Cellular Signalling 24 (2012) 931–942

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Cellular Signalling

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Spermine, a molecular switch regulating EGFR, integrin β3, Src, and FAK scaffolding

Ramesh M. Ray a,⁎, Chunying Li b, Sujoy Bhattacharya a, Anjaparavanda P. Naren a, Leonard R. Johnson a

a Department of Physiology, The University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN, 38163, United Statesb Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, 540 E. Canfield Avenue, Detroit, MI, 48201, United States

Abbreviations: ODC, ornithine decarboxylase; DFMthine; FRET, fluorescence resonance energy transfer.⁎ Corresponding author at: Department of Physiol

Health Science Center, 894 Union Ave., Memphis, TN 3901 448 7168; fax: +1 901 448 7126.

E-mail address: rray3@uthsc.edu (R.M. Ray).

0898-6568/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.cellsig.2011.12.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 December 2011Accepted 19 December 2011Available online 29 December 2011

Keywords:PolyaminesMigrationOrnithine Decarboxylase (ODC)Lamellipodia

Intracellular polyamine levels are highly regulated by the activity of ornithine decarboxylase (ODC), whichcatalyzes the first rate-limiting reaction in polyamine biosynthesis, producing putrescine, which is subse-quently converted to spermidine and spermine. We have shown that polyamines regulate proliferation, mi-gration, and apoptosis in intestinal epithelial cells. Polyamines regulate key signaling events at the level of theEGFR and Src. However, the precise mechanism of action of polyamines is unknown. In the present study, wedemonstrate that ODC localizes in lamellipodia and in adhesion plaques during cell spreading. Spermine reg-ulates EGF-induced migration by modulating the interaction of the EGFR with Src. The EGFR interacted withintegrin β3, Src, and focal adhesion kinase (FAK). Active Src (pY418-Src) localized with FAK during spreadingand migration. Spermine prevented EGF-induced binding of the EGFR with integrin β3, Src, and FAK. Activa-tion of Src and FAK was necessary for EGF-induced migration in HEK293 cells. EGFR-mediated Src activationin live HEK293 cells using a FRET based Src reporter showed that polyamine depletion significantly increasedSrc kinase activity. In vitro binding studies showed that spermine directly binds Src, and preferentially inter-acts with the SH2 domain of Src. The physical interaction between Src and the EGFR was severely attenuatedby spermine. Therefore, spermine acts as a molecular switch in regulating EGFR-Src coupling both physicallyand functionally. Upon activation of the EGFR, integrin β3, FAK and Src are recruited to EGFR leading to thetrans-activation of both the EGFR and Src and to the Src-mediated phosphorylation of FAK. The activationof FAK induced Rho-GTPases and subsequently migration. This is the first study to define mechanisticallyhow polyamines modulate Src function at the molecular level.

© 2011 Elsevier Inc. All rights reserved.

1. Introduction

The polyamines, spermidine and spermine, and their precursor,putrescine, are found in virtually all cells of higher eukaryotes [1]and are intimately involved in, and required for, cell growth and pro-liferation [2,3]. Intracellular polyamine levels are highly regulatedand depend primarily on the activity of ornithine decarboxylase(ODC), which catalyses the first rate-limiting step in polyamine bio-synthesis, the decarboxylation of ornithine to form the diamine pu-trescine [4]. An increase in ODC activity is one of the earliest eventsassociated with the induction of cellular proliferation, and depletionof polyamines by DL-α-difluoromethyl- ornithine (DFMO), a specificand irreversible inhibitor of ODC, attenuates trophic responses in tis-sues and cultured cells [5–7]. DFMO has been used to inhibit ODC anddeplete cells of polyamines in many studies, and has no effects exceptthose caused by the inhibition of ODC and the subsequent decrease in

O, DL-α-difluoromethyl orni-

ogy, University of Tennessee8163, United States. Tel.: +1

rights reserved.

intracellular polyamines [8,9]. The inhibitory effects of DFMO on cellproliferation, migration and apoptosis are prevented by the additionof exogenous polyamines [9–11]. We have shown that ECM-mediated signaling increased the autophosphorylation of EGFR, Src,and FAK in a polyamine dependent manner and regulated migrationand apoptosis in IEC-6 cells [12,13]. Furthermore, the activation ofEGFR, Src, and integrin β3 by DFMO were immediate and were in-stantaneously prevented by the addition of putrescine along withDFMO [14]. Based on these results, we predicted that polyaminesmight regulate interactions among these proteins at the proximityof the plasma membrane.

Cell migration is an essential event in cell growth and cancer me-tastasis [15]. Epidermal growth factor (EGF), a ligand for EGFR, pro-motes cancer cell migration and metastasis by activating multipledownstream protein kinases, such as c-Src [16], focal adhesion kinase[17], and p21-activated kinase [18]. The c-Src protein is composed ofan N-terminal myristylation sequence that directs the association ofproteins with the plasma membrane, a unique region where thegreatest sequence divergence among family members occurs, a Src-homology-3 (SH3) domain and an SH2 domain that mediate pro-tein–protein interactions, a kinase domain and a C-terminal regulato-ry domain [19]. Src is located in the cytoplasm, at cellular sites ofintegrin clustering (the so-called focal adhesions in fibroblasts) and

932 R.M. Ray et al. / Cellular Signalling 24 (2012) 931–942

at cadherin-mediated cell–cell adhesions in epithelial cells. Both theEGFR and c-Src are over-expressed in a variety of human tumors, in-cluding breast cancer, suggesting that these tyrosine kinases mayfunctionally interact and contribute to the progression of the disease[20,21]. FAK, a non-receptor tyrosine kinase, is activated and autop-hosphorylated at Y397 by binding to integrin complexes [22]. C-Srckinase then is recruited to the FAK complex by binding at phosphor-ylated Y397 through its SH2 domain [23]. Upon recruitment at Y397,Src phosphorylates multiple tyrosine residues and fully activates FAKand thereby, downstream signaling [24]. Although, EGF initiates sig-nal transduction between the EGFR and FAK, evidence for the directinteraction between these proteins is lacking. We have shown thatfibronectin-induced integrin activation is associated with EGFR phos-phorylation and downstream activation of ERK1/2 [14]. Since the in-hibition of Src by PP2 decreased the phosphorylation of the EGFRand integrin β3, it appears that the activation of the EGFR and integrinβ3 is linked by a common mechanism involving Src.

