sane-epkowa(5)

sane-epkowa - SANE backend for EPSON scanners

DESCRIPTION

The sane-epkowa library implements a SANE (Scanner Access Now Easy) backend that provides access to EPSON atbed scanners. This backend should be considered alpha-quality software! At present, the following scanners are known to work with this backend: Model: japan other ---------------- ------------------GT-6600U Perfection 610 GT-6700U Perfection 640U GT-7200U Perfection 1250 Perfection 1250 PHOTO GT-7300U Perfection 1260 Perfection 1260 PHOTO GT-7600S Perfection 1200S GT-7600U Perfection 1200U GT-7600UF Perfection 1200U PHOTO GT-7700U Perfection 1240U GT-8300UF Perfection 1660 PHOTO GT-8700 Perfection 1640SU GT-8700F Perfection 1640SU PHOTO GT-8200U Perfection 1650 GT-8200UF Perfection 1650 PHOTO GT-9300UF Perfection 2400 PHOTO GT-9400 Perfection 3170 PHOTO GT-9700F Perfection 2450 PHOTO GT-9800F Perfection 3200 PHOTO GT-F500 Perfection 2480 PHOTO GT-F520 Perfection 3490 PHOTO GT-F550 Perfection 2580 PHOTO GT-F570 Perfection 3590 PHOTO GT-F600 Perfection 4180 PHOTO GT-F650 Perfection V100 PHOTO GT-F670 Perfection V200 PHOTO GT-F700 Perfection V350 PHOTO GT-S600 Perfection V10 GT-X700 Perfection 4870 PHOTO GT-X750 Perfection 4490 PHOTO GT-X800 Perfection 4990 PHOTO GT-X900 Perfection V700/V750 ES-2000 Expression 1600 ES-2200 Expression 1680 ES-6000 GT-10000 ES-6000H GT-10000+ ES-6000HS ES-7000H GT-15000 ES-8500 Expression 1640XL ES-9000H GT-30000 ES-10000G Expression 10000XL ES-H300 GT-2500 Stylus CX2800/CX2900/ME200 PX-A550 Stylus CX3500/CX3600

2007-03-07

Stylus CX3700/CX3800/DX3800 Stylus CX3900/DX4000 Stylus CX4100/CX4200/DX4200 Stylus CX4300/CX4400/CX5500/CX5600/DX4400 Stylus CX4500/CX4600 PX-A650 Stylus CX4700/CX4800/DX4800 Stylus CX4900/CX5000/DX5000 CC-600PX Stylus CX5100/CX5200 Stylus CX5300/CX5400 PX-A720 Stylus CX5900/CX6000/DX6000 Stylus CX6300/CX6400 Stylus CX6500/CX6600 Stylus CX7300/CX7400/DX7400 Stylus CX7700/CX7800 PX-A740 Stylus CX8300/CX8400/DX8400 Stylus CX9300F/CX9400Fax/DX9400F PM-A700 Stylus Photo RX420/RX425/RX430 Stylus Photo RX500/RX510 PM-A750 Stylus Photo RX520/RX530 PM-A820 Stylus Photo RX560/RX580/RX590 PM-A840 Stylus Photo RX585/RX595/RX610 PM-A850 Stylus Photo RX600 PM-A870 Stylus Photo RX620/RX630 PM-A890 Stylus Photo RX640/RX650 PM-A940 Stylus Photo RX680/RX685/RX690 PM-A900 Stylus Photo RX700 PM-A920 PM-A950 PM-A970 PM-T960 PM-T990 LP-A500 AcuLaser CX11 AcuLaser CX21 LP-M5500 LP-M5600 PX-A620 For other scanners the software may or may not sanedevel@lists.alioth.debian.org to report successes or failures. work. Please send mail to

