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Mulholland Drive [Loseless-FLAC] OST [2001] - David Lynch

Tue, 21 Jul 2009 22:10:50

Mulholland Drive (2001) [Special Edition] DVDrip [Naomi Watts]

Thu, 04 Jun 2009 17:11:27

Mulholland_Drive_(2001).720P.HDVDRip.X264.TLF.ShareReactor

Thu, 07 May 2009 18:14:07

A critically acclaimed masterpiece from the mind of David Lynch, creator of 'Twin Peaks' and 'Inland Empire'. Los Angeles, city of angels. Amnesiac and wounded, a mysterious femme fatale wanders on the sinuous road of Mulholland Drive. She finds a shelter at Betty's house (Naomi Watts 'King Kong'). an aspiring actress just arrived from her hometown and in search of stardom in Hollywood. First of all intrigued by the stranger who calls herself Rita (Laura Elena Harring), Betty discovers that her handbag is dull of dollar bundles. The two women get to know each other better and decide to investigate in order to discover Rita's true identity.... This slick, sinister, psycho-sexual thriller comes from the darkest recesses of Hollywood, a Los Angeles of strange dreams, unrequited love and jealous burning rage. FRAME RATE.....23.976fps VIDEO CODEC.....X264 1093 4878kbps AUDIO CODEC.....DTS 1536kbps ASPECT RATIO.....1.86:1 RESOLUTION.....1280X 688

Mulholland Drive [TRACKERSURFER french]

Wed, 29 Apr 2009 23:11:40

Mulholland.Drive.2001.Swesub.DVDrip.Royskatt

Sat, 25 Apr 2009 05:12:07

Genre:Thriller/Drama Audio: Engelska/English Subs: Svenska/Swedish IMDB: 8,0 http://www.imdb.com/title/tt0166924/ Oscarnominerad (beste regi) thriller-drama fr??n 2001 av David Lynch med Justin Theroux och Naomi Watts. En kvinna och tv?? m??n f??rdas i en limousine i Los Angeles. I motsatt riktning k??r tv?? bilar i full fart mot limousinen. I den frontalkrock som f??ljer d??das alla utom just kvinnan. F??r henne inneb??r olyckan att hon r??ddas fr??n att skjutas av m??nnen i limousinen. Hon drabbas av minnesf??rlust och f??rvirrad snubblar hon nerf??r v??gen och tar skydd i en l??genhet. Till samma l??genhet anl??nder Betty, en ung kvinna som kommit f??r att uppfylla sina sk??despelardr??mmar i den mest glamor??sa av st??der. Hon m??ts av Rita, som den f??rvirrade kvinnan kallar sig och tillsammans s??ker de, den ena med en ??ventyrlig nyfikenhet och den andra med en instinktiv r??dsla, efter sanningen om Ritas identitet. Samtidigt f??r den unge och beg??vade regiss??ren Adam Kesher problem efter flera m??rkliga och hotfulla m??ten med den ena udda karakt??ren efter den andra. Alla vill de att han ger en roll ??t den unga och oerfarna sk??despelerskan Camilla Rhodes. Seeding is sharing Sharing is caring Do it for free Without any fee Uppladdaren blir glad om du seeder efter nerladdingen! Du var inte redd for att ladde ner - var heller inte redd for att seede s?? andre ochs?? f??r del av denne filmen! ENJOY *********** ********* ******* ******* ******** ** ** *********** *********** ** ** ** ** ** ** ** *** ** *********** *** ** ** ** ** ** ** ** ** * ** *** *** ** ** ******* ******* ***** ** * ** *** *** ** ** ** ** ** ** ***** ** * ** *** *** ** ** ** ** ** ** ** ** * ** *** *** ********* ** *** ** *** ******** ** *** *** ********* *********** ********** ******** *********** ** *********** ** ** ** ** *********** ** *** ** ** ** ** *** ********* *** ********** ******** *** ********* *** ** ** ** ** *** ** *** ** ** ** ** *** ** *** ** ** ** ** *** ********* *** *** *** ** *** *** .nu DEN B??STA TORRENTSIDAN P?? INTERNET. BES??KF??R MER FILMER

Mulholland Drive 2001 Swesub DVDrip Royskatt

Sat, 25 Apr 2009 05:11:53

Mulholland Drive.avi (2001) w/ Naomi Watts & Laura Harring

Mon, 13 Apr 2009 15:11:58

Mulholland.Drive.2001.720P.HDVDRip.X264-TLF

Wed, 25 Mar 2009 00:12:57

_____________________________________________________________________________ ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??? ????????? ????????? ????????? ?????? ????????? ????????? ????????? remozz 2009 ??? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? _____________________________________________________________________________ Release iNFO HDSOURCE.....1080p ts DIVX RELEASE.....02.08.09 SUPPLIER.....TEAM TLF RIPPER.....VawaV IMDB RATE.....8.0 FRAME RATE.....23.976fps VIDEO CODEC.....X264 1093 4878kbps AUDIO CODEC.....DTS 1536kbps ASPECT RATIO.....1.86:1 RESOLUTION.....1280X 688 RUNTIME.....147MiN GENERE..... Drama | Fantasy | Mystery | Thriller LANGUAGE..... English SUBSTITLE.....N/A iMDB LiNK: http://www.imdb.com/title/tt0166924/