Polyamines are reported to be involved in many actions of EGF.For instance, DFMO can prevent the inhibition of parietal cell acid se-cretion by EGF [25]. Polyamines inhibit EGFR tyrosine kinase activityin A-431 cells [26]. In a colon cancer cell line, Caco-2, polyamine up-take is stimulated by EGF and inhibited by genistein, a tyrosine phos-phorylation inhibitor [27]. The overexpression of ODC is oncogenic intransfected NIH/3T3 cells [28]. In L6 cells and fetal bovine myoblasts,EGF stimulates polyamine biosynthesis, suggesting that the biosyn-thesis of polyamines is important for the early events mediated byEGFR [29]. In this study, we identified a specific polyamine-proteininteraction, which is responsible for the polyamine effect on Src sig-naling in response to EGF. This is the first study to define mechanisti-cally how polyamines modulate Src function at the cellular andmolecular level.

2. Materials and methods

2.1. Cell lines, vectors and cloning

HEK293 cells were grown in DMEM-F12 (Invitrogen) supplementedwith 10% FBS. For FRET imaging, HEK293 cells were transfected withpcDNA3 (containing CFP-Src-YFP, a kind gift from Dr. Shu Chien at theUniversity of California-San Diego, La Jolla, California), and positive col-onies were selected using 400 mg/ml G418 and confirmed by fluores-cence microscopy. For pull-down and co-immunoprecipitationstudies, HEK293 cells were transfected with a lentiviral expression sys-tem encoding V5-tagged EGFR (tag at the C-terminus), and the positivecolonies were selected using 2.0 mg/ml puromycin. Various domains ofc-Src (mouse neuronal) were subjected to PCR with complimentaryoverhangs created by building appropriate 5′ extensions into theprimers according to the manufacturer's instructions (Novagen). ThePCR products were purified (Qiagen PCR purification kit) and treatedwith Ligation-independent cloning (LIC)-qualified T4 DNA polymerasein the presence of dATP to generate pTRIEX-4 and pET41 vector specificoverhangs (Novagen). Clones were selected and sequenced for confor-mation. pTRIEX-4 has an N-terminal his6- and an S-tag. pET41 has anN-terminal GST-, his6- and S-tag. The EGFR cDNA sequence was origi-nally cloned in pLenti6-V5-D-TOPO [30] but was separated from theV5 epitope by a stop codon. To remove the stop codon and place theEGFR sequence in-frame with the V5-epitope, an EGFR fragment fromnucleotide 2704 to 3630 that lacked a stop codon and was flanked byBstEII and Xho1 restriction sites was amplified by PCR and insertedinto the BstEII/XhoI digested pLenti6 V5-D-TOPO EGFR construct. Theresultant pLenti6-V5+D-TOPO EGFR construct was transfected into293FT cells (Invitrogen, Carlsbad, CA) and packaged into virus usingthe Virapower Lentivirus packaging system (Invitrogen, Carlsbad CA)according to the manufacturer's instruction. HEK293 cells were thentransduced with the virus at a multiplicity of infection no less than106 cfu/mL. Stable clones were selected using blasticidin at a

concentration of 5 mg/mL andmaintained at a blasticidin concentrationof 2 mg/mL. CA-Src, DN-Src plasmid and pUSE empty vector were pur-chased from Millipore (Billerica, MA).

2.2. Cell culture

The IEC-6 cell line (CRL-1592) was obtained from the AmericanType Culture Collection (Manassas, VA) and maintained in T-150flasks in Dulbecco's Minimal Essential Medium (DMEM) supplemen-ted with 10% FBS, 10 μg/ml insulin and 50 μg/ml gentamicin sulfate at37°C and 10% CO2. Stock cells were passaged once a week and medi-um was changed three times a week. Prior to an experiment cellswere trypsinized, counted using a Beckman Coulter counter andgrown for 3 days in dialyzed FBS in control medium and wereserum starved for 24 h prior to an experiment.

2.3. Cell migration assay

This assay was carried out as described previously [31]. Six wellplates containing a confluent monolayer of cells were marked in thecenter (along the diameter) with a marker. A wound was created per-pendicular to the mark by scraping the monolayer with a gel loadingmicro-tip. Plates were washed, and the wound area was capturedwith a charged coupled device (CCD) camera system using NIHImage (Version1.62) at the intersection of the marked line at 0 hand at the desired time point (7 h). The wound area covered duringmigration was measured using Image J (Version1.36b) software. Cellmigration was calculated as the wound area covered. Each experi-ment was carried out in triplicate and two observations wererecorded from each well (n=6).

2.4. FRET imaging microscopy and image analysis

FRET imaging and image analysis were performed as described be-fore [30]. Briefly, HEK293 cells (expressing stable CFP-Src-YFP) forFRET imaging were seeded in the 35-mm glass bottom culture dishes(MatTek) and grown for 48–72 h in DMEM (with 10% FBS) containingcontrol, 5 mM DFMO, 5 mM DFMO plus 10 μM putrescine (or 5 μMeach for spermidine, spermine, and putrescine), or 10 μM putrescineonly (or 5 μM each for spermidine, spermine, and putrescine), andthen continued to grow in serum-deprived medium containing theabove compounds for another 24 h before the FRET assay. Prior tothe FRET imaging, cells were washed twice with Hanks' balance saltsolution (HBSS) before being mounted on an Olympus microscopysystem for FRET imaging. Cells were maintained in HBSS (containingthe above-mentioned respective compounds) in the dark at roomtemperature with the addition of EGF as indicated. Images wererecorded with a cooled charged-coupled device camera HamamatsuORCA285 (Hamamatsu, Japan) mounted on the Olympus microscopeIX51 (U-Plan Fluorite 60×1.25 NA oil-immersion objective), and thesystem was controlled by SlideBook 4.1 software (Intelligent ImagingInnovations, Denver, CO) with ratio and FRET modules used to obtainand analyze the FRET images. Excitation light was provided by a300 W Xenon lamp and attenuated with a neutral density filter with50% light transmission. Images were captured using a JP4 CFP/YFP fil-ter set (Chroma, Brattleboro, VT) including a 430/25-nm (25-nmband-pass centered at 430-nm) excitation filter, a double dichroicbeam splitter (86002v2bs), and two emission filters (470/30-nm forCFP and 535/30-nm for FRET) alternated by a high-speed filter-changer Lambda 10–3 (Sutter Instruments, Novato, CA). Time-lapseimages were acquired with 4×4 binning mode, 200–400 ms expo-sure time, and 1-min intervals to reduce photo bleaching of the fluor-ophores. Acquired fluorescent images were background-subtracted,and multiple regions of interest (ROIs) on the cell periphery were se-lected for quantitative data analysis (~20–30 ROIs per cell, and 4–6cells per condition were averaged). The emission ratio images (CFP/