OPTIONS

The options the backend supports can either be selected through command line options to programs like scanimage or through GUI elements in xscanimage or xsane. Valid command line options and their syntax can be listed by using scanimage --help -d epkowa Not all devices support all options. Scan Mode The --mode switch selects the basic mode of operation of the scanner valid choices are Binary, Gray and Color. The Binary mode is black and white only, Gray will produce up to 256 levels of gray and Color means 24 bit color mode. Some scanners will internally use 36 bit color, the external interface however does only support 24 bits. The --dropout option determines which color lters are used to scan in Binary mode. Valid choices are None, Red, Green and Blue.
The --halftoning switch selects the mode that is used in Binary mode. Valid options are None, Halftone A (Hard Tone), Halftone B (Soft Tone), Halftone C (Net Screen), Dither A (4x4 Bayer), Dither B (4x4 Spiral), Dither C (4x4 Net Screen), Dither D (8x4 Net Screen), Text Enhanced Technology, Download pattern A, and Download pattern B. The --dropout switch selects the so called dropout color. Vald options are None, Red, Green and Blue. The default is None. The dropout color is used for monochrome scanning and selects the color that is not scanned. This can be used to e.g. scan an original with a colored background. The --brightness switch controls the brightness of the scan. Valid options are the numbers from -3 to 3. The default is 0. The larger the brightness value, the brighter the image gets. If a user dened table for the gamma correction is selected, the brightness parameter is not available. The --sharpness switch sets the sharpness of the image data. Valid options are the numbers from -2 to 2, with -2 meaning "Defocus", -1 "Defocus slightly", 0 "Normal", 1 "Sharpen slighly" and 2 "Sharpen". The --gamma-correction switch controls the scanne internal gamma correction. Valid options are "Default", "User dened", "High density printing" "Low density printing" and "High contrast printing". The --color-correction switch controls the scanner internal color correction function. Valid options are "No Correction", "Impact-dot printers", "Thermal printers", "Ink-jet printers" and "CRT monitors". The default is "CRT monitors". The --resolution switch selects the resolution for a scan. Many EPSON scanners will scan in any resulution between the lowest and highest possible value. The list reported by the scanner can be displayed using the "--help -d epkowa" parameters to scanimage. The --mirror option controls the way the image is scanned. By reading the image data from right to left the image is mirored. Valid options are "yes" and "no". The default is "no". The --speed option can improve the scan speed in monochrome mode. Valid options are "yes" or "no", the "yes" option will speed up the scan if this option is supported. The --auto-area-segmentation switch turns on the automatic area segmentation for monochrome scans. The scanner will try to determine which areas are text and which contain images. The image areas will be halftoned, and the text will be impoved. Valid options are "yes" and "no". The default is "yes". The --gamma-table parameter can be used to download a user dened gamma table. The options takes 256 values from the range 0-255. In color mode this option equally affects the red, green, and blue channel. The --red-gamma-table parameter can be used to download a user dened gamma table for the red channel. The valid options are the same as for --gamma-table. The --green-gamma-table parameter can be used to download a user dened gamma table for the green channel. The valid options are the same as for --gamma-table. The --blue-gamma-table parameter can be used to download a user dened gamma table for the blue channel. The valid options are the same as for --gamma-table. The color correction coefcients --cct-1 --cct-2 --cct-3. --cct-9 will install color correction

coefcients for the user dened color correction. Possible values are in the range -127.127.
The --preview option requests a preview scan. The frontend software automatically selects a low resolution. Valid options are "yes" and "no". The default is "no". The --preview-speed options will increase the scan speed if this is supported by the scanner. Valid options are "yes" and "no", the default is "no".
The geometry options -l -t -x -y control the scan area: -l sets the top left x coordinate, -t the top left y coordinate, -x selects the width and -y the height of the scan aea. All parameters are specied in milimeters. The --quick-format option lets the user select a scan area with predened sizes. Valid parameters are "CD", "A5 portrait", "A5 landscape", "Letter", "A4" and "max". The default is "max", which selects the largest possible area. The --source option selects the scan source. Valid options depend on the installed options. The default is "Flatbed". The --auto-eject option will eject a page after scanning from the document feeder.

CONFIGURATION FILE

The conguration le /etc/sane.d/epkowa.conf species the device(s) that the backend will use. The current version only supports one scanner per EPSON backend. Possible connection types are: SCSI This is the default, and if nothing else is specied the backend software will open a given patch as SCSI device. More information about valid syntax for SCSI devices can be found in sane-scsi(5).
PIP - Parallel Interface The parallel interface can be congured in two ways: An integer number starting at the beginning of a line will be interpreted as the IO address of the parallel port. To make it clearer that a congured IO address is a parallel port the port address can be preceded by the string "PIO". The PIO connection does not use a special device le in the /dev directory. USB A device le that is preceded by the string "USB" is treated as a scanner connected via the Universal Serial Bus. The correct special device le has to be created prior to using it with Sane. See the USB documentation for more information about how to set up the USB subsystem and the required device les.
/usr/lib/sane/libsane-epkowa.a The static library implementing this backend. /usr/lib/sane/libsane-epkowa.so The shared library implementing this backend (present on systems that support dynamic loading).

ENVIRONMENT

SANE_DEBUG_EPKOWA If the library was compiled with debug support enabled, this environment variable controls the debug level for this backend. E.g., a value of 128 requests all debug output to be printed. Smaller levels reduce verbosity.

SANE_EPSON_CMD_LVL This allows to override the function or command level that the backend uses to communicate with the scanner. The function level a scanner supports is determined during the initialization of the device. If the backend does not recognize the function level reported by the scanner it will default to function level B5. Valid function levels are A1, A2, B1, B2, B3, B4, B5, B6, B7, B8 and F5. Use this feature only if you know what you are doing!

SEE ALSO

sanescsi(5), scanimage(1), xscanimage(1), xsane(1), saneepson(5)
When used with "scanimage -T" the backend hangs after sucessfully completing the tests. It is necessary to powercycle the scanner to get the communication between backend and scanner going again. Sometimes the scanner is not initialized correctly. The problem can be resolved by killing the program and restarting it again.

UNSUPPORTED DEVICES

The backend may be used with EPSON scanners that are not yet listed under the list of supported devices. A scanner that is not recognized may default to the function level B3, which means that not all functions that the scanner may be capable of are accessible. If the scanner is not even recognized as an EPSON scanner it is probably because the device name eported by the scanner is not in the correct format. Please send this information to the backend maintainer (email address is in the AUTHORS le).devices. A scanner that is not recognized may default to the function level B3, which means that not all functions that the scanner may be capable of are accessible. If the scanner is not even recognized as an EPSON scanner it is probably because the device name eported by the scanner is not in the correct format. Please send this information to the backend maintainer (email address is in the AUTHORS le).

