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Shiro Suetsugu

Publications and source records attributed to Shiro Suetsugu.

17 recordsLinked to original sources
PubMed2005

Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells.

WAVEs (WASP-family verprolin-homologous proteins) regulate the actin cytoskeleton through activation of Arp2/3 complex. As cell motility is regulated by actin cytoskeleton rearrangement and is required for tumor invasion and metastasis, blocking actin polymerization may be an effective strategy to prevent tumor dissemination. We show that WAVEs, especially WAVE2, are essential for invasion and metastasis of melanoma cells. Malignant B16F10 mouse melanoma cells expressed more WAVE1 and WAVE2 proteins and showed higher Rac activity than B16 parental cells, which are neither invasive nor metastatic. The effect of WAVE2 silencing by RNA interference (RNAi) on the highly invasive nature of B16F10 cells was more dramatic than that of WAVE1 RNAi. Membrane ruffling, cell motility, invasion into the extracellular matrix, and pulmonary metastasis of B16F10 cells were suppressed by WAVE2 RNAi. WAVE2 RNAi also had a profound effect on invasion induced by a constitutively active form of Rac (RacCA). In addition, ectopic expression of both RacCA and WAVE2 in B16 cells resulted in further increase in the invasiveness than that observed in B16 cells expressing only RacCA. Thus, WAVE2 acts as the primary effector downstream of Rac to achieve invasion and metastasis, suggesting that suppression of WAVE2 activity holds a promise for preventing cancer invasion and metastasis.

Actins↗
PubMed2004

Regulation of actin cytoskeleton by mDab1 through N-WASP and ubiquitination of mDab1.

Migration of cells is critical to development of the central nervous system. Reelin, which was identified from the reeler mutant mice having a defect in the multilamellar structure of the brain, is thought to be a key signalling molecule that functions as a cue for determination of cell position. mDab1 (mouse Disabled homologue 1) functions downstream of Reelin. However, the mechanism by which mDab1 regulates cell migration during brain development is unknown. In the present paper, we show that mDab1 associates with N-WASP (neuronal Wiskott-Aldrich syndrome protein) in vitro and in brains of embryonic mice. mDab1 activates N-WASP directly, and induces actin polymerization through the Arp2/3 (actin-related protein 2/3) complex. mDab1 induces formation of filopodia when it is overexpressed in COS-7 cells. This filopodium formation is dependent on N-WASP, because expression of an N-WASP mutant that cannot induce Arp2/3-complex-mediated actin polymerization suppressed filopodium formation. The PTB (phosphotyrosine-binding) domain of mDab1 binds to N-WASP via the NRFY (Asn-Arg-Phe-Tyr) sequence close to the CRIB (Cdc42/Rac-interactive binding) motif of N-WASP and activates N-WASP in vitro. When mDab1 is phosphorylated by Fyn kinase in COS-7 cells, mDab1 is ubiquitinated in a Cbl-dependent manner, and mDab1 does not induce filopodium in the presence of activated Fyn. These findings suggest that mDab1 regulates the actin cytoskeleton through N-WASP, which is negatively regulated by phosphorylation-mediated ubiquitination of mDab1.

Actins↗
PubMed2004

N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor.

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.

Actins↗
PubMed2004

WAVE/Scars in platelets.

Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolin-homologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. The 3 WAVE isoforms were distributed in the actin-rich Triton X-100-insoluble pellets following platelet aggregation induced by thrombin receptor-activating peptide. We also found that all 3 WAVE isoforms are substrates for calpain in vivo and in vitro. Although portions of these 3 isoforms were commonly distributed in the actin- and actin-related protein 2 and 3 (Arp2/3)-rich edge of the lamellipodia in spreading platelets, only WAVE2 remained in the cell fringe following detergent extraction or fixation of the cells. Finally, by mass spectrometry, we found that the proteins, which reportedly interact with WAVE/Scars, are present in platelets. These data suggest that the 3 WAVE isoforms exhibit common and distinct features and may potentially be involved in the regulation of actin cytoskeleton in platelets.

Actin Cytoskeleton↗
PubMed2004

PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia.

