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. 2012 Apr;24(4):931-42.
doi: 10.1016/j.cellsig.2011.12.016. Epub 2011 Dec 29.

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

Affiliations

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

Ramesh M Ray et al. Cell Signal. 2012 Apr.

Abstract

Intracellular polyamine levels are highly regulated by the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting reaction in polyamine biosynthesis, producing putrescine, which is subsequently converted to spermidine and spermine. We have shown that polyamines regulate proliferation, migration, and apoptosis in intestinal epithelial cells. Polyamines regulate key signaling events at the level of the EGFR and Src. However, the precise mechanism of action of polyamines is unknown. In the present study, we demonstrate that ODC localizes in lamellipodia and in adhesion plaques during cell spreading. Spermine regulates EGF-induced migration by modulating the interaction of the EGFR with Src. The EGFR interacted with integrin β3, Src, and focal adhesion kinase (FAK). Active Src (pY418-Src) localized with FAK during spreading and migration. Spermine prevented EGF-induced binding of the EGFR with integrin β3, Src, and FAK. Activation of Src and FAK was necessary for EGF-induced migration in HEK293 cells. EGFR-mediated Src activation in live HEK293 cells using a FRET based Src reporter showed that polyamine depletion significantly increased Src kinase activity. In vitro binding studies showed that spermine directly binds Src, and preferentially interacts with the SH2 domain of Src. The physical interaction between Src and the EGFR was severely attenuated by spermine. Therefore, spermine acts as a molecular switch in regulating EGFR-Src coupling both physically and functionally. Upon activation of the EGFR, integrin β3, FAK and Src are recruited to EGFR leading to the trans-activation of both the EGFR and Src and to the Src-mediated phosphorylation of FAK. The activation of FAK induced Rho-GTPases and subsequently migration. This is the first study to define mechanistically how polyamines modulate Src function at the molecular level.

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Figures

Fig. 1
Fig. 1. Role of Src in the EGF-induced migration
A, confluent monolayers of IEC-6 cells were wounded with a gel loading tip in the center of the plates, washed and left untreated or treated with 10 nM EGF in the presence and absence of 10 μM PP2. B, migration was calculated as described in the methods. Values are means ± SEMs of triplicates. * Significantly different compared to UT group. C, following attachment and spreading of cells, coverslips were washed, fixed, permeabilized and processed for immunolocalization for the pY418-Src and FAK as described in the methods. Representative images from three observations are shown. D, confluent monolayers of IEC-6 cells were wounded with a gel loading tip in the center of the plates, washed and left untreated or treated with 10 nM EGF in the presence and absence of 2.5μM FAK14 (FAK inhibitor). Migration was calculated as described in the methods. Values are means ± SEMs of triplicates. * Significantly different compared to UT group. E, Confluent monolayers of IEC-6 cells stably expressing empty vector, constitutively active (CA), and dominant negative (DN) Src were wounded with a gel loading tip in the center of the plates, washed and left untreated (UT) or treated with 10 nM EGF. Migration was expressed as percent untreated. Values are means ± SEMs of triplicates. * Significantly different compared to UT group, # significantly different compared to cells expressing empty vector and treated with EGF.
Fig. 2
Fig. 2. Spermine inhibits EGF-induced migration
A, confluent monolayers of IEC-6 cells were wounded with a gel loading tip in the center of the plates, washed and left untreated or treated with 10 nM EGF in the presence and absence of 10 μM of spermine or triethylenetetramine (TETA). Migration was calculated as described in the methods. Values are means ± SEMs of triplicates. * Significantly different compared to UT group. B, serum starved IEC-6 cells were plated in DMEM containing dFBS and were allowed to attach and spread. Cells were washed, fixed and processed for localization of ODC and f-actin as described in methods. A cropped image of a cell showing ODC localization in lamellipodia is shown (upper panel), A group of cells showing ODC and F-actin in lamellipodia and focal plaques (lower panel). Representative images from three experiments are shown.
Fig. 3
Fig. 3. Spermine prevents interaction of EGFR with integrin β3, Src, and FAK
Confluent monolayers of IEC-6 cells were washed and left untreated or treated with 10 nM EGF in the presence and absence of 10 μM of spermine for 10 min. A, Monolayers were washed and lysates were subjected to immunoprecipitation using anti-EGFR antibody. Membranes were probed for the detection of integrin β3, Src, and FAK. Membranes were striped and probed for pY-EGFR and total-EGFR using specific antibodies. Blots shown are representative of three observations. B, Whole cell lysates were analyzed by western blot using FAK, integrin β3 and Src-specific antibodies. Representative blots from three experiments are shown.
Fig. 4
Fig. 4. Polyamines prevent Src activation in live cells
A, confluent monolayers of HEK293 cells were wounded with a gel loading tip in the center of the plates, washed and left untreated or treated with 10 nM EGF in the presence and absence of 10 μM PP2 and 10 μM FAK14 for 20h. Migration was calculated as described in the methods. Values are means ± SEMs of triplicates. * Significantly different compared to UT group, and # significantly different compared to cells treated with EGF. B, panel a, the CFP/FRET emission ratio images in response to EGF in HEK293 cells overexpressing a Src reporter pretreated with DFMO (5 mM) and/or polyamines (5 mM each for spermidine, spermine, and putrescine) for 3–4 days before the FRET imaging. panel b, the time courses of normalized CFP/FRET emission ratio of the Src reporter. The arrows indicate the addition of EGF (first arrow: 50ng/ml, the second arrow: 250ng/ml). panel c, the bar graphs of the normalized CFP/FRET emission ratio at the time point (20 min) after EGF addition (n=4–6).
Fig. 4
Fig. 4. Polyamines prevent Src activation in live cells
A, confluent monolayers of HEK293 cells were wounded with a gel loading tip in the center of the plates, washed and left untreated or treated with 10 nM EGF in the presence and absence of 10 μM PP2 and 10 μM FAK14 for 20h. Migration was calculated as described in the methods. Values are means ± SEMs of triplicates. * Significantly different compared to UT group, and # significantly different compared to cells treated with EGF. B, panel a, the CFP/FRET emission ratio images in response to EGF in HEK293 cells overexpressing a Src reporter pretreated with DFMO (5 mM) and/or polyamines (5 mM each for spermidine, spermine, and putrescine) for 3–4 days before the FRET imaging. panel b, the time courses of normalized CFP/FRET emission ratio of the Src reporter. The arrows indicate the addition of EGF (first arrow: 50ng/ml, the second arrow: 250ng/ml). panel c, the bar graphs of the normalized CFP/FRET emission ratio at the time point (20 min) after EGF addition (n=4–6).
Fig. 5
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 between His-S-SH2 and HisS-SH3 domains of Src with BSA-spermine. The mixture was pulled down using S-beads, and blotted with anti-spermine serum. Representative blots from three 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 down using glutathione beads, and blotted with anti-spermine serum. A representative blot from three independent observations is shown.
Fig. 6
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-tagged EGFR. 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 Src and 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 independent observations 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, pairwise 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 shown from three independent observations.
Fig. 7
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. Activated FAK 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 preventing 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 spreading and migration.

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