Biomedical subjects

Takeshi Imamura

Publications and source records attributed to Takeshi Imamura.

13 recordsLinked to original sources

PubMed2003

Differential ubiquitination defines the functional status of the tumor suppressor Smad4.

Smad4 is an essential signal transducer of all transforming growth factor-beta (TGF-beta) superfamily pathways that regulate cell growth and differentiation, and it becomes inactivated in human cancers. Receptor-activated (R-) Smads can be poly-ubiquitinated in the cytoplasm or the nucleus, and this regulates their steady state levels or shutdown of the signaling pathway. Oncogenic mutations in Smad4 and other Smads have been linked to protein destabilization and proteasomal degradation. We analyzed a panel of missense mutants derived from human cancers that map in the N-terminal Mad homology (MH) 1 domain of Smad4 and result in protein instability. We demonstrate that all mutants exhibit enhanced poly-ubiquitination and proteasomal degradation. In contrast, wild type Smad4 is a relatively stable protein that undergoes mono- or oligo-ubiquitination, a modification not linked to protein degradation. Analysis of Smad4 deletion mutants indicated efficient mono- or oligo-ubiquitination of the C-terminal MH2 domain. Mass spectrometric analysis of mono-ubiquitinated Smad4 MH2 domain identified lysine 507 as a major target for ubiquitination. Lysine 507 resides in the conserved L3 loop of Smad4 and participates in R-Smad C-terminal phosphoserine recognition. Mono- or oligo-ubiquitinated Smad4 exhibited enhanced ability to oligomerize with R-Smads, whereas mutagenesis of lysine 507 led to inefficient Smad4/R-Smad hetero-oligomerization and defective transcriptional activity. Finally, overexpression of a mutant ubiquitin that only leads to mono-ubiquitination of Smad4 enhanced Smad transcriptional activity. These data suggest that oligo-ubiquitination positively regulates Smad4 function, whereas poly-ubiquitination primarily occurs in unstable cancer mutants and leads to protein degradation.

Amino Acid Sequence↗

PubMed2003

Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads.

Smad ubiquitin regulatory factor (Smurf) 1 binds to receptor-regulated Smads for bone morphogenetic proteins (BMPs) Smad1/5 and promotes their degradation. In addition, Smurf1 associates with transforming growth factor-beta type I receptor through the inhibitory Smad (I-Smad) Smad7 and induces their degradation. Herein, we examined whether Smurf1 negatively regulates BMP signaling together with the I-Smads Smad6/7. Smurf1 and Smad6 cooperatively induced secondary axes in Xenopus embryos. Using a BMP-responsive promoter-reporter construct in mammalian cells, we found that Smurf1 cooperated with I-Smad in inhibiting BMP signaling and that the inhibitory activity of Smurf1 was not necessarily correlated with its ability to bind to Smad1/5 directly. Smurf1 bound to BMP type I receptors via I-Smads and induced ubiquitination and degradation of these receptors. Moreover, Smurf1 associated with Smad1/5 indirectly through I-Smads and induced their ubiquitination and degradation. Smurf1 thus controls BMP signaling with and without I-Smads through multiple mechanisms.

Activin Receptors, Type I↗

PubMed2003

Glypican-3, overexpressed in hepatocellular carcinoma, modulates FGF2 and BMP-7 signaling.

The Glypican (GPC) family is a prototypical member of the cell-surface heparan sulfate proteoglycans (HSPGs). The HSPGs have been demonstrated to interact with growth factors, act as coreceptors and modulate growth factor activity. Here we show that based on oligonucleotide array analysis, GPC3 was upregulated in hepatocellular carcinoma (HCC). By northern blot analysis, GPC3 mRNA was found to be upregulated in 29 of 52 cases of HCC (55.7%). By Western blot analysis carried out with a monoclonal anti-GPC3 antibody we generated, the GPC3 protein was found to be overexpressed in 6 hepatoma cell lines, HepG2, Hep3B, HT17, HuH6, HuH7 and PLC/PRF/5, as well as 22 tumors (42.3%). To investigate the role of overexpressed GPC3 in liver cancer, we analyzed its effects on cell growth of hepatoblastoma-derived cells. Overexpression of GPC3 modulated cell proliferation by inhibiting fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 7 (BMP-7) activity. An interaction of GPC3 and FGF2 was revealed by co-immunoprecipitation, while GPC3 was found to inhibit BMP-7 signaling through the Smad pathway by reporter gene assay. The modulation of growth factors by GPC3 may help explain its role in liver carcinogenesis. In addition, the ability of HCC cells to express GPC3 at high levels may serve as a new tumor marker for HCC.