Polarized cell movement is triggered by the development of a PtdIns(3,4,5)P(3) gradient at the membrane, which is followed by rearrangement of the actin cytoskeleton. The WASP family verprolin homologous protein (WAVE) is essential for lamellipodium formation at the leading edge by activating the Arp2/3 complex downstream of Rac GTPase. Here, we report that WAVE2 binds to PtdIns(3,4,5)P(3) through its basic domain. The amino-terminal portion of WAVE2, which includes the PtdIns(3,4,5)P(3)-binding sequence, was localized at the leading edge of lamellipodia induced by an active form of Rac (RacDA) or by treatment with platelet-derived growth factor (PDGF). Production of PtdIns(3,4,5)P(3) at the cell membrane by myristoylated phosphatidylinositol-3-OH kinase (PI(3)K) is sufficient to recruit WAVE2 in the presence of dominant-negative Rac and latrunculin, demonstrating that PtdIns(3,4,5)P(3) alone is able to recruit WAVE2. Expression of a full-length mutant of WAVE2 that lacks the lipid-binding activity inhibited proper formation of lamellipodia induced by RacDA. These results suggest that one of the products of PI(3)K, PtdIns(3,4,5)P(3), recruits WAVE2 to the polarized membrane and that this recruitment is essential for lamellipodium formation at the leading edge.

Amino Acid Sequence↗
PubMed2004

FBP11 regulates nuclear localization of N-WASP and inhibits N-WASP-dependent microspike formation.

WASP family proteins are involved in cortical actin cytoskeleton reorganization. Neural Wiskott-Aldrich syndrome protein (N-WASP), a ubiquitously expressed WASP homologous protein, directly binds with Cdc42, activating Arp2/3 complex. In this study, we show that N-WASP-dependent microspike formation is inhibited by formin binding protein 11 (FBP11). Endogenous FBP11 localizes with nuclear-speckles, and co-localization of N-WASP and FBP11 was observed when they were co-expressed. Epidermal growth factor (EGF) induced actin-microspike formation in COS7 cells. However, transient expression of FBP11 suppressed N-WASP-dependent actin-microspike formation by trapping N-WASP in the nucleus. These results indicate that FBP11 regulates localization of N-WASP, thus negatively regulating the function of N-WASP in the cytoplasm.

Actin-Related Protein 2↗
PubMed2003

Focal adhesion kinase regulation of N-WASP subcellular localization and function.

N-WASP is a member of the WASP family of proteins that regulate actin cytoskeleton remodeling. FAK is a cytoplasmic tyrosine kinase implicated in integrin signaling during cell migration. Here we identify a direct interaction between N-WASP and FAK and show that N-WASP is phosphorylated by FAK at a conserved tyrosine residue, Tyr(256). We found that phosphorylation of Tyr(256) affected N-WASP nuclear localization, suggesting that phosphorylation of N-WASP by FAK may regulate its activity in vivo by altering its subcellular localization. We also showed that the nuclear localization of N-WASP is dependent on its being in the open conformation either after its activation by Cdc42 or the truncation of the C-terminal VCA domain. Phosphorylation of Tyr(256) of N-WASP could reduce its interaction with nuclear importin NPI-1, which might be responsible for its decreased nuclear localization. Lastly, we show that phosphorylation of Tyr(256) plays an important role in promoting cell migration. Together, these results suggest a novel regulatory mechanism of N-WASP by tyrosine phosphorylation and subcellular localization and its potential role in the regulation of cell migration.

Animals↗
PubMed2003

WAVE2 is required for directed cell migration and cardiovascular development.

WAVE2, a protein related to Wiskott-Aldrich syndrome protein, is crucial for Rac-induced membrane ruffling, which is important in cell motility. Cell movement is essential for morphogenesis, but it is unclear how cell movement is regulated or related to morphogenesis. Here we show the physiological functions of WAVE2 by disruption of the WAVE2 gene in mice. WAVE2 was expressed predominantly in vascular endothelial cells during embryogenesis. WAVE2-/- embryos showed haemorrhages and died at about embryonic day 10. Deficiency in WAVE2 had no significant effect on vasculogenesis, but it decreased sprouting and branching of endothelial cells from existing vessels during angiogenesis. In WAVE2-/- endothelial cells, cell polarity formed in response to vascular endothelial growth factor, but the formation of lamellipodia at leading edges and capillaries was severely impaired. These findings indicate that WAVE2-regulated actin reorganization might be required for proper cell movement and that a lack of functional WAVE2 impairs angiogenesis in vivo.

Animals↗
PubMed2003

Translocation of N-WASP by nuclear localization and export signals into the nucleus modulates expression of HSP90.