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FRET) were generated at different time points on a pixel-by-pixelbasis by the SlideBook ratio module and normalized through dividingall ratios by the emission ratio right before the addition of reagents,thereby setting the basal emission ratio to 1, as formulated below,

R ¼ Emission intensity of CFP imageEmission intensity of FRET image

The ratio (R) was normalized as Rt/R0, where Rt is the ratio at timepoint t and R0 is the ratio at time point=0 (right before the additionof first test compound). The cell images are presented in pseudocolorto highlight the changes in the ratio of CFP/FRET fluorescenceintensity.

2.5. ELISA assay

Different amounts of the GST, or GST-Src (100 μl) in TBS were ap-plied to duplicate wells and incubated for 1 h at 22 °C. The plateswere washed three times with 200 μl/well of TBS containing 0.1%Tween 20 (TBST), followed by blocking with 200 μl of TBST containing1% BSA at 22 °C for 1 h. After decanting the blocking solution, 100 μl ofBSA-conjugated spermine in TBST was added and incubated with theimmobilized GST or GST-Src for 3 h at 22 °C (or at 4 °C overnight). Theplates were washed three times with TBST, and then blocked againwith 200 μl of TBST containing 1% BSA at room temperature for20 min. 100 μl/well of the Primary Antibody (anti-Spermine anti-body; 1:10,000 dilution in TBST/1% BSA) was added and incubatedfor 1 h at 22 °C. Plates were washed three times with TBST, followedby incubation with 100 μl/well of the secondary antibody (goat anti-rabbit HRP-conjugated, 1:20,000 dilution in TBST/1% BSA) for 1 h at22 °C. After three washes with TBST, 100 μl of the 1-Step™ SlowTMB-ELISA substrate was added to each well, and incubated atroom temperature in the dark for 5–30 min. The reaction was stoppedby adding 100 μl of 2 M sulfuric acid (H2SO4) to each well. The absor-bance of each well was measured at 450 nM using a microplate read-er (Molecular Devices, CA).

2.6. Pair-wise binding assay

For pair-wise binding between the SH-2 domain of His-S-Src orGST-His-S-Src and BSA-conjugated spermine, lysis buffer (200 μl)was added into a 1.5 ml microfuge tube containing 5 mg of BSA-spermine. Purified His-S-Src SH domain (20 mg) or GST fusion pro-teins (0–40 mg) were added and mixed at 22 °C for 30 min. To thismix, 20 ml S-beads or glutathione beads were added and mixed for16 h at 4 °C. The mixture was washed 3 times with lysis buffer fol-lowed by centrifugation at 3000 ×g for 1 min. 15 μl of 5X-samplebuffer was added to the tubes containing complexes and subjectedto western blot analysis. Blots were probed using anti-spermineserum (1:2000; Chemicon). For pair-wise binding between GST orGST-Src SH2 domain and EGFR-V5 in the presence of BSA-conjugated spermine (BSA-Spermine), lysis buffer (200 μl) wasadded into a 1.5 ml microfuge tube containing various amounts ofBSA-spermine. Purified GST or GST-Src-SH2 (5 mg) were added andmixed at 22 °C for 30 min, followed by the addition of purified theEGFR-V5 (200 ng), and the mixture was incubated for another 2 h.Then, to this mixture, 20 ml glutathione beads were added andmixed for 16 h at 4 °C. The mixture was washed 3 times and pro-cessed for western blot analysis as described above. Blots wereprobed using anti-V5 IgG (1:5000).

2.7. Western blotting

Protein samples were separated by SDS-PAGE and transferred toPVDF (polyvinylidene difluoride) membranes. The membranes werethen blocked with either 5% bovine serum albumin (BSA) or blocking

grade non-fat dry milk made in tris-buffered saline containing 0.1%Tween 20. Appropriate primary and secondary antibodies were usedto detect the proteins of interest by enhanced chemiluminescence de-tection reagents.

2.8. Immunocytochemistry

Immunolocalization studies of proteins were done as describedpreviously [32,33]. Cells seeded on poly-L-lysine-coated glass cover-slips placed in 24-well plates were allowed to attach and spread.Cells were fixed with 3.7% para-formaldehyde for 15 min, washedtwice with DPBS, permeabilized with 0.1% Triton X-100 for 10 min,and washed again with PBS. Blocking was carried out with 2% BSAfor 20 min followed by a two hour incubation with the appropriateprimary antibody. The coverslips were washed with PBS, followedby incubation with an appropriate fluorescent dye-conjugated sec-ondary antibody. The coverslips were mounted on glass slides andphotographed using a Nikon Diaphot inverted fluorescence micro-scope with appropriate filters.

2.9. Pull-down assay

Pull-down assays were performed as described before [34–36].Briefly, HEK293 cells overexpressing the EGFR-V5 were lysed inlysis buffer (PBS/0.2% Triton X100+protease inhibitors). Cell lysateswere mixed at 4 °C for 15 min followed by centrifugation at16,000 ×g for 10 min at 4 °C. GST or GST-Src-SH2 domain or -SH3 do-main were added and mixed with the clear supernatant and incubat-ed for 30–60 min at 4 °C followed by additional incubation for 2 hwith glutathione Sepharose beads (20 μl). Thereafter, the mixtureswere spun at 800 ×g for 2 min, and the beads were washed threetimes with the same lysis buffer before the proteins were elutedfrom beads with Laemmli sample buffer (containing 2.5% β-mercaptoethanol). Eluates were separated on 4–15% gel and immu-noblotted with anti-V5 IgG.