Aged↗

PubMed2003

Cdc42 is a Rho GTPase family member that can mediate insulin signaling to glucose transport in 3T3-L1 adipocytes.

We investigated the role of cdc42, a Rho GTPase family member, in insulin-induced glucose transport in 3T3-L1 adipocytes. Microinjection of anti-cdc42 antibody or cdc42 siRNA led to decreased insulin-induced and constitutively active G(q) (CA-G(q); Q209L)-induced GLUT4 translocation. Adenovirus-mediated expression of constitutively active cdc42 (CA-cdc42; V12) stimulated 2-deoxyglucose uptake to 56% of the maximal insulin response, and this was blocked by treatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin, or LY294002. Both insulin and CA-G(q) expression caused an increase in cdc42 activity, showing that cdc42 is activated by insulin and is downstream of G alpha(q/11) in this activation pathway. Immunoprecipitation experiments showed that insulin enhanced a direct association of cdc42 and p85, and both insulin treatment and CA-cdc42 expression stimulated PI3-kinase activity in immunoprecipitates with anti-cdc42 antibody. Furthermore, the effects of insulin, CA-G(q), and CA-cdc42 on GLUT4 translocation or 2-deoxyglucose uptake were inhibited by microinjection of anti-protein kinase C lambda (PKC lambda) antibody or overexpression of a kinase-deficient PKC lambda construct. In summary, activated cdc42 can mediate 1) insulin-stimulated GLUT4 translocation and 2) glucose transport in a PI3-kinase-dependent manner. 3) Insulin treatment and constitutively active G(q) expression can enhance the cdc42 activity state as well as the association of cdc42 with activated PI3-kinase. 4) PKC lambda inhibition blocks CA-cdc42, CA-G(q), and insulin-stimulated GLUT4 translocation. Taken together, these data indicate that cdc42 can mediate insulin signaling to GLUT4 translocation and lies downstream of G alpha(q/11) and upstream of PI3-kinase and PKC lambda in this stimulatory pathway.

3T3 Cells↗

PubMed2003

Chromosomal region maintenance 1 (CRM1)-dependent nuclear export of Smad ubiquitin regulatory factor 1 (Smurf1) is essential for negative regulation of transforming growth factor-beta signaling by Smad7.

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces translocation of Smad7 to the cytoplasm. Smurf1 then associates with the transforming growth factor (TGF)-beta type I receptor, TbetaR-I, enhancing turnover. However, the mechanism of nuclear export of Smad7 by Smurf1 has not been elucidated. Here we identified a functional nuclear export signal (NES) in a C-terminal region of Smurf1. In transfected cells, the Smurf1-Smad7 complex was accumulated in the cytoplasm by the nuclear export receptor, CRM1; this action was prevented by treatment with leptomycin B, a specific inactivator of CRM1 function. A green fluorescence protein fusion protein containing the C-terminal NES motif of Smurf1, located in the cytoplasm, accumulated in the nucleus following treatment with leptomycin B. Moreover, Smurf1 was shown to bind physically to CRM1 through NES, and nuclear export of the Smurf1-Smad7 complex was prevented by mutations of Smurf1 within the NES. Finally, the Smurf1 NES mutant reduced inhibition by Smad7 of the transcriptional activation induced by TGF-beta. These results thus suggest that CRM1-dependent nuclear export of Smurf1 is essential for the negative regulation of TGF-beta signaling by Smad7.

Active Transport, Cell Nucleus↗

PubMed2003

Regulation of TGF-beta signaling and its roles in progression of tumors.

Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor of most types of cells; therefore, perturbations of TGF-beta signaling are believed to result in progression of various tumors. On the other hand, TGF-beta has been shown to act as an oncogenic cytokine through induction of extracellular matrices, angiogenesis, and immune suppression. A wide variety of effects of TGF-beta are mediated by physical interaction of signal transducer Smad proteins with various transcription factors. Among these, Runx3 plays a pivotal role in prevention of gastric cancer. TGF-beta signaling is regulated by various mechanisms in the cytoplasm and nucleus. Inhibitory Smads (I-Smads) repress TGF-beta signaling mainly by interacting with activated TGF-beta receptors. Smad ubiquitin regulatory factors (Smurfs) play important roles in facilitating the inhibitory signals induced by I-Smads. In addition, the transcriptional co-repressors c-Ski and SnoN interact with Smads, and repress transcription induced by TGF-beta. Abnormalities of these regulators of TGF-beta signaling may thus participate in the progression of various tumors.

Animals↗

PubMed2003

Insulin-induced GLUT4 translocation involves protein kinase C-lambda-mediated functional coupling between Rab4 and the motor protein kinesin.

Insulin stimulates glucose transport by promoting translocation of GLUT4 proteins from the perinuclear compartment to the cell surface. It has been previously suggested that the microtubule-associated motor protein kinesin, which transports cargo toward the plus end of microtubules, plays a role in translocating GLUT4 vesicles to the cell surface. In this study, we investigated the role of Rab4, a small GTPase-binding protein, and the motor protein KIF3 (kinesin II in mice) in insulin-induced GLUT4 exocytosis in 3T3-L1 adipocytes. Photoaffinity labeling of Rab4 with [gamma-(32)P]GTP-azidoanilide showed that insulin stimulated Rab4 GTP loading and that this insulin effect was inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 or expression of dominant-negative protein kinase C-lambda (PKC-lambda). Consistent with previous reports, expression of dominant-negative Rab4 (N121I) decreased insulin-induced GLUT4 translocation by 45%. Microinjection of an anti-KIF3 antibody into 3T3-L1 adipocytes decreased insulin-induced GLUT4 exocytosis by 65% but had no effect on endocytosis. Coimmunoprecipitation experiments showed that Rab4, but not Rab5, physically associated with KIF3, and this was confirmed by showing in vitro association using glutathione S-transferase-Rab4. A microtubule capture assay demonstrated that insulin stimulation increased the activity for the binding of KIF3 to microtubules and that this activation was inhibited by pretreatment with the PI3-kinase inhibitor LY294002 or expression of dominant-negative PKC-lambda. Taken together, these data indicate that (i) insulin signaling stimulates Rab4 activity, the association of Rab4 with kinesin, and the interaction of KIF3 with microtubules and (ii) this process is mediated by insulin-induced PI3-kinase-dependent PKC-lambda activation and participates in GLUT4 exocytosis in 3T3-L1 adipocytes.

3T3 Cells↗

PubMed2002

Two short segments of Smad3 are important for specific interaction of Smad3 with c-Ski and SnoN.

c-Ski and SnoN are transcriptional co-repressors that inhibit transforming growth factor-beta signaling through interaction with Smad proteins. Among receptor-regulated Smads, c-Ski and SnoN bind more strongly to Smad2 and Smad3 than to Smad1. Here, we show that c-Ski and SnoN bind to the "SE" sequence in the C-terminal MH2 domain of Smad3, which is exposed on the N-terminal upper side of the toroidal structure of the MH2 oligomer. The "QPSMT" sequence, located in the vicinity of SE, supports the interaction with c-Ski and SnoN. Sequences similar to SE and QPSMT are found in Smad2, but not in Smad1. The N-terminal MH1 domain and linker region of Smad3 protrude from the N-terminal upper side of the MH2 oligomer toroid. Smurf2 induces ubiquitin-dependent degradation of SnoN, since it appears to be located close to SnoN through binding to the linker region of Smad2. In contrast, transcription factors Mixer and FoxH3 (FAST1) bind to the bottom side of the Smad3 MH2 toroid; therefore, c-Ski does not affect the interaction of Smads with these transcription factors. Our findings thus demonstrate the stoichiometry of how multiple molecules can associate with the Smad oligomers and how the Smad-interacting proteins functionally interact with each other.

Activin Receptors, Type I↗

PubMed2002

Smurf1 regulates the inhibitory activity of Smad7 by targeting Smad7 to the plasma membrane.