Mulholland Drive (David Lynch) - Movie Script

Sun, 22 Mar 2009 14:16:39

Good screenplay. Here's the science bit: Identification of the 200-kDa protein as a nonmuscle myosin IIA heavy chain by microsequencing. The SDS-PAGE-purified 200-kDa protein (1.5 ??g) immunoprecipitated with an EMS1-actin complex from the NIH 3T3 cells (see Fig. 1A) was digested by trypsin (29), and the resulting peptides were separated by high-performance liquid chromatography (28) and identified as the fragments of murine nonmuscle myosin IIA heavy chain by the microsequencing procedures previously described (2, 5, 28, 29) and comparison with the amino acid sequences of the rat and human nonmuscle myosin IIA heavy chains that were determined previously (3, 36, 40, 44) (Table 1). View this table: [in this window] [in a new window] TABLE 1. The 200-kDa protein immunoprecipitated with an EMS1-F-actin complex is murine myosin IIA heavy chaina Preparation of muscle actin and HS1 or EMS1 fragments. Actin was purified from acetone powder of rabbit skeletal muscle (Sigma Chemicals) as described previously (41). Full-length human HS1 and the actin-binding domain (residues 82 to 192) were produced in bacteria as thrombin-cleavable glutathione S-transferase (GST) fusion proteins by subcloning the corresponding PCR DNAs (EcoRI fragments) into the vector pGEX-2TH. Full-length human EMS1-GST fusion protein (17) and other GST fusion proteins were affinity purified by glutathione beads (24). F-actin cross-linking assay. F-actin cross-linking activity of EMS1 or HS1 was measured by the superprecipitation procedure (17) with a slight modification in the presence or absence of PIP2. Purified GST-fusion proteins of EMS1 or HS1 were preincubated with PIP2 on ice for 30 min. F-actin was then added into the above-mentioned mixtures and incubated for another 30 min on ice. The mixture was then centrifuged at 15,000 ?? g for 10 min at 4??C, and the resultant pellets were subjected to SDS-PAGE. The Coomassie blue-stained gels were then subjected to densitometry for quantitation of actin bands. Under these conditions, the control GST alone caused no cross-linking of F-actin. PIP2-binding assay. Purified GST fusion proteins of EMS1 or HS1 were fixed onto a nitrocellulose membrane with a dot blot apparatus. The membrane was then blocked with 10% skim milk and washed (47). The membrane was then incubated with 10 mg of PIP2 per ml in TSBT (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% bovine serum albumin, and 0.02% Tween 20) at 25??C for 30 min. After being washed, the membrane was incubated with the 1:1,000 rabbit antiserum against PIP2 (7) at 25??C for 60 min, washed three times, and immunoblotted with 1:10,000 goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase (Bio-Rad, Richmond, Calif.) at 25??C for 60 min. The membrane was incubated with enhanced chemiluminescence detection solution (Amersham, Little Chalfont, United Kingdom) at 25??C for 1 min and exposed to XAR5 film (Kodak, Rochester, N.Y.) for 2 min. The density of each dot was quantitated by a densitometer. Under these conditions, the control GST alone did not bind PIP2. Construction of plasmids expressing human HS1 (wild type and mutants) in mammalian cells. For expression of wild-type HS1 and its mutants in mammalian cells, EcoRI DNA fragments containing the HindIII site followed by a Kozak consensus sequence (AAGCTTGCCGCCACCATG) at the 5' end and encoding full-length human HS1 (HS1F) or its SH3-negative (HS1-SH3) actin-binding domain-negative mutants (HS1ad) and HS1-EMS1 chimera (HEaH) were prepared by PCR and subcloned into the retroviral vector pMV7 as described previously (24). The orientation of the inserts was determined by HindIII digestion (a second HindIII site is located 100 bp downstream of the EcoRI site in the vector). The resultant plasmids were purified by a Plasmid Maxi Kit Qiagen (Hilden, Germany) according to the manufacturer's instructions and used for transfection. Each plasmid expresses a neomycin G418 resistance selectable marker in addition to HS1 (wild type or mutants). Effect of wild-type HS1 or its mutants on the colony-forming ability of v-Ha-Ras transformants in soft agar. v-Ha-Ras-transformed NIH 3T3 cells were transfected with HS1F or other plasmids as complexes with liposomes (31). A parallel transfection with the vector alone was also carried out as a negative control. The resultant transfectants were cloned in the presence of 400 ??g of G418 per ml (24). The whole-cell lysate of each G418-resistant clone was then subjected to immunoblotting with the rabbit antiserum against a human HS1 peptide (residues 306 to 320) (51) for selecting out the clones that overexpress the wild-type HS1 or its mutants. The colony formation in a soft agar (anchorage-independent growth) of these HS1 transfectants in comparison with that for the clones with the control vector alone was examined by incubating 1,000 cells/plate at 37??C for 18 days under standard culture conditions (24). The colonies were stained with 0.005% crystal violet and counted. Autokinase assay for v-Ha-Ras GTPase. Unlike normal Ras GTPases, v-Ha-Ras GTPase, which uniquely contains Thr at position 59, is autophosphorylated at this position during the hydrolysis of GTP (11). According to the previously described autokinase assay procedures (4), the cell lysate containing 100 ??g of protein prepared from the parental v-Ha-Ras transformants or HS1 transfectants was immunoprecipitated with the anti-Ras antibody Y13-259, and then each immunoprecipitate resuspended in the kinase buffer (4) was subjected to the autokinase assay with 10 nM [-32P]GTP at 37??C for 30 min and to SDS-15% PAGE. The autophosphorylated v-Ha-Ras band was visualized by autoradiography. F-actin binding assay. The assay was performed according to the F-actin cosedimentation procedure (49). Briefly, F-actin (4 ??M) was mixed with the fibroblast lysate containing the native full-length HS1 or HS1-EMS1 chimera. The mixture (100 ??l) was kept on ice for 60 min and then centrifuged at 100,000 ?? g for 30 min at 4??C in a Beckman model TL-100 ultracentrifuge. The resultant supernatants and pellets were subjected to SDS-PAGE, followed by immunoblotting with the antibody against HS1. RESULTS AND DISCUSSION Top Previous Next References Ras-induced disruption of an EMS1-actomyosin complex. Microinjection of oncogenic Ras mutants into normal fibroblasts causes the disruption of actin stress fibers, followed by the induction of membrane ruffles in half an hour (1). To understand how Ras causes such a rapid change in the actomyosin complexes, presumably without any change in the levels of its protein