AUTHOR

EPSON AVASYS Corporation

JBC Papers in Press. Published on June 7, 2004 as Manuscript M402767200
The Adaptor Protein Nck1 Mediates Endothelin A Receptor-regulated Cell Migration through the Cdc42-dependent c-Jun N-terminal Kinase Pathway
Downloaded from www.jbc.org by guest, on June 6, 2011
Yuki Miyamoto1, Junji Yamauchi1, 2, Norikazu Mizuno1, and Hiroshi Itoh1,*
From the 1Department of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan;
Department of Neurobiology, Stanford University School of Medicine, Stanford, California

94305-5125, USA.

To whom correspondence should be addressed. Telephone: +81-743-72-5440. Fax:
+81-743-72-5449. E-mail: hitoh@bs.naist.jp.
Running Title: Nck1-mediated Regulation of Cell Migration and JNK by ET-1
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Abstract

Cell migration plays key roles in physiological and pathological phenomena, such as development and oncogenesis. The adaptor proteins Grb2, CrkII, and Nck1 are composed only of a single Src homology (SH) 2 domain and some SH3 domains, giving specificity to each signal transduction pathway. However, little is known about the relationships between their adaptor proteins and cell migration, which are regulated by the G protein-coupled receptor (GPCR). Here, we show that Nck1, but not Grb2 or CrkII, mediates the inhibition of cell migration induced by the endothelin-1 (ET-1) and endothelin A (ETA) receptor. The small interference RNA and dominat negative mutants of Nck1 inhibited the ET-1-induced inhibition. Although overexpression of wild-type Nck1 was detected in the cytosol and did not affect cell migration, expression of the myristoylation signal sequence-conjugated Nck1 was in the membrane and induced activation of Cdc42 and c-Jun N-terminal kinase (JNK), inhibiting cell migration. Taken together, these results suggest that the ETA receptor transduces the signal of inhibition of cell migration through Cdc42-dependent JNK activation by using Nck1. These findings provide new insights into the GPCR-mediated regulation of cell migration.

Introduction

Cell migration plays key roles in development and oncogenesis. During development, organized cell migration is essential for proper tissue formation (1). However, unregulated cell migration is observed in a pathological process, such as oncogenesis, which involves, specifically, invasion and metastasis. The elucidation of the molecular signaling mechanisms that positively and negatively regulate migration is critical in understanding
the development process as well as these diseases. In many cases of the migration process, cells show directed movement (called chemotaxis) toward soluble chemoattractants. A number of chemoattractants have been identified: chemokines, lipid mediators, growth factors, and cytokines (1, 2). Chemokines bind and activate their cognate chemokine receptors, which belong to a large family of G protein-coupled receptors (GPCRs) characteristic of the seven-transmembrane structure (3-6). In contrast, we have only limited information about the inhibitors of chemotaxis, i.e. chemorepellents, which are also the ligands of GPCRs. These include bioactive lipids, thrombin, metastin and opioid peptide (7-10). However, the mechanism by which
GPCRs inhibit migration is not fully understood so far. Adaptor proteins, such as Nck1, Grb2, and CrkII, consist primarily of a single Src homology (SH) 2 domain and various numbers of SH3 domains and do not have other functional motifs (11, 12). The SH2 domain recognizes tyrosine-phosphorylated proteins, and the SH3 domain associates with the PXXP motif-containing proteins (13). These
domains provide a site to couple the distinct molecules to the core machinery that regulates the cellular function and gives specificity to the signal transduction. Among them, Grb2 provides a well-characterized example of how adapter proteins of this group function to transduce signals. Grb2 constantly associates with a guanine-nucleotide exchange factor (GEF) Sos of Ras GTPases via the SH3 domains (14). Following growth factor stimulation, Grb2 binds to tyrosine-phosphorylated receptor tyrosine kinases and/or other adaptor proteins, such as Shc, via its SH2 domain. As a result, Sos is translocated to the plasma

membranes, leading to Ras-dependent activation of extracellular signal-regulated protein kinase (ERK), a subfamily of mitogen-activated protein kinases (MAPKs) (15, 16). We demonstrated that the endothelin-1 (ET-1) and endothelin A (ETA) receptor activate Cdc42 of Rho GTPases, which in turn stimulate the signaling cascade of c-Jun N-terminal kinase (JNK), a subfamily of MAPKs (17, 18). This signaling pathway is involved in the inhibition of cell migration (17, 18). To further investigate the mechanism whereby the ETA receptor inhibits cell migration, the effects of various dominant-negative mutants on cell migration were assayed on the transient transfection system using human epithelial 293T cells. Here, we show that the Nck1, but not Grb2 or CrkII, is a critical regulator in the chemorepellent signaling pathway coupling the ETA receptor to Cdc42-dependent activation of JNK. Additionally, membrane-targeted Nck1 inhibited cell migration. These results suggest that Nck1 functions as a mediator of the chemorepellent signaling downstream of the ETA receptor.