2.10. Co-immunoprecipitation

Confluent serum starved IEC-6 cells left untreated or treated withEGF were exposed to 10 μM spermine for 5–15 min and washed withDPBS. Cell lysates were subjected to immunoprecipitation using EGFRantibody at 4 °C. Immunoprecipitates were washed twice with TBST,and the proteins were eluted from beads with Laemmli sample buffer.Eluates were analyzed by western blotting to detect the levels of Src,integrin β3, EGFR, pY-EGFR, and FAK proteins. The HEK293 cells over-expressing EGFR-V5 were lysed in lysis buffer containing 15 μM poly-amines consisting of 5 μM each of spermidine, spermine, andputrescine for 10 min at 4 °C. The lysate was centrifuged (16,000 ×g,10 min, 4 °C), and the clear supernatant was used to immunoprecipi-tated the EGFR using immobilized anti-V5 IgG beads (or non-immune) in a 15 ml tube. Tube contents were mixed at 4 °C for 16 hand washed 3 times with lysis buffer (3000 ×g, 1 min each). Thebeads were transferred to a Spin Column (Pierce Chem. Co) andwashed with lysis buffer (100 mM Tris-0.2% TX-100) followedby10 mM Tris-0.2% TX-100, and eluted the antigen using 100 μl ofelution buffer (Glycine 100 mM, pH 2.2 containing 0.2% TX-100).The pH of the eluate was neutralized using 10 ml of 1 M Tris–HCl,pH 8.8 and subjected to western blotting. Blots were probed usinganti-Src and V5 IgG.

2.11. Data analysis and statistics

Results are presented as means±SEMs for the FRET, ELISA, andmigration studies. Western blots and examples of immunocytochem-istry shown are representative of at least three observation. Statistical

934 R.M. Ray et al. / Cellular Signalling 24 (2012) 931–942

analysis was performed using Student's t-test. A value of Pb0.05, orPb0.01 was considered significant.

3. Results

3.1. Role of Src and spermine in EGF-induced migration

Our earlier results suggested that the observed effects of poly-amines on migration and apoptosis might be mediated through the

Fig. 1. Role of Src in the EGF-induced migration. A, confluent monolayers of IEC-6 cells weretreated with 10 nM EGF in the presence and absence of 10 μM PP2. B, migration was calcula* Significantly different compared to UT group. C, following attachment and spreading of celfor the pY418-Src and FAK as described in the Materials and methods. Representative imwounded with a gel loading tip in the center of the plates, washed and left untreated or tMigration was calculated as described in the Materials and methods. Values are means±SEMof IEC-6 cells stably expressing empty vector, constitutively active (CA), and dominant negaand left untreated (UT) or treated with 10 nM EGF. Migration was expressed as percent untgroup, # significantly different compared to cells expressing empty vector and treated with

interaction of polyamines with either integrin-β3, the EGFR, or Src[12–14]. We have also shown that EGF increased the phosphorylationof Src (pY418-Src) and, thereby, its activation in IEC-6 cells [14]. EGFsignificantly decreased the wound width compared to that seen inuntreated monolayers (Fig. 1A). Furthermore, inhibition of Src byPP2 almost completely prevented EGF-induced migration and alsoinhibited basal migration (Fig. 1A and B). Although, PP2 had no effecton EGF-induced ERK1/2 activation [14], it completely preventedEGF-induced migration suggesting that Src activity is essential for

wounded with a gel loading tip in the center of the plates, washed and left untreated orted as described in the Materials and methods. Values are means±SEMs of triplicates.ls, coverslips were washed, fixed, permeabilized and processed for immunolocalizationages from three observations are shown. D, confluent monolayers of IEC-6 cells werereated with 10 nM EGF in the presence and absence of 2.5 μM FAK14 (FAK inhibitor).s of triplicates. * Significantly different compared to UT group. E, Confluent monolayerstive (DN) Src were wounded with a gel loading tip in the center of the plates, washedreated. Values are means±SEMs of triplicates. * Significantly different compared to UTEGF.

935R.M. Ray et al. / Cellular Signalling 24 (2012) 931–942

migration and that MEK/ERK may regulate downstream processes ac-tivated by Src. Activated Src strongly localized with focal adhesion ki-nase (FAK) during spreading of these cells (Fig. 1C). The inhibition offocal adhesion kinase activity by F14 significantly inhibited EGF-induced migration (Fig. 1D). Since pharmacological inhibitors mayhave non-specific effects on other kinases, we examined EGF-induced migration in IEC-6 cells transfected with constitutively active(CA) or dominant negative (DN) Src. IEC-6 cells lines stably expres-sing CA-Src and DN-Src were characterized and reported in a previousstudy by our laboratory [37]. Consistent with observations reported inFig. 1B, EGF increased migration in cells transfected with empty vec-tor. Cells expressing CA-Src increased basal migration, which was fur-ther increased in response to EGF (Fig. 1E). Conversely, expression ofDN-Src blocked basal and EGF-induced migration, suggesting that Srcplays a critical role in the regulation of migration.

Since spermine prevented the activation of Src by EGF and DFMOand eliminated the protection against apoptosis conferred by EGF

Fig. 2. Spermine inhibits EGF-induced migration. A, confluent monolayers of IEC-6 cells weretreated with 10 nM EGF in the presence and absence of 10 μM of spermine or triethyleneteValues are means±SEMs of triplicates. * Significantly different compared to UT group. B, sattach and spread. Cells were washed, fixed and processed for localization of ODC and f-actlocalization in lamellipodia is shown (upper panel), A group of cells showing ODC and F-acexperiments are shown.

and DFMO in IEC-6 cells [14], we examined the effect of spermineon EGF-induced migration to understand the early signaling eventsregulated by polyamines. EGF increased migration, measured aswound healing, about 3 fold compared to the untreated monolayer(Fig. 2A). Addition of 5 μM spermine significantly decreased EGF-induced migration (Fig. 2A). The specificity of the effect of sperminewas confirmed by using triethylenetetramine (TETA), which has thesame number of amine groups but a shorter carbon skeleton. Unlikespermine, TETA failed to block EGF-induced migration (Fig. 2A). Wealso confirmed that 5 μM spermine had no apoptotic effects onthese cells (data not shown). Our recent study demonstrated thatSrc activation is essential for the attachment and spreading of cells[12], and results in Fig. 2B show that cells formed large lamellipodia,F-actin stress fibers, and focal plaques during spreading. In thesecells, ODC localized in the lamellipodia as well as with the focal pla-ques (Fig. 2B, arrows), suggesting the involvement of polyaminesduring attachment and spreading. These data suggest that Src plays

wounded with a gel loading tip in the center of the plates, washed and left untreated ortramine (TETA). Migration was calculated as described in the Materials and methods.erum starved IEC-6 cells were plated in DMEM containing dFBS and were allowed toin as described in the Materials and methods. A cropped image of a cell showing ODCtin in lamellipodia and focal plaques (lower panel). Representative images from three