components, we have examined the interaction of EMS1 with F-actin in normal and v-Ha-Ras-transformed NIH 3T3 cells. EMS1 is an F-actin cross-linking SH3 protein with 550 amino acids (17). With an antibody specific for EMS1, proteins associated with EMS1 were immunoprecipitated from the cytosol of both normal and Ras-transformed cells and separated by SDS-PAGE. In normal cells, EMS1 forms a stable complex with F-actin and an additional protein of 200 kDa, whereas in Ras-transformed cells, such a complex fails to coprecipitate with EMS1 (Fig. 1A). Furthermore, overexpression of HS1 in the Ras transformants restores the complex with EMS1 and suppresses Ras-induced malignancy as described later. Under these conditions, either F-actin or the 200-kDa protein is not coprecipitated with protein A beads alone, nonimmune serum, or an antibody specific for HS1 (data not shown), confirming that these two proteins are coprecipitated only when they form a complex with EMS1. Since EMS1 is an F-actin cross-linker, even a few EMS1 molecules per actin filament consisting of an average of 1,000 actin monomers are sufficient for the anti-EMS1 to coprecipitate the F-actin 200-kDa protein complex. Ras has no effect on the expression level of either EMS1 or actin (Fig. 1B), indicating that Ras causes a disassembly of the EMS1-actin filament complex. View larger version (26K): [in this window] [in a new window] FIG. 1. Ras induces the disruption of an EMS1-actomyosin complex. (A) Proteins immunoprecipitated with EMS1. Normal (N), Ras-transformed (R), and HS1-transfected Ras-transformed (H) NIH 3T3 cells stained with Coomassie blue. HC, a protein of 200 kDa comigrating with myosin II heavy chain; AC, actin of 42 kDa. (B) No effect of Ras transformation on the expression levels of myosin II heavy chain (HC), actin (AC), and EMS1 (E). (Top panel), Coomassie-blue stained gel of the lysates from normal (N) and Ras-transformed (R) NIH 3T3 cells. (Bottom panel) Immunoblot showing the level of EMS1 in normal (N) and Ras-transformed (R) NIH 3T3 cells. EMS1 had a size of 85 kDa. Fifty micrograms of protein was loaded in each lane. (C) Effect of ATP. Dissociation of HC from the actin-EMS1 complex in the presence of 5 mM ATP (+). (D) Effect of SCH51344. Both myosin IIA heavy chain (HC) and actin (AC) were immunoprecipitated with EMS1 from Ras-transformed cells treated with SCH51344 (+) but not with cells without () drug treatments. Ig, immunoglobulin. We suspected that the 200-kDa protein is a heavy chain of a nonmuscle myosin II isozyme, because it is one of the major cytoplasmic proteins in the fibroblasts and comigrates with the heavy chain of skeletal muscle myosin (SDS-PAGE). In support of this notion, 5 mM ATP, which dissociates any myosin from actin filament (23), causes a dissociation of the 200-kDa protein from the EMS1-actin complex (Fig. 1C). Finally, the microsequencing of several tryptic peptides derived from the 200-kDa protein confirmed that it is the heavy chain of a nonmuscle myosin II type A (Table 1). The cytoplasmic myosin II level is not affected by Ras transformation (Fig. 1B). This notion was further confirmed by immunoblotting with an antibody specific for myosin IIA heavy chain (data not shown). Thus, it is clear that Ras transformation is associated with the disruption of an EMS1-actomyosin II complex, and the reversion of Ras transformation by HS1 overexpression restores the ability of EMS1 to bind actomyosin II. This is the first biochemical demonstration of the disruption by Ras of the specific interaction of actomyosin with ubiquitous cellular proteins such as EMS1. However, so far, no clear-cut change has been detected in the overall intracellular localization pattern of EMS1 upon Ras transformation by fluorescent EMS1 staining (44a). SCH51344, which blocks Rac-induced membrane ruffling suppresses Ras-induced disruption of the EMS1-actomyosin II complex. We have shown previously that the drug SCH51344 blocks both Ras/Rac-induced membrane ruffling and malignant transformation and stimulates stress fiber formation (21, 46). However, this drug has no effect on either Raf-induced activation of MEK/mitogen-activated protein kinase (extracellular signal-regulated protein kinases) or Rac-induced activation of JNK kinase pathways which are not essential for membrane ruffling (21, 46). Interestingly, the treatment of v-Ha-Ras-transformed cells with 50 ??M SCH51344 completely restores the EMS1-actomyosin II complex (Fig. 1D), clearly indicating that Ras requires a component(s) acting downstream of Rac to both disrupt the EMS1-actomyosin II complex and induce membrane ruffling. PIP2 inhibits the F-actin cross-linking activity of either EMS1 or HS1 in vitro. The G protein Rac that is activated by Ras through PI-3 kinase (35) induces overproduction of PIP2 by activating PI-4 and PI-5 kinases (14). Interestingly, as shown in Fig. 2A, we found that both EMS1 and HS1 contain a putative PIP2-binding motif (R/K YG V/I E/D R/K D R/K) in their N-terminal 37-amino-acid repeats (20, 39, 54) as do other F-actin cross-linking proteins, such as alpha-actinin and vinculin (8, 9), and F-actin-severing proteins, such as gelsolin and cofilin (18, 53). In fact, PIP2 clearly inhibits the F-actin cross-linking activity of EMS1 in a dose-dependent manner in vitro (Fig. 2B). While the F-actin cross-linking activity of HS1 requires higher concentrations, the cross-linking is inhibited more effectively by PIP2 (Fig. 2C). As expected, HS1's apparent affinity for PIP2 appears to be at least several fold higher than that of EMS1 (Fig. 3A). EMS1 contains only one putative PIP2-binding motif, whereas HS1 contains three (Fig. 2A). View larger version (36K): [in this window] [in a new window] FIG. 2. PIP2 inhibits F-actin cross-linking activity of both EMS1 and HS1. (A) The putative PIP2-binding motifs (in boxes) within the actin-binding repeats (underlined) of EMS1 and HS1. Asterisks indicate the critical basic residues for PIP2 binding. (B) Inhibition by PIP2 of EMS1-induced F-actin cross-linking. F-actin alone or a mixture of F-actin and EMS1 with or without PIP2 was centrifuged under conditions in which only the cross-linked F-actin was significantly precipitated. PIP2, at 100 ??g/ml, completely abolished the F-actin cross-linking activity of EMS1. The data presented are the averages of three duplicated samples. (C) Inhibition by PIP2 of HS1-induced F-actin cross-linking. Much less PIP2 (25 ??g/ml) was sufficient to inhibit the F-actin cross-linking activity of HS1. Under these conditions, GST alone did not cross-link F-actin. The data are the averages of two duplicated samples. View larger version (30K): [in this window] [in a new window] FIG. 3. (A) HS1 has a much higher affinity for PIP2 than EMS1. PIP2-binding assay was performed with GST fusion proteins of full-length HS1 or EMS1 fixed on a nitrocellulose membrane. Under these conditions, GST alone did not bind PIP2. The data are the averages of three duplicated samples. (B) HS1 reverses the effect of PIP2 that inhibits the F-actin cross-linking activity of EMS1. HS1 (0.1 ??M) that does not cause F-actin cross-linking abolished the inhibition by PIP2 (200 ??g/ml) of EMS1-induced F-actin cross-linking, as the assay was performed under the essentially same conditions as those described in the legend for Fig. 2B. The data presented are the averages of two duplicated samples. HS1 rescues EMS1 from the PIP2 effect that blocks its F-actin cross-linking. Since the F-actin cross-linking activity of HS1 is much weaker than that of EMS1, HS1 (0.1 ??M) does not significantly cross-link F-actin (0.6 ??M), whereas EMS1 at the same concentration is a strong actin cross-linker (Fig. 3B). In the presence of both HS1 and EMS1 (0.1 ??M), PIP2, even at 200 ??g/ml, fails to inhibit F-actin cross-linking by EMS1 (Fig. 3B). These observations clearly indicate that the high-affinity PIP2 binder HS1 is able to abolish completely the action of PIP2 that inhibits the F-actin cross-linking activity of EMS1 at least in vitro. These in vivo and in vitro effects of the PIP2-binding protein HS1 and the Rac blocker SCH51344 on the EMS1-actin interaction support the notion that PIP2 mediates Ras-induced disruption of the EMS1-actomyosin II complex, at least in part, as a factor acting downstream of Rac. HS1 reverses Ras-induced malignant phenotype. Two distinct PIP2-binding or F-actin cross-linking proteins, alpha-actinin and vinculin, suppress SV40-induced malignant transformation of NIH 3T3 cells when they are overexpressed (6, 13). Furthermore, a mutant of gelsolin, the PIP2-binding or F-actin-severing or -capping protein, also suppresses v-Ha-Ras-induced malignancy of the same fibroblasts (30). In addition, we have shown that NF2/Merlin, which is closely related to the Ezrin-Radixin-Moesin (ERM) family of PIP2-binding and F-actin-capping proteins (16, 38), reverses v-Ha-Ras-induced malignancy (43). These previous observations prompted us to examine the anticancer potential of the newly identified PIP2-binding and F-actin cross-linking protein HS1. v-Ha-Ras-transformed fibroblasts were transfected with a DNA encoding full-length human HS1 with a retroviral vector (24). Several neomycin-resistant HS1-overexpressing clones were screened by immunoblot analysis with an antibody specific for the HS1 epitope which is not present in EMS1 (Fig. 4A). Unlike the parental Ras transformants, none of HS1 overexpressors forms any focus at their confluence (data not shown), suggesting that HS1 restores contact inhibition of growth. The colony-forming ability in soft agar (anchorage-independent growth) of these HS1-overexpressing clones is greatly reduced (by 70 to 80%) compared with that of the parental or vector-alone-transfected v-Ha-Ras transformants (Table 2). This reduction was not due to any loss of v-Ha-Ras gene expression, because all HS1 overexpressors show the basically same autophosphorylating activity of v-Ha-Ras as that of the parental cells (Fig. 4B). These observations clearly indicate that overexpression of HS1 suppresses Ras transformation. View larger version (57K): [in this window] [in a new window] FIG. 4. Characterization of HS1 overexpressors derived from v-Ha-Ras-transformed NIH 3T3 cells. (A) Screening of HS1 overexpressors by immunoblot analysis. The supernatants (50 ??l each) of cell lysates prepared from G418-resistant transfectants were subjected to SDS-PAGE and blotted by the antiserum against the human HS1 fragment. L, blotted with 1:1,000 antiserum; lanes 2, 4, 7, 9, and 12 correspond to the full-length HS1 overexpressors no. 2, 4, 7, 11, and 14, respectively. R, blotted with 1:2,000 antiserum; lanes 1, 2, and 5 correspond to the full-length HS1 overexpressors no. 15, 16, and 11. Arrow, HS1 of 75 kDa. (B) Autophosphorylation of v-Ha-Ras GTPase in HS1 revertants. Arrowhead, v-Ha-Ras GTPase of 21 kDa. There was no significant reduction in the v-Ha-Ras GTPase level of Ras-transformed cells transfected with HS1F (full-length HS1). View this table: [in this window] [in a new window] TABLE 2. Full-length HS1 suppresses anchorage-independent growth of v-Ha-RAS transformation Both the SH3 domain and the F-actin- and PIP2-binding motifs of HS1 are required for the anti-Ras action. Full-length HS1 contains several distinct functional domains (Fig. 5): (i) the N-terminal three repeats of 37 amino acids, each of which binds F-actin (25, 26) and contains a putative PIP2-binding motif, (ii) the Pro-rich domain (PLLP) that binds the SH3 domain of the Tyr-kinase Lck (42), (iii) two critical Tyr residues at positions 378 and 397, whose phosphorylation by the kinase Syk is required for the apoptosis of B lymphocytes (52), (iv) a nuclear localization sequence (residues 263 to 274), and (v) the C-terminal SH3 domain, deletion of which causes the nuclear localization of HS1 (15, 26). As summarized in Table 3, we found that deletion of either the SH3 domain or the entire F-actin- and PIP2-binding motifs abolishes the ability of HS1 to suppress Ras-induced malignancy, although both deletion mutants are expressed as much as the full-length HS1 (data not shown). Here we describe the biological properties of only the following two distinct mutants of HS1 lacking all three actin-binding 37-amino-acid repeats of HS1 (ACT) that no longer bind either F-actin or PIP2. View larger version (10K): [in this window] [in a new window] FIG. 5. Schematic structures of HS1 and EMS1. Numbers indicate amino acid residues. Arrowheads indicate only the 37-amino-acid motifs that bind F-actin. P, putative PIP2-binding motif; NLS, nuclear localization sequence; PLLP, Pro-rich domain; Y, critical Tyr residue. View this table: [in this window] [in a new window] TABLE 3. SH3 and actin-binding domains are required for HS1 to suppress Ras-induced malignancy The N-terminal F-actin-binding domain of HS1 (residues 82 to 192) contains three unique tandem repeats, and each repeat comprises 37 amino acids and binds F-actin (25, 26). Although the corresponding domain of EMS1 (residues 83 to 193) also contains three similar tandem repeats of 37 amino acids possessing 60 to 70% sequence identity with the HS1 repeats, this EMS1 domain does not bind F-actin. Instead, only the fourth 37-amino-acid repeat of EMS1 (residue 194 to 230) binds the F-actin (25, 26). EMS1 appears