N-WASP regulates the actin cytoskeleton through activation of the Arp2/3 complex. N-WASP localizes at the cell periphery, where it controls actin polymerization downstream of signal molecules such as adapter proteins, Cdc42, Src family kinases, and phosphoinositides. N-WASP also localizes in the nucleus; however, the role of N-WASP in the nucleus is unclear. Here, we show that localization of N-WASP is controlled through phosphorylation by Src family kinases in which phosphorylated N-WASP is exported from the nucleus in a nuclear export signal (NES) and leptomycin B-dependent manner. N-WASP had nuclear localization signal (NLS) at its basic region and NES close to the phosphorylation site by Src family kinases, indicating that phosphorylation controls the accessibility to the NES through conformational changes. Increased levels of unphosphorylated N-WASP in the nucleus suppressed expression of HSP90 and transcription from a heat shock element (HSE). N-WASP bound heat shock transcription factor (HSTF) and enhanced the HSTF association with HSE. In addition, nuclear N-WASP was present in the protein complex that associates with HSE, suggesting that N-WASP participates in suppression of HSP90 transcription. Increased levels of unphosphorylated N-WASP also decreased the activities of Src family kinases in cells but not in experiments in vitro with pure N-WASP and Fyn. Because HSP90 is essential for the activities of Src family kinases, these results suggest that localization of N-WASP modulates Src kinase activity by regulating HSP90 expression.

Active Transport, Cell Nucleus↗
PubMed2003

Essential role of the C. elegans Arp2/3 complex in cell migration during ventral enclosure.

Migration of cells through the reorganization of the actin cytoskeleton is essential for morphogenesis of multicellular animals. In a cell culture system, the actin-related protein (Arp) 2/3 complex functions as a nucleation core for actin polymerization when activated by the members of the WASP (Wiskott-Aldrich syndrome protein) family. However, the regulation of cell motility in vivo remains poorly understood. Here we report that homologues of the mammalian Arp2/3 complex and N-WASP in Caenorhabditis elegans play an important role in hypodermal cell migration during morphogenesis, a process known as ventral enclosure. In the absence of one of any of the C. elegans Arp2/3 complex subunits (ARX-1, ARX-2, ARX-4, ARX-5, ARX-6 or ARX-7) or of N-WASP (WSP-1), hypodermal cell migration led by actin-rich filopodia formation is inhibited during ventral enclosure owing to the reduction of filamentous actin formation. However, there is no effect on differentiation of hypodermal cells and dorsal intercalation. Disruption of the function of ARX-1 and WSP-1 in hypodermal cells also resulted in hypodermal cell arrest during ventral enclosure, suggesting that their function is cell autonomous. WSP-1 protein activated Arp2/3-mediated actin polymerization in vitro. Consistent with these results, the Arp2/3 complex and WSP-1 colocalized at the leading edge of migrating hypodermal cells. The stable localization of WSP-1 was dependent on the presence of Arp2/3 complex, suggesting an interaction between the Arp2/3 complex and WSP-1 in vivo.

Actin-Related Protein 2↗
PubMed2003

Regulation of cortical actin networks in cell migration.

The actin cytoskeleton is a primary determinant of cell shape and motility. Studies on actin regulatory proteins are now coupled with studies of the signal transduction that directs actin cytoskeleton reorganization, and we have gained insights into how external stimuli such as chemoattractants drive changes in actin cytoskeleton. Chemoattractants regulate actin regulatory proteins such as the Arp2/3 complex through WASP family proteins, ADF/cofilin downstream of LIM-kinase, and various other phosphoinositide-dependent or -independent pathways. Through branching of actin filaments, Arp2/3 complex-dependent actin polymerization is suffcient to generate the force necessary for protrusion.

Actin Depolymerizing Factors↗
PubMed2003

Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration.

Cell migration is driven by actin polymerization at the leading edge of lamellipodia, where WASP family verprolin-homologous proteins (WAVEs) activate Arp2/3 complex. When fibroblasts are stimulated with PDGF, formation of peripheral ruffles precedes that of dorsal ruffles in lamellipodia. Here, we show that WAVE2 deficiency impairs peripheral ruffle formation and WAVE1 deficiency impairs dorsal ruffle formation. During directed cell migration in the absence of extracellular matrix (ECM), cells migrate with peripheral ruffles at the leading edge and WAVE2, but not WAVE1, is essential. In contrast, both WAVE1 and WAVE2 are essential for invading migration into ECM, suggesting that the leading edge in ECM has characteristics of both ruffles. WAVE1 is colocalized with ECM-degrading enzyme MMP-2 in dorsal ruffles, and WAVE1-, but not WAVE2-, dependent migration requires MMP activity. Thus, WAVE2 is essential for leading edge extension for directed migration in general and WAVE1 is essential in MMP-dependent migration in ECM.

Actin Cytoskeleton↗
PubMed2002

Small GTPase Rah/Rab34 is associated with membrane ruffles and macropinosomes and promotes macropinosome formation.

