WIP regulates N-WASP-mediated actin polymerization and filopodium formation.

Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.

Rho family GTPase Cdc42 is essential for the actin-based motility of Shigella in mammalian cells.

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP-depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP but not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP-actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein-tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG-N-WASP-Arp2/3 complex formed on intracellular Shigella.

A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein, WISH, induces Arp2/3 complex activation independent of Cdc42.

We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.

Profilin enhances Cdc42-induced nucleation of actin polymerization.

We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-l-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-l-proline. Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex.

Mitogenesis in response to PDGF and bombesin abolished by microinjection of antibody to PIP2.

The turnover of phosphatidylinositol 4,5-bisphosphate (PIP2) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including mitogens, but decisive evidence for the idea has not been obtained. In the present study, a monoclonal antibody to PIP2 was microinjected into the cytoplasm of NIH 3T3 cells before or after exposure to mitogens. The antibody completely abolished nuclear labeling with [3H]thymidine induced by platelet-derived growth factor and bombesin, but not by fibroblast growth factor, epidermal growth factor, insulin, or serum. The findings strongly suggest that PIP2 breakdown is crucial in the elicitation and sustaining of cell proliferation induced by some types of mitogens such as platelet-derived growth factor and bombesin.

Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis.

Endocytic proteins such as epsin, AP180, and Hip1R (Sla2p) share a conserved modular region termed the epsin NH2-terminal homology (ENTH) domain, which plays a crucial role in clathrin-mediated endocytosis through an unknown target. Here, we demonstrate a strong affinity of the ENTH domain for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. With nuclear magnetic resonance analysis of the epsin ENTH domain, we determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin Lys76 --> Ala76, with an ENTH domain defective in phosphoinositide binding, blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and PtdIns(4,5)P2 is essential for endocytosis mediated by clathrin-coated pits.

Requirement of phospholipase Cdelta4 for the zona pellucida-induced acrosome reaction.

Several phospholipase C (PLC) isoforms have been found in male and female mammalian gametes, and splicing isoforms of PLCdelta4 are predominantly expressed in testis. Here we report that male mice in which the PLCdelta4 gene had been disrupted either produced few small litters or were sterile. In vitro fertilization studies showed that insemination with PLCdelta4-/- sperm resulted in significantly fewer eggs becoming activated and that the calcium transients associated with fertilization were absent or delayed. PLCdelta4-/- sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida. These data demonstrate that PLCdelta4 functions in the acrosome reaction that is induced by the zona pellucida during mammalian fertilization.

Rb plays a role in survival of Abl-dependent human tumor cells as a downstream effector of Abl tyrosine kinase.

The retinoblastoma (Rb) gene product is a tumor suppressor that is mutated or inactivated in many types of human cancers. Although Rb is known to be an upstream negative regulator of Abl protein tyrosine kinase, we propose here that Rb also functions as a downstream effector of Abl that plays a positive role in survival of Abl-dependent human tumor cells, including Bcr/Abl-positive chronic myelogenous leukemia (CML). We show that Rb is constitutively phosphorylated at tyrosine in Abl-dependent tumor cells, and that Abl phosphorylates Rb specifically at Y805 within the C-terminal domain of the molecule. We also show that ectopic expression of Rb induces apoptosis in Abl-dependent tumor cells by inhibiting the Abl tyrosine kinase activity, and that Rb-induced apoptosis is compromised by Abl-catalysed phosphorylation of Rb at Y805. Furthermore, the silencing of endogenous Rb by RNA interference induced apoptosis in Abl-dependent tumor cells. Thus, our findings suggest that Abl-catalysed tyrosine phosphorylation of Rb is necessary for survival of Abl-dependent human tumor cells, and raises the possibility that this phosphorylated Rb can be a molecular target for cancer therapy aimed at inducing apoptosis of Abl-dependent tumor cells, such as Bcr/Abl-positive CML.

Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments.

