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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Apr 25;103(18):6901–6906. doi: 10.1073/pnas.0509719103

Phosphorylation of TNF-α converting enzyme by gastrin-releasing peptide induces amphiregulin release and EGF receptor activation

Qing Zhang *, Sufi M Thomas †, Vivian Wai Yan Lui †, Sichuan Xi †, Jill M Siegfried *,‡, Huizhou Fan §, Thomas E Smithgall ‡,, Gordon B Mills ‖, Jennifer Rubin Grandis *,†,‡,**
PMCID: PMC1458991  PMID: 16641105

Abstract

G protein-coupled receptors induce EGF receptor (EGFR) signaling, leading to the proliferation and invasion of cancer cells. Elucidation of the mechanism of EGFR activation by G protein-coupled receptors may identify new signaling paradigms. A gastrin-releasing peptide (GRP)/GRP receptor-mediated autocrine pathway was previously described in squamous cell carcinoma of head and neck. In the present study, we demonstrate that TNF-α converting enzyme (TACE), a disintegrin and metalloproteinse-17, undergoes a Src-dependent phosphorylation that regulates release of the EGFR ligand amphiregulin upon GRP treatment. Further investigation reveals the phosphatidylinositol 3-kinase (PI3-K) as the intermediate of c-Src and TACE, contributing to their association and TACE phosphorylation. phosphoinositide-dependent kinase 1 (PDK1), a downstream target of PI3-K, has been identified as the previously undescribed kinase to directly phosphorylate TACE upon GRP treatment. These findings suggest a signaling cascade of GRP-Src-PI3-K-PDK1-TACE-amphiregulin-EGFR with multiple points of interaction, translocation, and phosphorylation. Furthermore, knockdown of PDK1 augmented the antitumor effects of the EGFR inhibitor erlotinib, indicating PDK1 as a therapeutic target to improve the clinical response to EGFR inhibitors.

Keywords: G protein-coupled receptor, squamous cell carcinoma of head and neck

The observation that epithelial cancers are characterized by overexpression of epidermal growth factor receptors (EGFR) has led to the development of therapeutic strategies that block this growth factor receptor. In a population of nonsmall cell lung cancer (NSCLC) patients with activating mutations in the EGFR, the responses to EGFR targeting therapy have been striking (1, 2). However, the frequency of response in patients without activating EGFR mutations in NSCLC and other cancers has been limited but not absent (3–5). The basis for these modest clinical responses in most patients, despite robust activity observed in preclinical models, is not completely understood. In addition to direct activation of EGFR by ligands, EGFR can be transactivated by G protein-coupled receptors (GPCRs) in multiple cell types including fibroblasts, smooth muscle cells, neurons, and tumor cells (6–9). Upon EGFR activation, tumor cells demonstrate increased proliferation, invasion, survival, and chemotherapy resistance (10).

The mechanisms underlying GPCR-induced EGFR signaling involves both intracellular and extracellular pathways (11). EGFR activation by GPCRs has been proposed to be mediated by Src family kinases, phosphatidylinositol 3-kinase (PI3-K) and/or PKC signaling (11). Src family kinases have been reported to regulate GPCR ligand-induced EGFR phosphorylation in the colon cancer cell line Caco-2, gastric epithelial cells RGM1, Cos-7 cells, GT1–7 neuronal cells, lung cancer cell 201T, and head and neck cancer cells (12–16). PI3-K also has been implicated in GPCR and EGFR crosstalk (17–19). In addition to intracellular molecules involved in GPCR-induced EGFR activation, growing evidence suggests that transmembrane metalloproteases mediate EGFR proligand shedding in response to GPCR ligands (8, 20–23). Cumulative results indicate that the metalloproteinase involved in EGFR proligand cleavage is both cell type- and GPCR ligand-specific. Matrix metalloproteinases 2 and 9 have been reported to mediate estrogen receptor/GPCR ligand-induced EGFR ligand cleavage in breast cancer cells and gonadotropic cells, respectively (24, 25), whereas a disintegrin and metalloprotease (ADAM) 10 was implicated in bombesin or lysophosphatidic acid-induced heparin-binding-EGF cleavage and EGFR activation in Cos-7 cells (23). TNF-α converting enzyme (TACE) has been shown to mediate carbachol or lysophosphatidic acid-induced proamphiregulin release and downstream EGFR and mitogen-activated protein kinase (MAPK) activation in squamous cell carcinoma of head and neck (SCCHN) cells (26). However, the precise intracellular signaling cascade coupling GPCR stimulation to metalloproteinase activation and EGFR signaling remains incompletely understood.