Macropinocytosis is an efficient process for the uptake of nutrients and solute macromolecules into cells from the external environment. Macropinosomes, which are surrounded by actin, are formed from the cell surface membrane ruffles and migrate toward the cell center. We have cloned the entire coding sequence of a member of the Rab family small GTPases, Rah/Rab34. It lacked a consensus sequence for GTP-binding/GTPase domain. Although wild-type Rah exhibited extremely low GTPase activity in vitro, it exerted appreciable GTPase activity in vivo. In fibroblasts, Rah was colocalized with actin to the membrane ruffles and membranes of relatively large vesicles adjacent to the ruffles. These vesicles were identified as macropinosomes on the basis of several criteria. Rah and Rab5 coexisted in some, but not all, macropinosomes. Rah was predominantly associated with nascent macropinosomes, whereas Rab5 was present in endosomes at later stages. The number of macropinosomes in the cells overexpressing Rah increased about 2-fold. The formation of macropinosomes by the treatment of platelet-derived growth factor or phorbol ester was also facilitated by Rah but suppressed by a dominant-negative Rah. Rah-promoted macropinosome formation was retarded by dominant-negative mutants of Rac1 and WAVE2, which are essential for membrane ruffling. These results imply that Rah is required for efficient macropinosome formation from the membrane ruffles.

Amino Acid Sequence↗
PubMed2002

Visualization and force measurement of branching by Arp2/3 complex and N-WASP in actin filament.

To determine whether the Arp2/3 complex activated by N-WASP (VCA) branches actin filaments at the side (side branching), or at the barbed (B-)end (end branching) of the mother filaments, we have directly observed the branching process of actin filaments and examined single-molecule unbinding under optical microscope. We found that side branching was predominant, though not exclusive. At the initial stage of polymerization, the branching at the B-end occurred and subsequently the side branching started to occur. In either type of branching, the mother and daughter filaments elongated at nearly the same rate (growing type). Independently of the stage of polymerization, branching due to the direct coupling of filaments with an acute angle to the mother filaments (a coupling type) occurred. Phalloidin suppressed the growing type of branching but not the coupling type, implying that actin monomers are required for the former but not the latter. We found, by single molecule measurements using optical tweezers, that the Arp2/3 complex attaches to the side of actin filaments and the N-WASP appears to detach from the actin-Arp2/3 complex at 6-7 pN.

Actin-Related Protein 2↗
PubMed2002

Sustained activation of N-WASP through phosphorylation is essential for neurite extension.

Neurite extension is a key process for constructing neuronal circuits during development and remodeling of the nervous system. Here we show that Src family tyrosine kinases and proteasome degradation signals synergistically regulate N-WASP in neurite extension. Src family kinases activate N-WASP through tyrosine phosphorylation, which induces Arp2/3 complex-mediated actin polymerization. Tyrosine phosphorylation of N-WASP also initiates its degradation through ubiquitination. When neurite growth is stimulated in culture, degradation of N-WASP is markedly inhibited, leading to accumulation of the phosphorylated N-WASP. On the other hand, under culture conditions that inhibit neurite extension, but favor proliferation, the phosphorylated N-WASP is degraded rapidly. Collectively, neurite extension is regulated by the balance of N-WASP phosphorylation (activation) and degradation (inactivation), which are induced by tyrosine phosphorylation.

Acetylcysteine↗
PubMed2002

Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading.

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by forming an actin comet tail. The VirG (IcsA) pro-tein expressed at one pole of the bacterium recruits neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP family, which in turn stimulates actin-related protein (Arp) 2/3 complex-mediated actin polymerization. As all the WASP family proteins induce actin polymerization by recruiting Arp2/3 complex, we investigated their involvement in Shigella motility. Here, we show that VirG binds to N-WASP but not to the other WASP family proteins. Using a series of chimeras obtained by swapping N-WASP and WASP domains, we demonstrated that the specificity of VirG to interact with N-WASP lies in the N-terminal region containing the pleckstrin homology (PH) domain and calmodulin-binding IQ motif of N-WASP. A conformational change in N-WASP was important for the VirG-N-WASP interaction, as elimination of the C-terminal acidic region, which is responsible for the intramolecular interaction with the central basic region of N-WASP, affected the specific binding to VirG. We observed that, in haematopoietic cells such as macrophages, polymorphonuclear leucocytes (PMNs) and platelets, WASP was predominantly expressed, whereas the expression of N-WASP was greatly suppressed. Indeed, unlike Listeria, Shigella was unable to move in macrophages at all, although the movement was restored as N-WASP was expressed ectopically. Thus, our findings demonstrate that N-WASP is a specific ligand of VirG, which determines the host cell type allowing actin-based spreading of Shigella.

Actins↗