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT-type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-beta type I receptor (TbetaR-I) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1.Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(deltaC2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(deltaC2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface TbetaR-II.TbetaR-I complex. Moreover, although Smurf1(deltaC2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of TbetaR-I and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-beta signaling pathway.

Activin Receptors, Type I↗

PubMed2002

Phosphatidylinositol 3-kinase is required for insulin-stimulated tyrosine phosphorylation of Shc in 3T3-L1 adipocytes.

The interactions between the phosphatidylinositol 3-kinase (PI 3-kinase) and Ras/MAPK kinase pathways have been the subject of considerable interest. In the current studies, we find that epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) lead to rapid phosphorylation of Shc (maximum at 1-2 min), whereas insulin-mediated Shc phosphorylation is relatively delayed (maximum at 5-10 min), suggesting that an intermediary step may be necessary for insulin stimulation of Shc phosphorylation. The Src homology-2 (SH2) domain of Shc is necessary for PDGF- and EGF-mediated Shc phosphorylation, whereas the phosphotyrosine binding (PTB) domain is critical for the actions of insulin. Because the Shc PTB domain can interact with phospholipids, we postulated that PI 3-kinase might be a necessary intermediary step facilitating insulin-stimulated phosphorylation of Shc. In support of this, we found that the PI 3-kinase inhibitors, wortmannin and LY294002, blocked insulin-stimulated but not EGF- or PDGF-stimulated Shc phosphorylation. Furthermore, overexpression of a dominant negative PI 3-kinase construct (p85N-SH2) blocked insulin, but not EGF- or PDGF-induced Shc phosphorylation. All three growth factors cause localization of Shc to the plasma membrane, but only the effect of insulin was inhibited by wortmannin, supporting the view that PI 3-kinase-generated phospholipids mediate insulin-stimulated Shc phosphorylation. Consistent with this, expression of a constitutively active PI 3-kinase (p110(C)(AAX)) increased membrane localization of Shc, and this was completely blocked by wortmannin. A mutant Shc with a disrupted PTB domain (Shc S154) did not localize to the membrane in p110(C)(AAX)-expressing cells or after insulin stimulation and was not phosphorylated by insulin. In summary, 1) PI 3-kinase is a necessary early step in insulin-stimulated Shc phosphorylation, whereas the effects of EGF and PDGF on Shc phosphorylation are independent of PI 3-kinase. 2) PI 3-kinase-stimulated generation of membrane phospholipids can localize Shc to the plasma membrane through the Shc PTB domain facilitating phosphorylation by the insulin receptor.

3T3 Cells↗

PubMed2002

Functional heterogeneity of bone morphogenetic protein receptor-II mutants found in patients with primary pulmonary hypertension.

Germline mutations in the BMPR2 gene encoding bone morphogenetic protein (BMP) type II receptor (BMPR-II) have been reported in patients with primary pulmonary hypertension (PPH), but the contribution of various types of mutations found in PPH to the pathogenesis of clinical phenotypes has not been elucidated. To determine the biological activities of these mutants, we performed functional assays testing their abilities to transduce BMP signals. We found that the reported missense mutations within the extracellular and kinase domains of BMPR-II abrogated their signal-transducing abilities. BMPR-II proteins containing mutations at the conserved cysteine residues in the extracellular and kinase domains were detected in the cytoplasm, suggesting that the loss of signaling ability of certain BMPR-II mutants is due at least in part to their altered subcellular localization. In contrast, BMPR-II mutants with truncation of the cytoplasmic tail retained the ability to transduce BMP signals. The differences in biological activities among the BMPR-II mutants observed thus suggest that additional genetic and/or environmental factors may play critical roles in the pathogenesis of PPH.