to form a homodimer for cross-linking F-actin (17). Only these three F-actin-binding 37-amino-acid motifs of HS1 and the single F-actin-binding motif of EMS1 contain a putative PIP2-binding motif (Fig. 2A). To examine whether the three F-actin binding motifs of HS1 are involved in the anti-Ras action of HS1, we have generated an HS1-EMS1 chimera in which the whole F-actin-binding domain (residues 82 to 192) of HS1 is replaced by the corresponding domain of EMS1 (residues 83 to 193), which contains the highly homologous three 37-amino-acid motifs but does not bind F-actin. Unlike the wild-type HS1, the HS1-EMS1 chimera overexpressed in v-Ha-Ras-transformants fails to bind F-actin (Fig. 6A). The HS1-EMS1 chimera does not suppress the colony-forming ability of Ras transformants in soft agar (Table 3). Similarly, an internal deletion mutant of HS1 that lacks all three actin-binding motifs (ACT) no longer suppresses Ras transformation (Table 3). These observations clearly indicate that the actin-binding domain of HS1 is also essential for the anti-Ras action. A reverse HS1 mutant alone, consisting of only three actin-binding 37-amino-acid motifs, binds PIP2 (Fig. 6B), strongly supporting the notion that the putative PIP2-binding motifs within these actin-binding repeats are responsible for the PIP2 binding. Thus, it remains to be clarified whether F-actin-binding or PIP2-binding activity (or both) is required for the anti-Ras action of HS1. View larger version (23K): [in this window] [in a new window] View larger version (14K): [in this window] [in a new window] FIG. 6. Characterization of HS1 mutant (HS1ad) lacking the actin-binding domain and HS1-EMS1 chimera (HEaH). (A) F-actin binds the full-length HS1 but not the HS1-EMS1 chimera. v-Ha-Ras-transformed NIH 3T3 transfectants (106 cells) that overexpress the wild-type HS1 (lane 1 to 4) or chimera (lane 5 to 8) were disrupted, and the cleared cell lysate was incubated with (+) or without () F-actin and then centrifuged at 100,000 ?? g. The immunoblot of resultant supernatants (lanes 1, 3, 5, and 7) and pellets (lanes 2, 4, 6, and 8) indicates that the wild-type HS1 coprecipitates with F-actin (lane 4), whereas the chimera does not coprecipitate with F-actin (lane 8). (B) Actin-binding domain of HS1 binds PIP2. A GST fusion protein of actin-binding domain (residues 82 to 192) of HS1 fixed on a nitrocellulose membrane was subjected to a PIP2-binding assay as described in the legend for Fig. 3A. The actin-binding domain alone (HS1A) is sufficient to bind PIP2, as is the full-length HS1 (HS1F). The data presented are the averages of two duplicated samples. Interestingly, like HS1, all other actin-binding tumor suppressors, such as alpha-actinin, vinculin, NF2, tensin, and gelsolin or cofilin mutants, bind PIP2 (26, 27). However, a mutant of cofilin (Gln112 Gln114) that no longer binds F-actin but still binds PIP2 is able to suppress Ras-induced malignancy (26, 27). In addition, we have previously demonstrated that microinjection of the antibody specific for PIP2 into Ras-transformed NIH 3T3 cells inhibits their proliferation and reverts their morphology to the normal flat phenotype, although these effects are both partial and transient (7), suggesting an essential role for PIP2 in Ras transformation. It would be of great interest to examine whether other PIP2-binding proteins, such as alpha-actinin and vinculin, also restore the EMS1-actomyosin II complex in Ras-transformed cells and suppress Ras transformation. To test more directly the notion that the PIP2 binding alone is sufficient for both restoring the EMS1-actomyosin II complex and suppressing the malignancy, we are currently examining the effects of PIP2-binding antibiotics such as neomycin, which inhibits thrombin-induced actin polymerization in vivo (22), presumably by blocking the PIP2-induced uncapping of actin filament plus-ends (14). If some PIP2-binding drugs or peptides prove to have potent anticancer activities, the screening for various other PIP2-binding compounds or PI-4 and PI-5 kinase inhibitors such as ribofuranosyl derivatives of echiguanine analogs (37) could lead to the development of novel anticancer therapeutics useful for the treatment of Ras-associated tumors, which represent more than 30% of all human cancers. ACKNOWLEDGMENTS Top Previous Next References We are grateful to Rob Michalides for his gift of human EMS1 cDNA, Henk van Damme and Vera van Buuren for their studies on intracellular localization of EMS1, Anjali Tikoo for her unpublished information on the anti-Ras action of F-actin cappers and cofilin mutants, and Tony Burgess for his consistent support and encouragement throughout this study. The work has been supported in part by a grant from the Anti-Cancer Council of Victoria. FOOTNOTES * Corresponding author. Mailing address: Ludwig Institute for Cancer Resch., P.O. Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia. Phone: 613-9341-3155. Fax: 613-9341-3104. E-mail: hiroshi.maruta@ludwig.edu.au. REFERENCES Top Previous 1. Bar-Sagi, D., and J. Feramisco. 1986. Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by Ras proteins. Science 233:1061-1065[Abstract/Free Full Text]. 2. Bleasby, A. J., and J. C. Wootton. 1990. Construction of validated, non-redundant composite protein sequences in a protein database. Protein Eng. 3:153-159[Abstract/Free Full Text]. 3. Choi, O. H., C. S. Park, K. Itoh, R. S. Adelstein, and M. Beaven. 1996. Cloning of cDNA encoding rat myosin II-A heavy chain and evidence for the absence of myosin II-B heavy chain in cultured rat mast (RBL-2H3) cells. J. Muscle Res. Cell Motil. 17:69-77[Medline]. 4. D'Abaco, G., R. Whitehead, and A. W. Burgess. 1996. Synergy between APC (Min) and an activated Ras mutation is sufficient to induce colon carcinomas. Mol. Cell. Biol. 16:884-891[Abstract]. 5. Eng, J. K., A. McCormack, and J. Yates. 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Am. Soc. Mass Spectrom. 5:976-989. 6. Fernandez, J. L. R., B. Geiger, D. Salomon, I. Sabanay, M. Zoeller, and A. Ben-Ze'ev. 1992. Suppression of tumorigenicity in SV40-transformed cells after transfection with vinculin cDNA. J. Cell Biol. 119:427-438[Abstract/Free Full Text]. 7. Fukami, K., K. Matsuoka, O. Nakanishi, A. Yamakawa, S. Kawai, and T. Takenawa. 1988. Antibody to PIP2 inhibits oncogene induced mitogenesis. Proc. Natl. Acad. Sci. USA 85:9057-9061[Abstract/Free Full Text]. 8. Fukami, K., K. Furuhashi, M. Inagaki, T. Endo, S. Hatano, and T. Takenawa. 1992. Requirement of PIP2 for alpha-actinin function. Nature 359:150-152[Medline]. 9. Fukami, K., T. Endo, M. Imamura, and T. Tekenawa. 