Materials and Methods

Materials--- Mouse monoclonal antibodies against active phosphorylated JNK (Thr183/Tyr185), active phosphorylated ERK (Thr202/Tyr204), and rabbit polyclonal antibodies against JNK and ERK were purchased from Cell Signaling Technology, Inc (Beverly, MA). A mouse monoclonal antibody against MBP and rabbit polyclonal antibodies against JNK1 and c-Src were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibodies against Cdc42 and Nck1 were purchased from BD Biosciences Transduction Laboratories (San Jose, CA). A mouse antibody against Aequorea victoria green fluorescence protein (GFP) was obtained from Medical and Biological Laboratories (Nagoya, Japan). A mouse monoclonal antibody against tubulin was purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse and anti-rabbit IgG antibodies conjugated with horseradish peroxidase were obtained from Amersham Biosciences (Buckinghamshire, UK). SP600125, U0126, SB203580, and PP1 were purchased from Biomol (Plymouth Meeting, PA), and Clostridium difficile toxin B was from Carbiochem-Novabiochem (San Diego, CA). Endothelin-1 and epidermal growth factor (EGF) were purchased from the Peptide Institute, Inc. (Osaka, Japan) and Roche Diagnostics Co. (Indianapolis, IN), respectively. Plasmids --- Wild-type Nck1, Grb2 and CrkII were amplified from a mouse brain cDNA library and subcloned into the mammalian MBP-tag expression vector pCMV-MBP. Wild-type Nck1 was also ligated into the pEGFP-N3 (BD Biosciences Clontech, Franklin Lakes, NJ) to make pEGFP-Nck1-N3. To produce dominant negative mutants of Nck1 and CrkII, the conserved arginine residue of the FLVRES sequence in the SH2 domain was

changed into lysine, making Nck1R308K and CrkIIR38K, or the first tryptophan residue of the characteristic tryptophan doublet of the SH3 domain was changed to lysine, creating Nck1W38K, Nck1W143K, Nck1W229K, CrkIIW169K, and CrkIIW275K. These residues are identified as being essential for binding to their ligands (19). The amino acid substitutions were performed by the overlap extension method based on polymerase chain reaction (PCR) with mutant oligonucleotides. These mutants were ligated into the pCMV-MBP. Mutations in all three Nck1 SH3 domains (designed SH3All in this paper) were carried out by sequentially repeating the above procedure. The fragments of Grb2 SH2 lacking the SH2 domain (amino acids 60-158) and Grb2 SH3 lacking the SH3 domain (amino acids 1-60 and 159-218) of Grb2 were inserted into pCMV-MBP. For the myristoylated products, the 14 amino-acid c-Src myristoylation signal was first synthesized by PCR (19). The PCR product was digested and cloned into pEGFP-N3 (BD Biosciences Clontech, Franklin Lakes, NJ) to make the pEGFP-myr-N3 plasmid. Nck1 was subcloned into pEGFP-myr-N3. pCMV-FLAG-RhoT19N, pCMV-FLAG-RacT17N,
pCMV-FLAG-Cdc42T17N, and the Escherichia coli expression plasmid encoding the Cdc42-binding domain (CRIB) of Pak were constructed as described previously (20, 21). pUSE-CA-Src (a constitutively activated mutant of c-Src) was purchased from Upstate Inc. (Charlottesville, VA). pME-ETA receptor-EGFP was generously provided by Dr. T. Sakurai (Tsukuba University, Tsukuba, Japan) (22). Cell culture and transfection --- Human epithelial 293T cells were maintained in Dulbecco's modified Eagles medium (DMEM) containing 100 g/ml kanamycin and 10% heat-inactivated fetal bovine serum. The cells were cultured at 37 oC in a humidified
atmosphere containing 5% CO2. Plasmid DNAs were transfected into cells by the calcium-phosphate precipitation method (18). Transfection efficiency typically exceeded 80% using the pEGFP-C1 (BD biosciences Clontech) as a control plasmid in 293T cells. The final amount of transfected DNA for 60-mm dish was adjusted to 15 g by pCMV. The pME-ETA receptor-EGFP (0.3 g) was cotransfected with the dominant negative mutants of pCMV-MBP-adaptor proteins (1-3 g) or pCMV-FLAG-Rho GTPases (1 g) into 293T cells. In some cases, cells were transfected with 1 g of pUSE-CA-Src, 1 g of
pEGFP-Nck1-N3, or 1 g of pEGFP-myr-Nck1-N3. The medium was replaced 24 h after transfection, and cells were starved in a serum-free medium. Cells were pretreated with SP600125 (100 nM, 45 min), U0126 (10 M, 1 h), SB203580 (10 M, 1 h), PP1 (1 M, 16 h), or ToxinB (0.2 ng/ml, 16 h) before stimulation with ET-1 (100 nM, 20-30 min) or EGF (10 ng/ml, 10 min). siRNA preparation and transfection --- RNA oligonucleotides were synthesized by Dharmacon, Inc. (Lafayette, CO). GFP siRNA was used as a negative control. The siRNAs were transfected into 293T cells using LipofectamineTM 2000 transfection reagent (Invitrogen Co., Carlsbad, CA), according to manufacturers protocol. Immunoprecipitation and immunoblotting --- After the addition of ET-1 or EGF, cells were lysed in 600 l of a lysis buffer (20 mM HEPES-NaOH (pH 7.5), 3 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethane sulfonylfluoride, 1 g/ml leupeptin, 1 mM EGTA, 1 mM Na3VO4, 10 mM NaF, 20 mM -glycerophosphate, and 0.5% NP-40) per 60-mm dish (18). Immunoprecipitation was carried out as described previously (18). The immunoprecipitates or the expressed proteins in the cell lysates were denatured and