Fig. 3. Spermine prevents interaction of EGFR with integrin β3, Src, and FAK. Confluentmonolayers of IEC-6 cells were washed and left untreated or treated with 10 nM EGF inthe presence and absence of 10 μM of spermine for 10 min. A, Monolayers werewashed and lysates were subjected to immunoprecipitation using anti-EGFR antibody.Membranes were probed for the detection of integrin β3, Src, and FAK. Membraneswere striped and probed for pY-EGFR and total-EGFR using specific antibodies. Blotsshown are representative of three observations. B, Whole cell lysates were analyzedby western blot using FAK, integrin β3 and Src-specific antibodies. Representativeblots from three experiments are shown.

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a key role in regulating downstream signal transmission during mi-gration and also suggest the possibility that spermine modulates Srcactivation. We therefore, examined the binding of Src with the EGFRin cells treated with EGF for various time intervals in the presenceand absence of spermine. EGF activated the EGFR and binding of Srcto the EGFR was detected at 10 min (Fig. 3A). EGFR activation was ev-ident by increased phosphorylation (pY-EGFR), which was associatedwith decreased EGFR levels due to internalization and degradation.Spermine prevented the binding of the EGFR with Src at 10 min(Fig. 3A) without affecting Src expression (Fig. 3B). Furthermore,spermine decreased EGF-induced internalization and degradation ofEGFR and consequently led to the accumulation of pY-EGFR. Wereported earlier that EGF rapidly increased the phosphorylation ofEGFR, integrin β3, Src, and FAK in IEC-6 cells [12,14]. Therefore, wedetermined binding of integrin β3 and FAK with the EGFR in cellstreated with EGF. Both FAK and integrin β3 binding with the EGFR in-creased in response to EGF. However, total FAK and integrin β3 pro-tein levels did not change (Fig. 3B). Spermine inhibited theassociation of EGFR with FAK and integrin β3 indicating that sper-mine binds either Src or integrin β3, and modulates their activitiesand the activities of the EGFR associated binding partners includingFAK and Src.

Fig. 4. Polyamines prevent Src activation in live cells. A, confluent monolayers of HEK293untreated or treated with 10 nM EGF in the presence and absence of 10 μM PP2 and 10 μMValues are means±SEMs of triplicates. * Significantly different compared to UT group, andemission ratio images in response to EGF in HEK293 cells overexpressing a Src reporter preand putrescine) for 3–4 days before the FRET imaging. Panel b, the time courses of normaliz(first arrow: 50 ng/ml, the second arrow: 250 ng/ml). Panel c, the bar graphs of the norma

3.2. Polyamine depletion augments EGF-induced Src activation in liveHEK293 cells

To demonstrate that EGF promotes cell migration via a Src and FAK-dependent manner; we tested the role of these protein kinases in EGF-induced migration in HEK293 cells. EGF significantly increasedmigration of HEK293 cells and pharmacologic inhibition of Src kinase(using PP2) blocked basal and EGF-induced migration (Fig. 4A), whichis similar to IEC-6 cells. Furthermore, inhibition of FAK (using FAK14)had no effect on basal migration, but blocked EGF-induced migration,suggesting that EGF-induced migration requires activation of Src andFAK (Fig. 4A). Upon confirming the role of Src and FAK in EGF-induced migration of HEK293 cells, we used a fluorescence resonanceenergy transfer (FRET) based reporter for Src kinase activity inHEK293 cells [34] as a tool to determine the effects of polyamines onSrc activity. This Src reporter is composed of CFP at the N-terminus,the SH2 domain of c-Src, a Src substrate peptide, and YFP at the C-terminus. The proximity of the N and C termini of the SH2 domain al-lows the juxtaposition of CFP and YFP to yield a high FRET. Upon Srcphosphorylation, the substrate peptide can bind to the phospho-peptide-binding pocket of the SH2 domain and separate YFP from CFP,thus decreasing FRET [34]. Phosphorylation of the Src reporter by acti-vated endogenous Src enhances CFP emission at the expense of YFPemission, leading to an increased cyan-to-yellow emission ratio (CFP/FRET or CFP/YFP). Because of the low efficiency of transfection in IEC-6 cells, HEK293 cells were transfected with the Src reporter and usedin this study. EGF induced a 5–10% emission ratio change in a dose-dependent manner [Fig. 4B, panels a–c], indicating Src phosphorylationin real time in live cells. Polyamine depletion by DFMO treatment eli-cited a 10–20% emission ratio change, demonstrating a significant in-crease in EGF-induced Src activity compared to the control (Fig. 4B,panels a–c). However, the phosphorylation of Src in response to EGFwas attenuated when cells were grown with exogenous polyaminesor DFMOplus exogenouspolyamines (Fig. 4B, panels a–c). It is notewor-thy that DFMO-treated cells demonstrated a higher basal level emissionratio than cells grown in control or DFMO plus exogenous polyaminescontaining media (Fig. 4B, panels a–c).