1994. Alpha-actinin and vinculin are PIP2-binding proteins involved in signaling by Tyr kinase. J. Biol. Chem. 269:1518-1522[Abstract/Free Full Text]. 10. Fukui, Y., T. Lynch, H. Brzeska, and E. D. Korn. 1989. Myosin I is located at the leading edges of locomoting Dictyostelium amoeba. Nature 341:328-331[Medline]. 11. Gibbs, J. B., R. Elis, and E. M. Scolnick. 1984. Autophosphorylation of v-Ha-Ras is modulated by amino acid 12. Proc. Natl. Acad. Sci. USA 81:2674-2678[Abstract/Free Full Text]. 12. Gimona, M., J. Kazzaz, and D. Helfman. 1996. Forced expression of tropo -myosin2 and 3 in v-Ki-Ras-transformed fibroblasts results in distinct phenotypic effects. Proc. Natl. Acad. Sci. USA 93:9618-9623[Abstract/Free Full Text]. 13. Glueck, U., D. Kwiatkowski, and A. Ben-Ze'ev. 1993. Suppression of tumorigenicity in SV40-transformed 3T3 cells transfected with alpha-actinin cDNA. Proc. Natl. Acad. Sci. USA 90:383-387[Abstract/Free Full Text]. 14. Hartwig, J., G. M. Bokoch, C. L. Carpenter, P. Janmey, L. Taylor, A. Toker, and T. Stossel. 1995. 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Tropomyosin-2 cDNA lacking the 3' untranslated region riboregulator induces growth inhibition of v-Ki-Ras-transformed fibroblasts. Mol. Biol. Cell 8:897-908[Abstract]. 20. Kitamura, D., H. Kaneko, Y. Miyazgoe, T. Ariyasu, and T. Watanabe. 1989. Isolation and characterization of a novel human gene expressed specially in the cells of hematopoietic lineage. Nucleic Acids Res. 17:9367-9379. 21. Kumar, C. C., C. Prorock-Rogers, J. Kelley, Z. Dong, J. J. Lin, L. Armstrong, H. F. Kung, M. J. Weber, and A. Afonso. 1995. SCH51344 inhibits Ras transformation by a novel mechanism. Cancer Res. 55:5106-5117[Abstract/Free Full Text]. 22. Lassing, I., and U. Lindberg. 1988. Evidence that the phosphatidylinositol cycle is linked to cell motility. Exp. Cell Res. 174:1-15[Medline]. 23. Maruta, H., and E. D. Korn. 1977. Acanthamoeba myosin II. J. Biol. Chem. 252:6501-6509[Free Full Text]. 24. Maruta, H., J. Holden, A. Sizeland, and G. D'Abaco. 1991. The residues of Ras and Rap proteins that determine their GAP specificities. J. Biol. Chem. 266:11661-11668[Abstract/Free Full Text]. 25. Maruta, H., and H. He. 1995. Cytoskeletal SH3 proteins. Biochemistry (Tokyo) 67:1210-1217. 26. Maruta, H. 1996. Downstream of RAS and actin-cytoskeleton, p. 139-180. In H. Maruta, and A. W. Burgess (ed.), Regulation of the RAS signaling network. Springer-Verlag, Heidelberg, Germany. 27. Maruta, H. 1997. F-actin cappers. Jpn. J. Cancer Chemother. 24:1442-1447. 28. Moritz, R. L., and R. J. Simpson. 1992. Application of capillary reversed-phase high-performance liquid chromatography to high-sensitive protein sequence analysis. J. Chromatogr. 599:119-130[Medline]. 29. Moritz, R. L., J. Eddes, G. Reid, and R. J. Simpson. 1996. S-pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: a rapid mass spectrometric method for identifying cysteine-containing peptides. Electrophoresis 17:907-917[Medline]. 30. Muellauer, L., H. Fujita, A. Shizaki, and N. Kuzumaki. 1993. Tumor-suppressive function of mutated gelsolin in Ras-transformed cells. Oncogene 8:2531-2536[Medline]. 31. Nur-E-Kamal, M. S. A., A. Sizeland, G. D'Abaco, and H. Maruta. 1992. Asn26, Glu31, Val45, and Tyr64 of Ras proteins are required for their oncogenicity. J. Biol. Chem. 267:1415-1418[Abstract/Free Full Text]. 32. Pollard, T. D., and J. A. Cooper. 1986. Actin and actin-binding proteins. A critical evaluation of mechanisms and function. Annu. Rev. Biochem. 55:987-1035[Medline]. 33. Prasad, G., R. Fuldner, and H. Cooper. 1993. Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the Ras oncogene. Proc. Natl. Acad. Sci. USA 90:7039-7043[Abstract/Free Full Text]. 34. Qiu, R. G., J. Che, D. Kirn, F. McCormick, and M. Symons. 1995. An essential role for Rac in Ras transformation. Nature 374:457-459[Medline]. 35. Rodriguez-Viciana, P., P. Warne, A. Khwaja, B. Marte, D. Pappin, P. Das, M. Waterfield, A. Ridley, and J. Downward. 1997. Role of PI-3 kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89:457-467[Medline]. 36. Saez, C. G., J. Myers, T. Shows, and L. Leinward. 1990. Human non-muscle II heavy chain mRNA: generation of diversity through alternative polyadenylylation. Proc. Natl. Acad. Sci. USA 87:1164-1168[Abstract/Free Full Text]. 37. Saito, Y., and K. Umezawa. 1997. Synthesis of echiguanine analogues and their ribofuranosyl glycosides that inhibit PI-4 kinase. Bioorg. Med. Chem. Lett. 7:861-864. 38. Sato, N., S. Yonemura, T. Obinata, S. Tsukita, and S. Tsukita. 1991. Radixin, an F-actin barbed end-capping protein, is concentrated at the cleavage furrow during cytokinesis. J. Cell Biol. 113:321-330[Abstract/Free Full Text]. 39. Schuuring, E., E. Verhoeven, S. Litvinov, and R. Michalides. 1993. The product of the EMS1 gene, amplified in human carcinomas, is homologous to a Src substrate and is localized in cell-substratum contact sites. Mol. Cell. Biol. 13:2891-2898[Abstract/Free Full Text]. 40. Simons, M., M. Wang, O. McBride, S. Kawamoto, K. Yamakawa, D. Gdula, R. S. Adelstein, and L. Weir. 1991. Human non-muscle myosin II heavy chains are encoded by two genes located on different chromosomes. Circ. Res. 69:530-539[Abstract/Free Full Text]. 41. Spudich, J. A., and S. Watt. 1971. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of tropomyosin-troponin complex with actin and proteolytic fragments of myosin. J. Biol. Chem. 246:4866-4871[Abstract/Free Full Text]. 42. Takemoto, Y., M. Furuta, X. K. Li, J. Strong-Sparks, and Y. Hashimoto. 1995. LchBP/HS1, expressed in hematopoietic lineage cells, directly associates with the SH3 domain of Lck. EMBO J. 14:3403-3414[Medline]. 43. Tikoo, A., V. Ramesh, J. Gusella, and H. Maruta. 1994. Anti-Ras function of neurofibromatosis type 2 gene product (NF2/Merlin). J. Biol. Chem. 269:23387-23390[Abstract/Free Full Text]. 44. Toothaker, L. E., D. Gonalez, N. Tung, R. Lemons, M. Lebeau, M. Arnaout, L. Clayton, and T. Tenen. 1991. Cellular myosin II heavy chain in human leukocytes: isolation of 5' cDNA clones, characterization of protein, chromosomal localization, and up-regulation during myeloid differentiation. Blood 78:1826-1833[Abstract/Free Full Text]. 44a. van Damme, H., V. van Buuren, and E. Schuuring. Unpublished observation. 45. Vandekerckhove, J., G. Bauw, A. Vancompernolle, B. Honore, and J. Celis. 1990. Gelsolin as one of the most prominent down-regulated markers of transformed human fibroblast and epithelial cells. J. Cell Biol. 111:95-102[Abstract/Free Full Text]. 46. Walsh, A. B., M. Dhanasekaran, D. Bar-Sagi, and C. C. Kumar. 1997. SCH51344-induced reversal of RAS-transformation is accompanied by the specific inhibition of the RAS and RAC-dependent cell morphology pathway. Oncogene 15:2553-2560[Medline]. 