then separated on SDS-polyacrylamide gels. The electrophoretically separated proteins were transferred to membranes, blocked, and immunoblotted as described previously (18). Images of protein bands in immunoblots were captured using Adobe Photoshop 5.0-J plug-in software and an EPSON GT-7300U scanner. The band intensity of kinases and Rho GTPases was semiquantified using NIH Image 1.61. The representatives of at least three separate experiments are shown in figures. MAPK assay --- Immunoprecipitated JNK or cell lysates were immunoblotted with an anti-phosphorylated JNK antibody or anti-phosphorylated ERK antibody (23). To compare the total amounts of MAPKs in the lysates, the immunoblottings were also performed using an anti-JNK or anti-ERK antibody. The levels of phosphorylated forms were normalized to the total amounts of kinases using NIH Image 1.61. Approximately, 10% of the total JNK were immunoprecipitated with anti-JNK antibody, and 20% of the immunoprecipitated JNK were phosphorylated after stimulation with ET-1. The representatives of four to six separate experiments are shown in figures. Pull-down assay --- To detect endogenous active GTP-bound Cdc42 in the cell lysates, we performed pull-down assays using recombinant GST-tagged PakCRIB (18). The levels of active GTP-bound Cdc42 were normalized to the total amounts of Cdc42 proteins. Cell migration assay --- Cell migration was measured using a 24-well Boyden chamber (Becton Dickinson Labware, Franklin Lakes, NJ) according to the manufacturer's protocol. Briefly, upper wells with polyethylene terephthalate filters (8 m pore size) were coated with 10 g/ml extracellular matrix E-C-L (Upstate Inc., Charlottesville, VA).
Serum-starved cells (2 x 105 cells in 500 l DMEM per well) were loaded into upper wells,
which were immediately plated in a chamber containing 165 nM ET-1 (750 l of DMEM per well). After incubation at 37 oC for 5 h, the upper filters were stained with a Diff-Quick staining kit (Biochemical Sciences Inc., Sterling Height, MI). Using an optical microscope, the number of migrated cells was counted in at least three independent experiments. Crude membrane preparation --- Briefly, the harvested cells were homogenized in an ice-cold buffer (5 mM Tris-HCl (pH 7.5), 250 mM sucrose, and 1 mM MgCl2). Nuclei and unbroken cells were separated from the cell extract by a centrifuge (700 x g, 4 oC, 10 min). The supernatant was sonicated and centrifuged (150,000 x g, 4 oC, 30 min). The supernatant was used as the postnuclear cytosol fraction, and the pellet was used as the crude membrane fraction (24). Statistical analysis --- Values shown represent the mean S.E.M. from at least three separate experiments. A Student's t-test was carried out for intergroup comparisons with the control (*, p<0.01).

Results

The ETA receptor induces the inhibition of cell migration via Cdc42/JNK --- Since we previously suggested that the ETA receptor inhibits cell migration through the JNK signaling pathway (17, 18), using the dominant negative mutant of JNK activator, MKK4K95R, we examined the effect of a JNK-specific inhibitor on cell migration. As shown in Figure 1A-C, ETA receptor-induced inhibition of cell migration was blocked only by SP600125 (JNK inhibitor), but not by U0126 (MEK1/2 inhibitor) or SB203580 (p38 MAPK inhibitor). Next, we investigated the involvement of Rho GTPases in this signaling pathway. As shown in Figure 1D, the dominant negative mutant of Cdc42, but not of RhoA or Rac1, attenuated ETA receptor-induced inhibition, indicating that Cdc42 functions downstream of the ETA receptor. Taken together with previous data (18), these results indicate that the Cdc42/JNK pathway plays a key role in the inhibition of cell migration induced by the ETA receptor. The ETA receptor induces the inhibition of cell migration through Nck1, but not through Grb2 or CrkII --- A growing number of studies have suggested that adaptor proteins play an important role in mediating the signaling pathway from receptor tyrosine kinases to Rho family-dependent regulation of actin cytoskeleton (25, 26). Cell migration is a complex cellular process that is regulated by a number of regulatory proteins and is driven by cytoskeletal reorganization. To explore the potential involvement of adaptor proteins in ETA receptor-induced inhibition of cell migration, we transiently transfected the dominant negative mutants of these adaptor proteins into cells. The cells transfected with
the plasmid encoding Grb2 SH2 or CrkIIR38K had no effect on ETA receptor-induced inhibition of cell migration (Figures 2A and 2B). In contrast, various SH3 domain-deficient mutants (Nck1W38K, W143K, W229K, and SH3All) and a SH2-deficient mutant (Nck1R308K) of Nck1 rescued the negative effect (Figure 2C). These results suggest that Nck1, but not Grb2 or CrkII, is necessary for the inhibition of cell migration induced by the ETA receptor. JNK activation induced by an ETA receptor is mediated by Nck1, but not by Grb2 or CrkII --- To examine whether an ETA receptor activates JNK through adaptor proteins, endogenous JNK was immunoprecipitated from the cell lysates and immunoblotted with an anti-phosphorylated JNK antibody, which recognizes the active state of JNK. As shown in Figure 3A, ETA receptor-induced JNK activation was blocked by co-transfection with the dominant negative mutants of Nck1 (Nck1W38K, W143K, W229K, SH3All, and R308K). However, co-transfection with dominant negative mutants of Grb2 (Grb2 SH3 and SH2) or CrkII (CrkllW169K, W275K, and R38K) had no effect on JNK activation elicited by the ETA receptor (Figures 3B and 3C). On the other hand, Grb2 SH3 inhibited EGF-induced ERK phosphorylation, and CrkIIR38K blocked EGF-induced JNK phosphorylation, in agreement with earlier findings (27, 28) (Figures 3D and 3E), whereas the dominant negative mutants of Nck1 had no effect on either EGF-induced JNK (Figure 3F) and ERK (data not shown) activation. These results indicate that Nck1, but not Grb2 or CrkII, is a specific component of the signaling pathway from the ETA receptor to JNK. The ETA receptor increases the GTP-bound form of Cdc42 through Nck1, but not through Grb2 or CrkII --- Next, using pull down assay, we investigated whether the ETA