3.3. Spermine interacts directly with Src and associates preferentiallywith the SH2 domain

Src is a 60 kDa protein comprised of SH2, SH3, and catalytic (ki-nase) domains (Fig. 5A). The domain structure accounts for its abilityto interact with various effector molecules and regulate signalingpathways. The SH2 domain is reported to interact with FAK [38] andEGFR [39], while the SH3 domain has been shown to bind to integrin[40]. Given that polyamines can affect Src kinase activity, we testedwhether polyamines could physically interact with Src. GST-Src fu-sion protein was purified as previously reported [35], and the purifiedGST-Src was recognized specifically by anti-Src monoclonal and poly-clonal antibodies in dot blots (data not shown). An ELISA-based pair-wise binding assay was performed to detect the interaction betweenGST-Src and BSA-conjugated spermine (BSA-spermine). Anti-spermine antibody was generated using BSA-spermine as an immu-nogen. Therefore, we used the same BSA-Spermine for the in vitrobinding and pull down assays. Pair-wise binding detects interactionsbetween two individually purified proteins [36]. In cells, precursorputrescine is rapidly converted to spermidine and subsequently tospermine. Therefore, we used spermine in binding assays. Our data

cells were wounded with a gel loading tip in the center of the plates, washed and leftFAK14 for 20 h. Migration was calculated as described in the Materials and methods.# significantly different compared to cells treated with EGF. B, panel a, the CFP/FRETtreated with DFMO (5 mM) and/or polyamines (5 mM each for spermidine, spermine,ed CFP/FRET emission ratio of the Src reporter. The arrows indicate the addition of EGFlized CFP/FRET emission ratio at the time point (20 min) after EGF addition (n=4–6).

937R.M. Ray et al. / Cellular Signalling 24 (2012) 931–942

Fig. 5. Spermine interacts directly with Src and associates preferentially with the SH2 domain. A, various domains of mouse neuronal c-Src. B, ELISA-based pair-wise binding of GST-Src with BSA-conjugated spermine (n=3). BSA-conjugated spermine was incubated with GST or GST-Src immobilized in the ELISA wells, followed by the incubation with anti-spermine serum. The absorbance was measured at 450 nM. C, PCR-cloned His-S-tagged or GST-tagged Src domain proteins resolved by SDS-PAGE. D, pair-wise binding betweenHis-S-SH2 and His-S-SH3 domains of Src with BSA-spermine. The mixture was pulled down using S-beads, and blotted with anti-spermine serum. Representative blots fromthree independent observations are shown. E, pair-wise binding between BSA-spermine and different doses of GST-His-S-SH2 domain of Src. The mixture was pulled downusing glutathione beads, and blotted with anti-spermine serum. A representative blot from three independent observations is shown.

938 R.M. Ray et al. / Cellular Signalling 24 (2012) 931–942

showed a dose dependent binding of GST-Src with BSA-spermineagainst BSA as the blank (Fig. 5B). Furthermore, we sought to definethe domain(s) of Src involved in the interaction with spermine. Inorder to determine the precise location of the spermine-binding siteon Src, we cloned the SH2 and SH3 domains of Src in pTriEx-4 andpET-41 vectors (Fig. 5C). The fusion proteins were expressed and puri-fied from bacteria, and we found that both the SH3 (amino acids1–150) and the SH2 (amino acids 150–270) domains were quite stable(Fig. 5C). Purified SH2- and SH3-domain containing fusion proteinswere incubated with BSA-Spermine in pair-wise binding assays. TheSH2-domain bound directly to spermine, while the SH3-domain lackedspermine binding (Fig. 5D). BSA alone did not bind to either the SH2- orSH3-domain. Dose-dependent binding (0–40 μg) of GST-SH2with BSA-spermine was observed, and maximal binding was seen with 20 μg ofGST-SH2 (Fig. 5E). These observations suggest that spermine binds di-rectly and preferentially to the SH2 domain of c-Src.

3.4. Physical association of EGFR with the Src-SH2 domain is inhibited byspermine

Accumulated evidence suggests that the mutual interaction be-tween activated EGFR and c-Src is critical to many EGFR-mediatedcellular functions [20,21]. Based on results shown in Fig. 5, it appears

that polyamines could disrupt both physical and functional couplingof EGFR and Src kinase. To test this, we generated a HEK293 cell linethat stably overexpressed V5-tagged EGFR in a lentiviral expressionsystem (Supplemental Fig. S1A, left panel) [41] and determinedEGFR expression in HEK293 cell and its phosphorylation by EGF.EGF increased EGFR phosphorylation in a dose-dependent fashion(Fig. 6A), which is similar to IEC-6 cells. Immunoprecipitation of EGFRfrom HEK293-EGFR-V5 cells that express endogenous Src incubated inthe presence or absence of spermine showed a significant reduction inthe amount of endogenous Src that co-immunoprecipitated with V5-tagged EGFR protein when cells were exposed to spermine (Fig. 6B). Asimilar decrease in Src binding with EGFR was evident when theEGFR-V5 cell extract was incubated in the presence of 15 μM poly-amines (5 μM each of putrescine, spermidine, and spermine) (Fig. 6BRight panel). We purified V5-tagged EGFR (Supplemental Fig. S1A,right panel) and confirmed its identity by MALDI TOF/Mass Spec (Sup-plemental Fig. S1B). GST or GST-SH2 and GST-SH3 domains of Src pro-teins were employed to pull-down EGFR-V5. We found that SH2domain of Src binds to the EGFR with the highest affinity (Fig. 6C), con-sistent with the previous report [39]. Next, we performed pair-wisebinding between purified EGFR-V5 and the purified GST-SH2 domainof Src in the presence of exogenous spermine. Our data demonstratedthat spermine exhibited a dose-dependent inhibitory effect on the di-rect interaction between EGFR and the SH2-domain of Src (Fig. 6D).

Fig. 6. Physical association of the EGFR with the Src-SH2 domain is inhibited by polyamines. A, EGFR phosphorylation in response to EGF in HEK293 cells overexpressing V5-taggedEGFR. The cells were subjected to EGF treatment for 30 min at 37 °C before being lysed in the lysis buffer. The overexpressed EGFR-V5 was immunoprecipitated with anti-V5 IgG,and immunoblotted with anti-pY-HRP (for phosphor-EGFR-V5). A representative blot from three independent observations is shown. B, co-immunoprecipitation of endogenous Srcand over-expressed EGFR in HEK293 cells in the presence or absence of 0.6 mM spermine or 15 μM polyamines (consisting of 5 μM each of spermidine, spermine, and putrescine).The cells were lysed in lysis buffer containing the above-mentioned compounds before being subjected to co-IP. Representative blots with densitometry from three independentobservations are shown. C, interaction between the EGFR and various domains of Src. EGFRs-V5 over-expressed in HEK293 cells were subjected to pull-down assay using GST-Src fusion proteins, and probed with anti-V5 IgG. Representative blots from three independent observations are shown. D, pair-wise binding between purified EGFR-V5(200 ng) and the GST-Src SH2 domain (5 μg) in the presence of polyamines (i.e., spermine conjugated to BSA; 0–300 μM). A representative blot and densitometry are shownfrom three independent observations.

