsal region of Xenopus embryos inhibited axis formation, which is known to be regulated by the Wnt signalling pathway. Daple also inhibited Dvl-induced secondary axis formation in Xenopus embryos. Daple binds to Dvl and functions as a negative regulator of the Wnt signalling pathway. [ABSTRACT FROM AUTHOR]

Investigates the activation of lymphoid enhancer factor (LEF) transcriptional activity by beta-arrestin1 (arr1). Function of the beta-arrestins; Role of beta-arr1 as a regulator of Dishevelled (Devl) protein-dependent LEF transcription; Interaction between beta-arr1 and Dvl in cells.

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]

Ral GDP dissociation stimulator (RalGDS), a putative effector protein of Ras, stimulated the GDP/GTP exchange reaction of the post-tanslationally lipid-modified but not the unmodified form of Ral in response to epidermal growth factor in COS cells. The RalGDS action on Ral was enhanced by an active form of Ras but not a Ras mutant which was not post-translationally modified in the cells. The RalGDS activity was inhibited by acidic membrane phospholipids such as phosphatidyl-inositol and phosphatidylserine but not by phosphatidylcholine or phosphatidylethanolamine in vitro. The post-translationally modified form but not unmodified form of Ras, Ral, and Rap were incorporated in liposomes consisting of these phospholipids. When Ral was incorporated alone in the liposomes, RalGDS did not stimulate the dissociation of GDP from Ral. When Ral was incorporated with the GTP-bound form of Ras in the liposomes, RalGDS stimulated the dissociation of GDP from Ral, while the GDP-bound form of Ras did not affect the RalGDS action. The Ras-dependent Ral activation through RalGDS required the Ras-binding domain of RalGDS. Rap, which shared the same effector loop as Ras, also stimulated the dissociation of GDP from Ral through RalGDS in the liposomes, although Rap did not enhance the RalGDS action in COS cells. Taken together with our previous observations that Ras recruits RalGDS to the membrane, these results indicate that the post-translational modifications of Ras and Ral are important for Ras-dependent Ral activation through RalGDS and that colocalization of Ras and Ral on the membrane is necessary for Ral activation in intact cells. [ABSTRACT FROM AUTHOR]

The distribution of ras-related small-molecular-mass guanine-nucleotide-binding regulatory proteins (SMG) of two insulin-secreting cell lines, RINm5F and HIT-T15, and of a catecholamine- secreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two-dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin-secreting cells, rho protein(s) that cannot be detected with the GTP-binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin-secreting cell line RINm5F and the catecholamine-secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT-T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion. [ABSTRACT FROM AUTHOR]

SUMO proteases possess two enzymatic activities to hydrolyze the C-terminal region of SUMOs (hydrolase activity) and to remove SUMO from SUMO-conjugated substrates (isopeptidase activity). SUMO proteases bind to SUMOs noncovalently, but the physiological roles of the binding in the functions of SUMO proteases are not well understood. In this study we found that SUMO proteases (Axam, SENP1, and yeast Ulp1) show different preferences for noncovalent binding to various SUMOs (SUMO-1, -2, -3, and yeast Smt3) and that the hydrolase and isopeptidase activities of SUMO proteases are dependent on their binding to SUMOs through salt bridge. Expression of Smt3 suppressed the phenotype of yeast mutant lacking smt3, which exhibits growth arrest, and the binding of Ulp1 to Smt3 was essential for this rescue activity. Although expression of an Smt3 mutant (smt3R64E(GG)), which conjugates to substrate but loses the ability to bind to Ulp1, rescued the phenotype of yeast lacking smt3 partially, the mutant cells showed an increment in the doubling time and a delay of desumoylation of Smt3-conjugated Cdc3. These results indicate that the noncovalent binding of SUMO protease to SUMO through salt bridge is essential for the enzymatic activities and that the balance between sumoylation and desumoylation is important for cell growth control. [ABSTRACT FROM AUTHOR]

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]