receptor activates Cdc42 through adaptor proteins. Following stimulation with ET-1, the GTP-bound active form of Cdc42 was dramatically increased, and this effect was blocked by the dominant negative mutants of Nck1 (Figure 4A). As shown in Figures 4B and 4C, the dominant negative mutants of Grb2 and CrkII had no effect on the ETA receptor-induced activation of Cdc42. Taken together, these results suggest that Nck1 mediates the ETA receptor-induced inhibition of cell migration involving Cdc42 and JNK. The membrane-bound form of Nck1 stimulates the inhibition of cell migration by increasing the activities of Cdc42 and JNK--- To examine whether Nck1 can inhibit cell migration through the Cdc42/JNK signaling pathway, we made a membrane-bound mutant of Nck1. We fused the myristoylation signal sequence of c-Src to the N-terminal of Nck1 (Figure 5A). To verify the effectiveness of the myristoylation signal sequence, transfected cells were homogenized and fractionated into crude membrane, cytosol, and nucleus. Wild-type Nck1 was detected in the cytosol fraction, and myr-Nck1 was mainly found in the membrane fraction (Figure 5A). As shown in Figure 5B, myr-Nck1 significantly inhibited cell migration; however wild-type Nck1 did not. Additionally, myr-Nck1 increased the activities of JNK and the GTP-bound form of Cdc42 (Figures 5C and 5D). Taken together, these results suggest that Nck1 mediates the activation of Cdc42 and JNK and inhibits cell migration. siNck1 inhibits the ETA receptor-induced inhibition of cell migration through the JNK pathway --- To confirm the requirement of Nck1 in the pathway from the ETA receptor to the inhibition of cell migration, we then carried out RNAi-mediated gene silencing using a synthetic 21-mer oligonucleotide RNA duplex (siRNA) of Nck1. As shown in Figure 6A,
we designed a pair of oligonucleotides (siNck1-1 or siNck1-2) corresponding to the sequence of human Nck1. Transfection of siNck1-1 or siNck1-2 into cells suppressed the expression of endogenous Nck1, but not tubulin, in a dose-dependent manner (Figure 6A). Since we observed a prominent silencing effect of siNck1-1 on endogenous Nck1, we used siNck1-1 in the following experiments. We investigated whether siNck1-1 affects the endogenous JNK activity stimulated by the ETA receptor. As shown in Figure 6B, ETA receptor-induced JNK activation was inhibited by siNck1-1. In contrast, the control siRNA (siGFP) had no effect on the JNK activation. Additionally, the distinct signaling, i.e. the EGF-induced phosphorylation of ERK, was not affected by siNck1-1 (data not shown). In parallel with JNK activity, increase of the active form of Cdc42 induced by the ETA receptor was inhibited by siNck1-1 (data not shown). Finally, we examined the effect of siNck1-1 on cell migration. As shown in Figure 6C, silencing of Nck1 partially rescued the ETA receptor-induced inhibition of cell migration. These results strongly suggested that Nck1 has an essential role in the ETA receptor-stimulated inhibition of cell migration through Cdc42 and JNK. Src kinase suppresses cell migration via the Nck1/Cdc42/JNK pathway --- We previously demonstrated that Src kinase acts upstream of the Cdc42 and JNK in the ETA receptor-signaling pathway (17, 18). To investigate the involvement of Nck1 in the CA-Src-induced inhibition of cell migration, the dominant negative mutants of Nck1 were co-transfected with CA-Src. As shown in Figure 7A, the CA-Src-induced inhibition of cell migration was completely rescued by Nck1SH3All or Nck1R308K. Additionally, CA-Src-induced JNK (Figure 7B) and Cdc42 (data not shown) activation was also inhibited

by these mutants. We previously demonstrated that Gq-coupled receptor induces the JNK activation in a Src-dependent manner (29). Additionally, ETA receptor-induced inhibition of cell migration was blocked by cotransfection of the G q inhibitors (regulator of G protein signaling (RGS) 4 or N-terminal domain of -adrenergic receptor kinase 1 ( ARKnt)) (data not
shown). Taken together, these results indicate that ETA receptor regulates cell migration mediated through Nck1 with Gq, Src, Cdc42, and JNK (Figure 8).