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These results clearly demonstrate that EGFR binds preferentially to theSH2-domain of Src kinase, and that this binding is inhibited by sper-mine. It is clear from this study that spermine binds the SH2-domainand modulates its interaction with its binding partners.

4. Discussion

There is agreement that the effects of polyamines depend on theirpositive charges. Putrescine, spermidine and spermine contain two,

three, or four amine groups, respectively, with pK values above 9, sothey are polycations at physiological pH and strongly bind to nega-tively charged macromolecules, particularly nucleic acids and pro-teins. The fact that these are not point charges, but are fixed along aflexible carbon chain, allows polyamines to interact with macromole-cules in structurally specific ways. Spermine, for example, binds tophosphate groups of the DNA helix, occupying the small groove andstabilizing the helix by binding the two strands together [42,43]. Atphysiologic pH, spermine stimulation is seen at NR1/NR2B subunits

940 R.M. Ray et al. / Cellular Signalling 24 (2012) 931–942

of NMDA receptors [44]. Ishihara and Ihara showed that spermineand spermidine blocked the strong inward rectifier K (+) channelKir2.1 [45]. Thus, direct interaction of polyamines with proteins mod-ulates functional properties. The interaction of spermidine/spermineacetyltransferase (SSAT) with α9β1 integrin at the receptor levelcaused a localized inhibition in the levels of polyamines and aug-mented potassium efflux and enhanced migration [46]. These studiesindicate that polyamines affect global as well as localized effects onmigration [47].

At this juncture it's important to discuss the concept of “poly-amine depletion”. DFMO is a perfect suicide inhibitor of ODC. Manyinvestigators have shown that its only effect is to block ODC, andthat the biological effects are all caused by a reduction in cellularpolyamines. We know this because in each instance supplying exog-enous putrescine, spermidine, or spermine along with DFMO pre-vented the biological effect of DFMO. Following DFMO treatment,putrescine disappears almost immediately, spermidine within 24 h,and spermine decreases to 40–50% of normal after 4 days [8,9]. Wehave described several instances where DFMO produces effects with-in an hour or two, NF-κB [48] and STAT-3 [49] activation, respectively,and within a few minutes, EGFR and Src phosphorylation [14]. Thecells still contain large amounts of polyamines, yet these early eventsare also prevented by exogenous polyamines and are due to poly-amine depletion. We believe that in these instances, it is the free ac-cessible, biologically active pool that is being depleted. Almost allcellular polyamines are bound to macromolecules due to their strongpositive charges, and most are present in granules or in the nucleus.This bound pool may well be important for certain cell functions,but newly synthesized, free polyamines appear to be essential aswell. This is best illustrated by the many experiments showing thatadministration of growth factors, trophic hormones, serum, and dam-aging agents cause ODC activity to increase from barely detectable tomaximum level within 3 h in cultured cells and in the appropriate tis-sues in vivo [5–7]. We believe that immediately upon exposure toDFMO, putrescine production ceases and any remainder is rapidlyconverted to spermidine and spermine and bound.

Over the years, we have demonstrated that polyamine depletioninhibits proliferation, apoptosis and migration of IEC-6 cells[10,12,14,37,50]. All of the terminal effects of polyamine depletionare regulated at the level of MEK/ERK and Src pathways, which con-verge on EGFR and integrin β3. We have shown that polyamine de-pletion achieved by growing cells in the presence of 5 mM DFMOfor 4 days significantly increases autophosphorylation of Src, integrinβ3, and EGFR without affecting the levels of these proteins [37]. In ad-dition, activation of integrin β3 trans-activated the EGFR and inducedERK1/2 activity in these cells. Furthermore, inhibition of ODC byDFMO significantly increased the phosphorylation of the EGFR, integrinβ3, and Src within 30 min, which was prevented by the addition of pu-trescine along with DFMO [14]. Thus, the EFGR and integrin β3 respondto instantaneous as well as prolonged changes in the intracellular poly-amine content. Since both the EGFR and integrin β3 activated Src[14,37], and since Src also caused the reciprocal activation of both re-ceptors, we predicted that polyamines might regulate Src by modulat-ing the interaction between Src, integrin β3 and the EGFR.

The protective effects of polyamine depletion on apoptosis are dueto the constitutive activation of antiapoptotic pathways. However, itis intriguing that the same pathways reported to increase migration,had inhibitory effects on migration in polyamine depleted cells. Theregulation of migration involves dynamic remodeling of the F-actincytoskeletal structure, and FAK is essential for the Tiam1-mediatedactivation of Rho-GTPases during migration [12]. We presume thatconstitutive activation of Src in polyamine-depleted cells may pre-vent dynamic turnover of FAK during migration. Therefore, it appearsthat the spatio-temporal changes in the levels of polyamines regulatethe turn over of Src-mediated FAK activity and, thereby, migration.Cell migration involves the coordinated activation and deactivation

of signaling proteins allowing the dynamic organization of F-actin,extension and retraction of laemilipodia, and focal adhesions. Thus,all of these processes are transient and spatial.

The pharmacological inhibition of Src and expression ofdominant-negative Src decreased basal as well as EGF-inducedmigra-tion (Fig. 1). In addition, basal migration increased in cells expressingconstitutively active Src (Fig. 1E) suggesting that Src is essential formigration in response to EGF and to migration in general. 10 μM sper-mine significantly inhibited EGF-induced migration while TETA didnot (Fig. 2A). Compared to spermine, TETA contains a shorter carbonbackbone with the same number of cationic charges (Fig. 2A). Theseobservations indicate that these effects of spermine are specific. Acti-vated Src (pY418) localized with FAK during cell spreading, and theinhibition of FAK by Fak-14 significantly decreased basal as well asEGF-induced migration. Furthermore, EGF increased migration ofHEK293 cells and pharmacologic inhibition of Src inhibited basaland EGF-induced migration (Fig. 4A). Inhibition of FAK by FAK14had no effect on basal migration but blocked EGF-induced migrationof HEK293 cells. These results further confirmed the roles of Src andFAK in migration (Figs. 1D, 4A). ODC protein was found precisely lo-calized at the leading edges of lamellipodia during spreading and atthe focal contact points in some cells indicating the involvement ofnewly synthesized polyamines in these processes (Fig. 2B).