Phosphatidylinositol 4,5-bisphosphate (PIP2) reorganizes actin filaments by modulating the functions of a variety of actin-regulatory proteins. Until now, it was thought that bound PIP2 is hydrolyzed only by tyrosine-phosphorylated phospholipase Cgamma (PLCgamma) after the activation of tyrosine kinases. Here, we show a new mechanism for the hydrolysis of bound PIP2 and the regulation of actin filaments by PIP2 phosphatase (synaptojanin). We isolated a 150-kDa protein (p150) from brains that binds the SH3 domains of Ash/Grb2. The sequence of this protein was found to be homologous to that of synaptojanin. The expression of p150 in COS 7 cells produces a decrease in the number of actin stress fibers in the center of the cells and causes the cells to become multinuclear. On the other hand, the expression of a PIP2 phosphatase-negative mutant does not disrupt actin stress fibers or produce the multinuclear phenotype. We have also shown that p150 forms the complexes with Ash/Grb2 and epidermal growth factor (EGF) receptors only when the cells are treated with EGF and that it reorganizes actin filaments in an EGF-dependent manner. Moreover, the PIP2 phosphatase activity of native p150 purified from bovine brains is not inhibited by profilin, cofilin, or alpha-actinin, although PLCdelta1 activity is markedly inhibited by these proteins. Furthermore, p150 suppresses actin gelation, which is induced by smooth muscle alpha-actinin. All these data suggest that p150 (synaptojanin) hydrolyzes PIP2 bound to actin regulatory proteins, resulting in the rearrangement of actin filaments downstream of tyrosine kinase and Ash/Grb2.

Role of phosphatidylinositol 4,5-bisphosphate in Ras/Rac-induced disruption of the cortactin-actomyosin II complex and malignant transformation.

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac-phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, we propose that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP2-binding molecules.

Different interactions of Grb2/Ash molecule with the NGF and EGF receptors in rat pheochromocytoma PC12 cells.

We have previously shown that nerve growth factor (NGF) induces a rapid and relatively continuous activation of Ras in rat pheochromocytoma PC12 cells while epidermal growth factor (EGF) activates Ras transiently, and that tyrosine kinase activity of the NGF receptor is essential for the activation of Ras (Muroya et al., Oncogene, 7, 277-281, 1992). In order to explore the signaling mechanism from tyrosine kinase to Ras activation in more detail, interactions between two adaptor molecules, Shc and Grb2/Ash, which contain Src homology regions, and their interactions with the NGF and EGF receptors were examined. Both NGF and EGF induced rapid tyrosine phosphorylation of Shc and its association with both the receptors and with Grb2/Ash. When cells were stimulated with EGF at 4 degrees C, the activation of Ras proceeded slowly and MAP kinase activation was quite low. Under such restricted conditions, tyrosine-phosphorylated Shc formed a complex with Grb2/Ash, suggesting that the complex formation may be one of the immediate early responses. In contrast to Shc, Grb2/Ash bound to EGF receptor but did not form a stable complex with the NGF receptor. These results suggest that there may be an alternative pathway for the activation of Ras in PC12 cells.

Meltrin alpha cytoplasmic domain interacts with SH3 domains of Src and Grb2 and is phosphorylated by v-Src.

Meltrin alpha/ADAM12 is a member of the ADAM/MDC family proteins characterized by the presence of metalloprotease and disintegrin domains. This protein also contains a single transmembrane domain and a relatively long cytoplasmic domain containing several proline-rich sequences. These sequences are compatible with the consensus sequences for binding the Src homology 3 (SH3) domains. To determine whether the proline-rich sequences interact with SH3 domains in several proteins, binding of recombinant SH3 domains to the meltrin alpha cytoplasmic domain was analysed by pull-down assays. The SH3 domains of Src and Yes bound strongly, but that of Abl or phosphatidylinositol 3-kinase p85 subunit did not. Full-length Grb2/Ash bound strongly, whereas its N-terminal SH3 domain alone did less strongly. Src and Grb2 in bovine brain extracts also bound to meltrin alpha cytoplasmic domain on affinity resin. Furthermore, immunoprecipitation with a monoclonal antibody to meltrin alpha resulted in coprecipitation of Src and Grb2 with meltrin alpha in cell extracts, suggesting that Src and Grb2 are associated in vivo with meltrin alpha cytoplasmic domain. This notion was also supported by the findings that exogenously expressed meltrin cytoplasmic domain coexisted with Src and Grb2 on the membrane ruffles. The C-terminal Tyr901 of meltrin alpha was phosphorylated both in vitro and in cultured cells by v-Src. These results may imply that meltrin alpha cytoplasmic domain is involved in a signal transduction for some biological function through the interaction with SH3-containing proteins.