Discussion

During embryogenesis, complex patterns of cell migration are essential for proper tissue formation. Bladt et al. recently demonstrated that the inactivation of Nck genes (Nck1 and Nck2) results in profound defects in mesoderm-derived embryonic structures (30). They provided genetic evidence for a role of Nck proteins in cell migration during embryogenesis using fibroblasts derived from Nck1-/- Nck2-/- embryos. In addition,
evidence indicates that endothelins and their receptors participate in the normal development of different neural crest lineages. Mice deficient in either ET-3/ETB receptor develop white spotted coats and an aganglionic megacolon due to the absence of neural crest-derived melanocytes and enteric neurons (31, 32). The phenotype of mice lacking the ETA receptor-mediated signaling causes malformations in the heart and pharyngeal arch-derived structures (33, 34). These data imply that both ET/ET receptor systems may participate in cell migration, which is essential for the normal development. Cardiovascular defects observed in the ETA receptor-deficient mice (33), as well as the Nck1-/- Nck2-/- defects in mesodermal-derived notochord (30), are in common with defects in the development of mesodermal structures in embryogenesis, which imply their role in cell migration during early development. Thus, we are investigating whether Nck1 is involved in the inhibition of cell migration induced by the ETA receptor. As a result, we show that Nck1 participates in a chemorepellent signaling pathway downstream of the ETA receptor. As far as we know, this is the first report of the role of the adaptor protein in the
regulation of cell migration downstream of GPCR. It is possible that the abnormal tissue morphogenesis observed in the ETA receptor-deficient mice depends, at least in part, upon Nck1. Cell migration includes multiple processes that are coordinately modulated by a number of regulatory proteins and driven by changes in the actin cytoskeleton (35, 36). Therefore, Nck1 could participate in the control of cell migration by binding and regulating signaling proteins involved in the rearrangement of the actin cytoskeleton. A genetic study

on Drosophila indicates that Dreadlocks (Dock), which is structurally related to the mammalian Nck genes (37), links tyrosine kinases to the actin cytoskeleton (38). Furthermore, in mammalian cells, it is also likely that Nck1 functions to couple the phosphotyrosine signals to the actin cytoskeleton (39, 40). For example, the SH2 domain of Nck1 binds the receptor tyrosine kinases, such as platelet-derived growth factor, EGF, and Eph receptors, as well as the tyrosine-phosphorylated docking proteins p62Dok-1 and p130Cas, both the substrate of Src family tyrosine kinases (39, 40). On the other hand, two Nck1 SH3 binding proteins, N-WASP and PAK1, regulate the actin cytoskeleton through Rac1- and Cdc42-dependent or -independent mechanisms (39, 40). In the present study, we suggested that Nck1 links Src kinase to the Cdc42/JNK cascade, which may be involved in the reorganization of the actin cytoskeleton. It remains to be investigated whether these known binding partners with Nck1 are involved in the ETA receptor signaling pathway in a Src/Cdc42/JNK-dependent manner. Rho GTPases act as molecular switches between active (GTP-bound) and inactive
(GDP-bound) states (41). Their activities are controlled positively by GEFs, which catalyze the replacement of GDP with GTP (42). GEFs may be the missing link between Nck1 and Cdc42 in the GPCR/JNK signaling pathway. Zhao et al. showed that the second SH3 domain (SH3[2]) of Nck1 associates with PAK, NIK, and WIP through their conserved motif, PXXPXRXXS (43). Recently, we identified a new signaling molecule, FRG, which functions as a specific GEF for Cdc42 (18). We thus analyzed whether FRG contains this Nck1 SH3[2]-binding motif. It was shown that FRG possesses a nearly identical sequence
to the Nck1 SH3[2]-binding motif. It would be interesting to examine whether FRG is a binding partner with the SH3 domain of Nck1 in the ETA receptor-signaling pathway. In this study, we demonstrated that the ETA receptor inhibits cell migration through the Src/Nck1/Cdc42/JNK pathway. On the basis of these findings, we summarized the proposed signaling pathway in Figure 8. A challenge for the future will be to define the roles of Nck1 in regulating the potential for crosstalks among the various signaling pathways involving FRG in the control of cell migration. Such studies might promote our understanding of the GPCR-regulated mechanism of the early process of development as well as oncogenesis.

Acknowledgements

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Footnotes

This work was partially supported by Grants-in-Aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (15370057) and by grants from the Yamanouchi Foundation, the Cell Science Research Foundation, and the Ono Medical Research Foundation.
The abbreviations used are CA, constitutively activated form; Cas, Crk-associated substrate; Dok, downstream of kinases; EGF, epidermal growth factor; ERK, extracellular signal-regulated protein kinase; ET, endothelin; ETA, endothelin A; FRG, Fgd-1-related Cdc42 guanine nucleotide exchange factor; GEF, guanine nucleotide exchange factor; GFP, green fluorescence protein; GPCR, G protein-coupled receptor; GST,

glutathione-S-transferase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MBP, maltose-binding protein; NIK, Nck-interacting kinase; PAK, p21-activated kinase; SH, Src homology; N-WASP, neural Wiskott-Aldrich syndrome protein; WIP, WASP-interacting protein.