Animals↗

PubMed2002

Insulin induces heterologous desensitization of G-protein-coupled receptor and insulin-like growth factor I signaling by downregulating beta-arrestin-1.

beta-Arrestin-1 mediates agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCRs) and is also essential for GPCR mitogenic signaling. In addition, insulin-like growth factor I receptor (IGF-IR) endocytosis is facilitated by beta-arrestin-1, and internalization is necessary for IGF-I-stimulated mitogen-activated protein (MAP) kinase activation. Here, we report that treatment of cells for 12 h with insulin (100 ng/ml) induces an approximately 50% decrease in cellular beta-arrestin-1 content due to ubiquitination of beta-arrestin-1 and proteosome-mediated degradation. This insulin-induced decrease in beta-arrestin-1 content was blocked by inhibition of phosphatidylinositol-3 kinase (PI-3 kinase) and MEK with wortmannin and PD98059, respectively. We also found a marked decrease in the association of beta-arrestin-1 with the IGF-IR and a 55% inhibition of IGF-I-stimulated MAP kinase phosphorylation. In insulin-treated, beta-arrestin-1-downregulated cells, there was complete inhibition of lysophosphatidic acid (LPA) or isoproterenol (ISO)-stimulated MAP kinase phosphorylation. This was associated with a decrease in beta-arrestin-1 association with the beta2-AR as well as a decrease in beta-arrestin-1-Src and Src-beta2-AR association. Ectopic expression of wild-type beta-arrestin-1 in insulin-treated cells in which endogenous beta-arrestin-1 had been downregulated rescued IGF-I- and LPA-stimulated MAP kinase phosphorylation. In conclusion, we found the following. (i) Chronic insulin treatment leads to enhanced beta-arrestin-1 degradation. (ii) This downregulation of endogenous beta-arrestin-1 is associated with decreased IGF-I-, LPA-, and ISO-mediated MAP kinase signaling, which can be rescued by ectopic expression of wild-type beta-arrestin-1. (iii) Finally, these results describe a novel mechanism for heterologous desensitization, whereby insulin treatment can impair GPCR signaling, and highlight the importance of beta-arrestin-1 as a target molecule for this desensitization mechanism.

3T3 Cells↗

PubMed2002

Protein phosphatase 2A forms a molecular complex with Shc and regulates Shc tyrosine phosphorylation and downstream mitogenic signaling.

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that carries out multiple functions. Although numerous observations suggest that PP2A plays a major role in downregulation of the mitogen-activated protein (MAP) kinase pathway, the precise mechanisms are unknown. To clarify the role of PP2A in growth factor (insulin, epidermal growth factor [EGF], and insulin-like growth factor 1 [IGF-1]) stimulation of the Ras/MAP kinase pathway, simian virus 40 small t antigen was expressed in Rat-1 fibroblasts which overexpress insulin receptors. Small t antigen is known to specifically inhibit PP2A by binding to the A PP2A regulatory subunit, interfering with the ability of PP2A to bind to its cellular substrates. Overexpressed small t protein was coimmunoprecipitated with PP2A and inhibited cellular PP2A activity but did not inhibit protein phosphatase 1 (PP1) activity. Insulin, IGF-1, and EGF stimulation also inhibited PP2A activity. Growth factor-stimulated Ras, Raf-1, MAP kinase, and mitogen-activated extracellular-signal-regulated kinase kinase (MEK) activities were elevated in small-t-antigen-expressing cells. Furthermore, Shc tyrosine phosphorylation and its association with Grb2 were also elevated in small-t-antigen-expressing cells. Expression levels of Shc, Ras, MEK, or MAP kinase and phosphorylation of insulin, EGF, and IGF-1 receptors were not altered. Interestingly, we found that PP2A associated with Shc in the basal state and dissociated in response to insulin and EGF and that this dissociation was inhibited by 65% in small-t-antigen-expressing cells. In addition, we found that PP2A associates with the phosphotyrosine-binding domain (PTB domain) of Shc and that phosphorylation of tyrosine 317 of Shc was required for PP2A-Shc dissociation. We conclude (i) that PP2A negatively regulates the Ras/MAP kinase pathway by binding to Shc, inhibiting tyrosine phosphorylation; (ii) that the Shc-PP2A association is mediated by the Shc PTB domain but the interaction is independent of phosphotyrosine binding, indicating a new molecular function for the PTB domain; (iii) that growth factor stimulation, or small-t-antigen expression, causes dissociation of the PP2A-Shc complex, facilitating Shc phosphorylation and downstream activations of the Ras/MAP kinase pathway; and (iv) that this defines a new mechanism of small-t-antigen action to promote mitogenesis.

Adaptor Proteins, Signal Transducing↗