Phosphoinositides, key molecules for regulation of actin cytoskeletal organization and membrane traffic from the plasma membrane.

Phosphoinositide plays a critical role not only in generating second messengers, such as inositol 1,4,5-trisphosphate and diacylglycerol, but also in modulating a variety of cellular functions including cytoskeletal organization and membrane trafficking. Many inositol lipid kinases and phosphatases appear to regulate the concentration of a variety of phosphoinositides in a specific area, thereby inducing spatial and temporal changes in their availability. For example, local concentration changes in phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in response to extracellular stimuli cause the reorganization of actin filaments and a change in cell shape. PI(4,5)P(2) uncaps the barbed end of actin filaments and increases actin nucleation by modulating a variety of actin regulatory proteins, leading to de novo actin polymerization. PI(4,5)P(2) also plays a key role in membrane trafficking processes. In endocytosis, PI(4,5)P(2) targets clathrin-associated proteins to endocytic vesicles, leading to clathrin-coated pit formation. On the contrary, PI(4,5)P(2) must be dephosphorylated when they shed clathrin coats to fuse endosome. Thus, through regulating actin cytoskeleton organization and membrane trafficking, phosphoinositides play crucial roles in a variety of cell functions such as growth, polarity, movement, and pattern formation.

Downregulated expression of the signaling molecules Nck, c-Crk, Grb2/Ash, PI 3-kinase p110 alpha and WRN during fibroblast aging in vitro.

An RT-PCR analysis was performed to examine changes in intracellular signal transducing molecules during in-vitro aging of human fibroblasts. Expression of Nck, c-Crk, Grb2/Ash, phosphoinositide (PI) 3-kinase p110 alpha and Werner's syndrome gene product WRN was noticeably reduced in late passage cells, showing a concurrent downregulation of a set of signaling molecules accompanying aging.

A defect of the myo-inositol maintenance mechanism in the lens of hereditary cataract mice.

The myo-inositol uptake system was studied in lenses of normal and hereditary cataract mouse. The normal mouse was able to accumulate myo-inositol continuously from medium and keep it in a high concentration. The specific myo-inositol uptake was dependent on temperature and it decreased in Ca(2+)-free medium. In contrast, specific uptake of myo-inositol reached a plateau after 15 min in the cataract mouse lens although initial incorporation was more rapid than that in normal mouse lens. This uptake system was not affected by temperature or Ca(2+) in the medium. The rate of myo-inositol efflux into the medium was more rapid in the cataract lens than that of the normal lens. It was shown that the low level of myo-inositol in the lens of hereditary cataract mouse was due to the defect of myo-inositol transport system and the enhanced efflux rate. These results suggest a dysfunction of the lens membrane.

myo-Inositol binding and transport in brush border membranes of rat kidney.

Using hypotonically treated brush border membranes, binding and transport of myo-inositol were examined. By hypotonic treatment, both total and non-specific uptake decreased significantly, but specific uptake was not affected. myo-Inositol release from membranes preloaded by incubation for 2 min was very rapid and about 98% of preloaded myo-inositol was released in 5 min of incubation. However, myo-inositol release from membranes preloaded by incubation for 20 min was fairly slow and 50% of myo-inositol remained in the membranes even after 10 min of incubation. Uptake of myo-inositol decreased by the increase of osmolarity in the medium. However, effect of osmolarity on the uptake was less significant when myo-inositol concentration was lower. Under conditions in which mainly binding occurred, myo-inositol binding to the membranes was measured. Two binding systems were demonstrated and high affinity site could bind 22 pmol/mg protein at most and the apparent Km value was 8.3 muM. Both binding and transport processes were dependent on Na+ and enhanced by Na+-gradient.