47. Wang, D. Z. M., M. S. A. Nur-E-Kamal, A. Tikoo, W. Montague, and H. Maruta. 1997. The GTPase and Rho GAP domains of p190-A, a tumor suppressor protein that binds the Ras GAP of 120 kDa, independently function as anti-Ras tumor suppressors. Cancer Res. 57:2478-2484[Abstract/Free Full Text]. 48. Weber, K., E. Lazarides, R. Goldman, A. Vogel, and R. Pollack. 1974. Localization and distribution of actin fibers in normal, transformed and revertant cells. Cold Spring Harbor Symp. Quant. Biol. 39:363-369[Abstract/Free Full Text]. 49. Wu, H., and T. Parsons. 1993. Cortactin, a Src substrate of 80/85 kDa, is an F-actin binding protein enriched in the cell cortex. J. Cell Biol. 120:1417-1426[Abstract/Free Full Text]. 50. Yahara, I., F. Harada, S. Sekita, K. Yoshihira, and S. Natori. 1982. Correlation between effects of 24 different cytochalasins on cellular structures and events and those on actin in vitro. J. Cell Biol. 92:69-78[Abstract/Free Full Text]. 51. Yamanashi, Y., M. Okada, T. Semba, T. Yamori, H. Umemori, S. Tsunasawa, K. Toyoshima, D. Kitamura, T. Watanabe, and T. Yamamoto. 1993. Identification of HS1 as a major substrate of Tyr kinase(s) upon B-cell antigen receptor-mediated signaling. Proc. Natl. Acad. Sci. USA 90:3631-3635[Abstract/Free Full Text]. 52. Yamanashi, Y., T. Fukuda, H. Nishizumi, T. Ianazu, K. Higashi, D. Kitamura, T. Ishida, H. Yamamura, T. Watanabe, and T. Yamamoto. 1997. Role of Tyr phosphorylation of HS1 in B-cell antigen receptor-mediated apoptosis. J. Exp. Med. 185:1387-1392[Abstract/Free Full Text]. 53. Yonezawa, N., Y. Homma, I. Yahara, H. Sakai, and E. Nishida. 1991. A short sequence responsible for both PIP2 binding and actin binding activities of cofilin. J. Biol. Chem. 266:17218-17221[Abstract/Free Full Text]. 54. Yu, F. X., H. Q. Sun, P. Janmey, and H. L. Yin. 1992. Identification of a PIP2-binding sequence in a G-actin binding domain of gelsolin. J. Biol. Chem. 267:14616-14621[Abstract/Free Full Text]. -------------------------------------------------------------------------------- Mol Cell Biol, July 1998, p. 3829-3837, Vol. 18, No. 7 0270-7306/98/$04.00+0 Copyright ?? 1998, American Society for Microbiology. All rights reserved. This article has been cited by other articles: El Sayegh, T. Y., Arora, P. D., Ling, K., Laschinger, C., Janmey, P. A., Anderson, R. A., McCulloch, C. A. (2007). Phosphatidylinositol-4,5 Bisphosphate Produced by PIP5KI{gamma} Regulates Gelsolin, Actin Assembly, and Adhesion Strength of N-Cadherin Junctions. Mol. Biol. Cell 18: 3026-3038 [Abstract] [Full Text] Zhu, J., Yu, D., Zeng, X.-C., Zhou, K., Zhan, X. (2007). Receptor-mediated Endocytosis Involves Tyrosine Phosphorylation of Cortactin. J. Biol. Chem. 282: 16086-16094 [Abstract] [Full Text] Hao, J.-J., Zhu, J., Zhou, K., Smith, N., Zhan, X. (2005). The Coiled-coil Domain Is Required for HS1 to Bind to F-actin and Activate Arp2/3 Complex. J. Biol. Chem. 280: 37988-37994 [Abstract] [Full Text] He, H., Pannequin, J., Tantiongco, J.-P., Shulkes, A., Baldwin, G. S. (2005). Glycine-extended gastrin stimulates cell proliferation and migration through a Rho- and ROCK-dependent pathway, not a Rac/Cdc42-dependent pathway. Am. J. Physiol. Gastrointest. Liver Physiol. 289: G478-G488 [Abstract] [Full Text] Brunati, A. M., Deana, R., Folda, A., Massimino, M. L., Marin, O., Ledro, S., Pinna, L. A., Donella-Deana, A. (2005). Thrombin-induced Tyrosine Phosphorylation of HS1 in Human Platelets Is Sequentially Catalyzed by Syk and Lyn Tyrosine Kinases and Associated with the Cellular Migration of the Protein. J. Biol. Chem. 280: 21029-21035 [Abstract] [Full Text] Chuang, Y.-y., Tran, N. L., Rusk, N., Nakada, M., Berens, M. E., Symons, M. (2004). Role of Synaptojanin 2 in Glioma Cell Migration and Invasion. Cancer Res. 64: 8271-8275 [Abstract] [Full Text] Pacholsky, D., Vakeel, P., Himmel, M., Lowe, T., Stradal, T., Rottner, K., Furst, D. O., van der Ven, P. F. M. (2004). Xin repeats define a novel actin-binding motif. J. Cell Sci. 117: 5257-5268 [Abstract] [Full Text] Di Ciano, C., Nie, Z., Szaszi, K., Lewis, A., Uruno, T., Zhan, X., Rotstein, O. D., Mak, A., Kapus, A. (2002). Osmotic stress-induced remodeling of the cortical cytoskeleton. Am. J. Physiol. Cell Physiol. 283: C850-C865 [Abstract] [Full Text] Mizutani, K., Miki, H., He, H., Maruta, H., Takenawa, T. (2002). Essential Role of Neural Wiskott-Aldrich Syndrome Protein in Podosome Formation and Degradation of Extracellular Matrix in src-transformed Fibroblasts. Cancer Res. 62: 669-674 [Abstract] [Full Text] Adjei, A. A. (2001). Blocking Oncogenic Ras Signaling for Cancer Therapy. JNCI J Natl Cancer Inst 93: 1062-1074 [Abstract] [Full Text] Wei, Q., Adelstein, R. S. (2000). Conditional Expression of a Truncated Fragment of Nonmuscle Myosin II-A Alters Cell Shape but Not Cytokinesis in HeLa Cells. Mol. Biol. Cell 11: 3617-3627 [Abstract] [Full Text] Weed, S. A., Karginov, A. V., Schafer, D. A., Weaver, A. M., Kinley, A. W., Cooper, J. A., Parsons, J. T. (2000). Cortactin Localization to Sites of Actin Assembly in Lamellipodia Requires Interactions with F-Actin and the Arp2/3 Complex. JCB 151: 29-40 [Abstract] [Full Text] Campbell, D. H., Sutherland, R. L., Daly, R. J. (1999). Signaling Pathways and Structural Domains Required for Phosphorylation of EMS1/Cortactin. Cancer Res. 59: 5376-5385 [Abstract] [Full Text] Funaki, M., Katagiri, H., Kanda, A., Anai, M., Nawano, M., Ogihara, T., Inukai, K., Fukushima, Y., Ono, H., Yazaki, Y., Kikuchi, M., Oka, Y., Asano, T. (1999). p85/p110-type Phosphatidylinositol Kinase Phosphorylates Not Only the D-3, but Also the D-4 Position of the Inositol Ring. J. Biol. Chem. 274: 22019-22024 [Abstract] [Full Text] Kapus, A., Szaszi, K., Sun, J., Rizoli, S., Rotstein, O. D. (1999). Cell Shrinkage Regulates Src Kinases and Induces Tyrosine Phosphorylation of Cortactin, Independent of the Osmotic Regulation of Na+/H+ Exchangers. J. Biol. Chem. 274: 8093-8102 [Abstract] [Full Text] Brunati, A. M., Donella-Deana, A., James, P., Quadroni, M., Contri, A., Marin, O., Pinna, L. A. (1999). Molecular Features Underlying the Sequential Phosphorylation of HS1 Protein and Its Association with c-Fgr Protein-tyrosine Kinase. J. Biol. Chem. 274: 7557-7564 [Abstract] [Full Text] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by He, H. Articles by Maruta, H. Search for Related Content PubMed PubMed Citation Articles by He, H. Articles by Maruta, H. -------------------------------------------------------------------------------- J. Bacteriol. J. Virol. Eukaryot. Cell Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals

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Mulholland Drive TITULO ORIGINAL Mulholland Drive DURACI??N 147 min. PA??S Estados Unidos DIRECTOR David Lynch GUI??N David Lynch M??SICA Angelo Badalamenti FOTOGRAF??A Peter Deming REPARTO Justin Theroux, Naomi Watts, Laura Elena Harring, Ann Miller, Robert Forster, Brent Briscoe, Jeannie Bates, Melissa George PRODUCTORA Coproducci??n USA-Francia; Les Films Alain Sarde / Asymetrical Production Intriga. Drama SINOPSIS: Betty, una joven aspirante a actriz que llega a Los ??ngeles para convertirse en estrella de cine, se aloja en el apartamento de su t??a. Dentro de la casa se encuentra con Rita, una mujer amn??sica que ha sufrido un accidente en Mulholland Drive. Juntas deciden investigar qui??n es Rita y c??mo lleg?? hasta all??.

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[DvdRip - Xvid - LD ita MP3] Mulholland Drive [www.master-italia.net]

Thu, 29 May 2008 06:18:30

[DvdRip - Xvid - LD ita MP3] Mulholland Drive [www.master-italia.net]

Tue, 27 May 2008 17:56:48

Mulholland Drive avi

Sun, 11 May 2008 08:25:34

Mulholland Drive avi

Sun, 11 May 2008 07:26:02

Mulholland.Drive.German.2001.DVDRiP.XViD.AC3

Wed, 02 Apr 2008 05:53:22

Mulholland Drive German 2001 DVDRiP XViD AC3

Fri, 28 Mar 2008 17:22:29

Titel..................: Mulholland Drive
Genre..................: Drama / Mystery / Thriller
Erscheinungsdatum......: 3 Januar 2002
Spieldauer.............: 2h 20min
Source (DVD/VHS/etc.)..: DVD
Codec..................: XviD
Videoformat............: Avi
Videobitrate...........: 987 Kbps
Audioformat............: AC3(6CH)
Audiobitrate...........: 448 Kbps
Sprache................: Deutsch
Aufl�sung..............: 720*400
Releasedatum...........: 27.03.08

Das h�bsche Landei Betty (Naomi Watts), frisch aus Ontario/Kanada angekommen, will Schauspielerin werden und Hollywood erobern. Da passt es ihr gerade recht, dass sie das Haus ihrer reichen Tante, ein alternder Filmstar, h�ten soll. Als sie sich in dem L.A.-Appartment einrichten will, steht pl�tzlich eine wildfremde Sch�nheit (Laura Elena Harring) unter ihrer Dusche. Sie ist v�llig verwirrt, wurde bei einem mysteri�sen Autounfall, den sie als einzige �berlebte, verletzt. Sie nennt sich Rita, kann sich aber an nichts erinnern, nicht einmal an ihren richtigen Namen. Statt die Polizei zu rufen, freundet sich Betty mit der geheimnisvollen Fremden mehr als an und versucht herauszufinden, wer sie ist.....

[DivX ITA] David Lynch - 2001 - Mulholland Drive.avi

Sun, 24 Feb 2008 07:27:32

[DVD5 Ita]MulHolland Drive[colombo-bt org]

Sun, 27 Jan 2008 21:21:52

[xvid-dvdrip.ac3-ita-eng]Mulholland Drive[ITA-ENG MP3-AC3][colombo-bt.org]

Fri, 11 Jan 2008 11:21:16

After a car wreck on the winding Mulholland Drive renders a woman amnesic, she and a perky Holliwood-hopeful search for clues and answers across Los Angeles in a twisting venture beyond dreams and reality.

Mulholland.Drive.German.2001.AC3.DVDRiP.iNTERNAL.XViD-PIEPclassix

Wed, 28 Nov 2007 02:18:42

Mulholland.Drive.German.2001.AC3.DVDRiP.iNTERNAL.XViD-PIEPclassix

Mon, 26 Nov 2007 01:22:42

Mulholland.Drive.2001.German.DL.720p.HDDVD.x264-SUBHD

Mon, 19 Nov 2007 01:12:05

Mulholland.Drive.HD DVDre.a1080.x264.DTS_hO

Mon, 05 Nov 2007 02:25:29

09.28.07.Mulholland.Drive.2001.HD-DVDRip.DTS-HD.DVD9.x264-CHD

Mon, 08 Oct 2007 06:23:19

09.28.07.Mulholland.Drive.2001.HD-DVDRip.DTS-HD.DVD9.x264-CHD

Tue, 02 Oct 2007 22:32:42

Angelo Badalamenti - Mulholland Drive OST

Mon, 10 Sep 2007 22:13:31

Mulholland.Drive.German.2001.DVDRip.AC3.XviD-r0ck3d

Sun, 05 Aug 2007 02:33:30

Mulholland.Drive.2001.DVDRiP.German.DL.x264-TheHC

Tue, 03 Jul 2007 10:30:47

Mulholland Drive.avi

Mon, 21 May 2007 20:41:27

Mulholland Drive.mov

Fri, 13 Apr 2007 02:43:14

Please continue to seed! Thanks!

[DivX - ITA] - Mulholland drive.avi

Fri, 13 Apr 2007 02:43:14

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Mullholland_Drive

Fri, 13 Apr 2007 02:42:54

Mullholland Drive - http://www.imdb.com/title/tt0166924

Mulholland Drive

Sun, 08 Apr 2007 21:40:47

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Mulholland Drive (2001) DVD-Rip avi

Sun, 18 Mar 2007 14:35:26

Mullholland Drive - http://www.imdb.com/title/tt0166924

Mulholland Drive [CD1] [2001] [DVDRip] [DivX]

Sun, 18 Mar 2007 14:21:28

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Mulholland Drive [CD2] [2001] [DVDRip] [DivX]

Sun, 18 Mar 2007 14:21:28

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David Lynch - Mulholland Drive (2001) [DVDrip AVC AAC][liDEL]

Sun, 18 Mar 2007 14:18:03

AVC&AAC release. sample and info files included. more releases at http://shurl.org/lidel-releases Show your appreciation and keep this torrent alive by staying connected once you've finished downloading.