Phosphorylation of Tyr 418 in the kinase domain activates Src andaugments its interaction with other signaling intermediates includingfocal adhesion kinase [51]. FAK activity is vital for the development oftisssues and regulates diverse cellular processes, including cell adhe-sion, migration, polarity, proliferation, and survival [52]. This range offunctions is evidence that FAK plays a fundamental role in integratingsignals from integrin and growth factor receptors to regulate the cel-lular responses mentioned above [51,52].

Immunoprecipitation of EGFR confirmed the binding of activatedEGFR with Src and integrin β3. Spermine almost completely pre-vented the interaction of EGFR with Src and integrin β3 (Fig. 3A).FAK was associated with phosphorylated pY-EGFR and spermine pre-vented this interaction (Fig. 3A). Although, growth factors activateFAK, direct binding of FAK with the activated EGFR has not been dem-onstrated. However, it is predicted that additional adaptor proteinsmight be involved in this process. The FRET based Src reporter assayalso confirmed that polyamine depletion increased Src activation,which was also prevented by polyamines (Fig. 4B, panels a-c). Immu-noprecipitation of the EGFR from HEK cells transfected with V5-EGFRshowed association of Src with the EGFR (Fig. 6B). Furthermore, thepresence of polyamines decreased the association between the EGFRand Src (Fig. 6B). EGFR-V5 pull down by GST-SH2 fusion protein,the decreased association of GST-SH2 with the EGFR in the presenceof polyamines, and the binding of spermine with SH2 domain of Srcsuggest that polyamines mediate the physical interaction betweenSrc and the EGFR (Figs. 5 and 6). Activated EGF receptors form hetero-meric complexes with multiple signaling and bridging molecules viaSH2-Tyr (p) interactions [53–55]. It has been shown that the SH2 do-main of the Src protein binds activated EGFR, and endogenous pp60c-src tightly associates with tyrosine-phosphorylated EGFR [39]. Thesefindings and other reports demonstrated that the c-Src SH2 domainis required for EGFR-mediated signal cascades [56] suggesting the di-rect interaction between the EGFR and c-Src in vivo. Previously, wehave demonstrated that polyamine depletion leads to increasedphosphorylation of the EGFR at tyrosine 1173, which is localized inthe intracellular domain, and of tyrosine 845, which is a putativephosphorylation site for Src, suggesting that under basal conditionspolyamines bind Src and prevent its interaction with the EGFR[53–55]. Thus, increased EGFR activation and thereby ERK1/2 phos-phorylation in polyamine depleted cells might result from increasedbinding of Src to the EGFR.

These observations provide direct evidence for the interaction ofthe EGFR and Src and the prevention of that interaction by

Fig. 7. A proposed model depicting the mechanism by which polyamines modulate EGFR-mediated signaling. EGF induced autophosphorylation of the EGFR recruits integrin β3,FAK and Src via the SH2 domain and, thereby, forms a macromolecular signaling complex. As a result, EGFR phosphorylates Src and enhances autophosphorylation of FAK. ActivatedFAK and Src induce downstream signaling cascades, which include PI3K, and Rho-GTPases. These pathways regulate migration. Polyamines bind to the SH2 domain of Src, thus pre-venting the physical interaction of EGFR with integrin β3 and the Src SH2 domain, and attenuates the Src-mediated FAK activation and thereby Rho-GTPases, essential for spreadingand migration.

941R.M. Ray et al. / Cellular Signalling 24 (2012) 931–942

polyamines, suggesting that polyamines modulate the interaction ofthe SH2 domain of Src with the EGFR. We examined the binding ofspermine with the purified GST-SH2 and GST-SH3 domains of Src. Itis clear from the results that the SH2 domain specifically binds sper-mine in a dose dependent fashion (Fig. 6).

Our results show that the activation of the EGFR increases trans-phosphorylation of integrin β3 allowing the recruitment of Src,which activates both the EGFR and integrin β3, amplifying the signal-ing cascade leading to the activation of downstream targets regulat-ing migration. Src and FAK activate the PI-3 kinase/Akt signalingaxis, which in turn recruits Tiam1 and other actin binding proteinsleading to increased activation of Rho-GTPases and cell migration.Our study demonstrates for the first time that spermine regulatesEGF-induced migration by preventing the formation of the scaffoldconsisting of EGFR, integrin β3, FAK and Src (Fig. 7). The single mostimportant finding in this study is the demonstration of the mecha-nism by which spermine regulates the EGFR/integrin β3/Src/FAK scaf-fold in intestinal epithelial cells. Spermine binds the SH2 domain ofSrc and prevents its interaction with activated EGFR and integrin β3and subsequently the phosphorylation of FAK.

The assembly and disassembly of focal adhesions (dynamic turn-over) is essential for migration. In the absence of polyamines staticfocal adhesions form due to the continual activation of Src which in-hibits migration. However, spermine binds the SH2 domain of Srcand prevents its association with the scaffold structure allowing thedisassembly of focal adhesions and the formation of lamellipodia. Ex-cess spermine in the vicinity of the scaffold mimics the effects of theSrc inhibitor, PP2. Thus, free intracellular spermine alters the

interaction of proteins and, thereby, acts as a molecular switch turn-ing on and off the signaling cascade required for migration.

Supplementary materials related to this article can be found on-line at doi:10.1016/j.cellsig.2011.12.016.

Acknowledgements

This publication was made possible by grants DK-052784 and DK-16505 (L.R.J.), and DK058545 and DK074996 (A.P.N.) from the Na-tional Institute of Diabetes and Digestive and Kidney Disease(NIDDK). Its contents are solely the responsibility of the authorsand do not necessarily represent the official views of the National In-stitute of Health. This work was also supported by the Thomas A.Gerwin endowment (L.R.J.).

We thank Dr. Shu Chien (University of California-San Diego, LaJolla, CA) for kindly providing CFP-Src-YFP construct and Dr.Alexander Levitzki (The Hebrew University of Jerusalem, Jerusalem,Israel) for GST-Src construct.

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