An inhibitor of inositol-phospholipid-specific phospholipase C.

Q12713 substance, a cyclic peptide from Actinomadura spp., strongly inhibited the enzyme activity of phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) but scarcely inhibited other phospholipases. Kinetic analysis demonstrated that the inhibition of PIP2-PLC activity was competitive with respect to phosphatidylinositol 4,5-bisphosphate. Calcium and magnesium ions had no significant effect on the inhibitory activity. On the contrary, potassium or rubidium ion was essential for the inhibitory activity. Furthermore, NaF and AlCl3-stimulated increase of phosphoinositides was decreased by Q12713 in the cultured 3T3 cells.

Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane.

The distribution of phosphoinositides and phosphatidic acid (PA) between the outer and inner layers of the human erythrocyte membrane was investigated by using two complementary methodologies: hydrolysis by phospholipase A2 (PLA2) and immunofluorescence detection with monoclonal antibodies against polyphosphoinositides. The contents of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and PA were decreased by 15-20% after 60 min incubation with PLA2, while that of phosphatidylinositol (PI) was increased. Studies with 32P-labelled cells revealed that PLA2 treatment led to indirect effects on the metabolism of these phospholipids. Therefore, the asymmetric distribution of phosphoinositides and PA was inferred from the data obtained in ATP-depleted erythrocytes. In these cells with arrested phosphoinositide metabolism, the asymmetric distribution of the major phospholipids was maintained: PLA2 hydrolyzed approx. 20% of PI, PIP2 and PA (but no PIP) indicating their localization in the outer layer of the membrane. This finding was confirmed by immunofluorescence studies with antibodies specific to each phosphoinositide. External addition of anti-PIP2 but not anti-PIP gave a positive reaction both in control and in ATP-depleted erythrocytes. A pretreatment of cells with PLA2 led to a decrease in the intensity of anti-PIP2 staining. These results demonstrate that significant fractions of PIP2, PI and PA are localized on the outer surface of the erythrocyte membrane.

Regulation of endocytosis by phosphatidylinositol 4,5-bisphosphate and ENTH proteins.

Clathrin-mediated endocytosis starts by a recruitment of endocytic proteins to the plasma membrane to induce invagination of lipid bilayer and subsequent vesicule formation. The recruitment of these components requires PtdIns(4,5)P2, a phosphoinositide on the plasma membrane. Although it is well known that the synthesis as well as the disruption of this lipid is important, recent studies have revealed the indispensable roles of direct interaction between PtdIns(4,5)P2 and the endocytic machinery. The ENTH domain is a newly found PtdIns(4,5)P2 binding unit conserved among endocytic proteins like epsins, AP180, and the Hip1/Sla2 family. This review focuses on the essential roles of PtdIns(4,5)P2 and its specific binding partner, the ENTH domain, in clathrin-mediated endocytosis.

Cellular distribution of polyphosphoinositides in rat hepatocytes.

The distribution of total phospholipids, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was studied in isolated rat hepatocytes: (i) by mass assay and isotopic labelling in the fractions of plasma membranes, microsomes, mitochondria and nuclei prepared from isolated hepatocytes and (ii) by immunolocalization of PIP2 with a specific antibody (kt3g) in whole hepatocytes and isolated nuclei. Mass measurement and isotopic labelling showed that PIP was distributed in all four fractions. PIP2 was present in the plasma membrane and the nuclei. In whole cells, PIP2 was also detected in the plasma membrane by immunolocalization with the anti-PIP2 antibody kt3g. In unpolarized single hepatocytes, PIP2 distributed evenly throughout the plasma membrane. However, in polarized cell couplets, PIP2 was the most often undetectable in the lateral domain between the cells, and distributed preferentially in the sinusoidal domain of the plasma membrane. These results suggest that hepatocytes segregate PIP2 in particular domains of their plasma membrane. In purified fractions of nuclei, immunolocalization experiments showed that PIP2 was present uniquely in the nuclear envelope.