Figure Legends

Figure 1. The ETA receptor-induced inhibition of cell migration involves Cdc42/JNK. Cell migration was measured in epithelial 293T cells transiently transfected with the plasmid encoding ETA receptor (A-D) and dominant negative mutants of Rho GTPases (D) using a Boyden chamber. Cells were pretreated with or without SP600125 (100 nM, 45 min) (A), U0126 (10 M, 1 h) (B), or SB203580 (10 M, 1 h) (C). After incubation at 37oC for 5 h
with (+) or without (-) ET-1 (165 nM), the cells attached to the filters were stained and analyzed under a microscope (A), and the number of stained cells was counted (A-D). Expression of ETA receptor (A-D) and the dominant negative mutants of Rho family GTPases (D) was shown. Data were evaluated using the Students t-test. Asterisks indicate p<0.01.
Figure 2. Involvement of adaptor protein Nck1 in the ETA receptor-induced inhibition of cell migration. Cells were transfected with the ETA receptor (A-C) and dominant negative mutants of Grb2 (A), CrkII (B), or Nck1 (C). After incubation for 5 h, the cells attached to the filters were stained and counted. Expression of ETA receptor and the dominant negative mutants of adaptor proteins was shown.
Figure 3. Nck1 is involved in ETA receptor-induced JNK activation. JNK activity (A, B, C, E, and F) and ERK activity (D) were measured as described in Materials and Methods.
(A-C) Cells cotransfected with the ETA receptor (A-C) and dominant negative mutants of Nck1 (A), Grb2 (B), or CrkII (C) were treated with ET-1 (100 nM, 20 min). Endogenous JNK was immunoprecipitated with anti-JNK antibody and blotted with anti-phosphorylated JNK antibody or anti-JNK antibody. The levels of JNK phosphorylation were quantified and normalized against the total immunoprecipitated JNK levels. (D-F) Cells transfected with the dominant negative mutants of adaptor proteins were treated with EGF (10 ng/ml, 10 min). Cell lysates were blotted with an anti-phosphorylated ERK or anti-ERK antibody. The levels of ERK or JNK phosphorylation were quantified and normalized against the total immunoprecipitated ERK or JNK levels. Expression of ETA receptor (A-C) and the dominant negative mutants of adaptor proteins (A-F) was shown.

Figure 4. Nck1 mediates the ETA receptor-induced activation of Cdc42. Cells were cotransfected with the ETA receptor (A-C) and dominant negative mutants of Nck1 (A), Grb2 (B), or CrkII (C). The amount of GTP-bound Cdc42 was assessed after the addition of ET-1 (100 nM, 30 min) with the pull-down assay using PAK-CRIB (A-C). The total
Cdc42 in the cell lysates was immunoblotted with anti-Cdc42 antibody. Expression of ETA receptor and the dominant negative mutants of adaptor proteins was shown.
Figure 5. Membrane-bound Nck1 induces the inhibition of cell migration. (A) Structure and expression of wild-type or myristoylation signal-tagged Nck1 (myr-Nck1). For the membrane-bound construct, the 14 amino-acid c-Src myristoylation signal was linked to the N-terminal of the wild type of Nck1. Lysates from cells transfected with wild-type or
myr-Nck1 were fractionated into crude membrane (m), cytosol (c), and nucleus (n). An equal amount (10 g of proteins) of each fraction was blotted with an anti-Nck1 antibody. (B) Cells were transfected with wild-type or myr-Nck1. After incubation for 5 h, the cells attached to the filters were stained and counted. (C-D) Cells were transfected with wild-type or myr-Nck1. The amount of phosphorylated JNK (C) or GTP-bound Cdc42 (D) was measured after stimulation of ET-1. Data were evaluated using the Students t-test. Asterisks indicate p<0.01.
Figure 6. Effects of siNck1 on the ETA receptor-induced inhibition of cell migration and Cdc42/JNK activation. (A) Sequence of the synthetic siRNA duplex targeting Nck1 (siNck1-1 or siNck1-2). The 2-nucleotide 3-overhang of 2-deoxythymidine is indicated as TT. The indicated amount of siRNAs was transfected using LipofectAMINE 2000 into cells. Cell lysates were immunoblotted with anti-Nck1 or anti-tubulin antibodies. (B) Cells were transfected with siNck1-1 or siGFP using Lipofectamine 2000. The phosphorylated JNK was detected after addition of ET-1. (C) Cells were transfected with siNck1-1 or siGFP. After incubation with ET-1, the migrating cells in the Boyden chambers were stained and counted. Data were evaluated using the Students t-test. Asterisks indicate p<0.01.
Figure 7. The Src kinase-induced inhibition of cell migration and activation of JNK involves Nck1. (A-B) Cells were cotransfected with CA-Src and dominant negative mutants of Nck1. After incubation for 5 h, the cells attached to the filters were stained and counted (A). The phosphorylated JNK was analyzed 20 min after stimulation with ET-1 (B).

Expression of CA-Src and the dominant negative mutants of Nck1 was shown.
Figure 8. Schematic model for the signaling pathway coupling the ETA receptor to the inhibition of cell migration. Details are described in the Discussion.