We previously reported that Src family kinases regulate gastrin-releasing peptide (GRP)-induced EGFR proligand cleavage leading to downstream EGFR and MAPK activation in SCCHN, which contributes to cell proliferation and invasion (15, 27). In addition, blockade of endogenous GRP by using the 2A11 antibody decreased SCCHN cell proliferation and tumor growth in vivo (28). Here we show that Src associates with TACE after GRP treatment of SCCHN cells. This association is accompanied by phosphorylation and translocation of Src and TACE to the cell membrane. Phosphorylation of TACE by GRP requires both Src family kinases and PI3-Ks. Further investigation identified phosphoinositide-dependent kinase 1 (PDK1) as the kinase that directly mediates GRP-induced TACE phosphorylation. Knockdown of PDK1 enhanced the antitumor effects of an EGFR inhibitor. These results implicate PDK1 as a therapeutic target in cancers where transactivation of EGFR by GPCR contributes to tumor progression.

Results

GRP Induces TACE and c-Src Association.

We previously demonstrated that Src family kinases contribute to GRP-induced EGFR and MAPK activation by facilitating the release of tethered EGFR ligands in SCCHN (15). EGFR ligand cleavage in response to activation of GPCRs can be mediated by several metalloproteases, including members of the ADAM family (8, 20, 21). Many ADAMs are rich in proline residues on their cytoplasmic domains, specifically PXXP consensus sequences, which enable them to interact with Src homology 3 domains in a variety of intracellular proteins (29). Indeed, TACE has been shown to contribute to thrombin and lysophosphatidic acid-induced EGFR activation (20, 26). We therefore examined whether Src family kinases contribute to EGFR ligand cleavage by physical association with TACE through Src homology 3 domain interaction.

To test whether TACE and c-Src can associate either constitutively or after GPCR activation, we transfected HEK-293 cells with a WT c-Src expression plasmid, followed by coimmunoprecipitation. In this model, TACE and c-Src association increases upon c-Src transfection and this association is specific upon TACE immunoprecipitation (Fig. 8 a and b, which is published as supporting information on the PNAS web site). To investigate the effect of GRP on the association between endogenous TACE and c-Src in a biologically relevant system, SCCHN cells (PCI-37A and PCI-15B) were treated with GRP, followed by TACE and c-Src coimmunoprecipitation. In both SCCHN cell lines, GRP increases the association between TACE and c-Src (Fig. 1 a and b).

Fig. 1.

Fig. 1.

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GRP induces TACE and c-Src association. (a and b) Representative SCCHN cells (PCI-37A or PCI-15B) were treated with GRP (400 nM) for 10 min, followed by immunoprecipitation (IP) with TACE and immunoblotting (IB) for c-Src. The membrane was stripped to immunoblot for TACE to ensure equivalent loading. (c) PCI-15B cells were plated on coverslips, followed by serum starvation for 2 days. Upon incubation with GRP (400 nM) for 5 min, cells were double-stained with anti-TACE (green) and anti-c-Src (red) antibody. Arrows indicate the redistribution of TACE and c-Src to the cell membrane upon GRP stimulation.

GPCR agonist angiotensin II has been reported to induce TACE redistribution to the apical membrane, followed by EGFR phosphorylation (30). We next examined whether GRP could induce TACE and c-Src association by intracellular translocation of these proteins by using confocal microscopy. In the absence of GRP, TACE and c-Src are present mainly in the cytoplasm. Short-term treatment with GRP induces a coordinate redistribution of both Src and TACE to the cell periphery (Fig. 1c). EGFR extracellular antibody was used to costain cells with TACE to confirm TACE translocation to the membrane upon GRP treatment (Fig. 9, which is published as supporting information on the PNAS web site). These results suggest that GRP induces association of TACE and c-Src, in conjunction with translocation to the cell membrane, where TACE can cleave EGFR proligands.

TACE Is the Metalloproteinase Involved in GRP-Induced EGFR Proligand Cleavage.

TACE has been implicated in thrombin and lysophosphatidic acid-induced cleavage of proamphiregulin with subsequent EGFR and MAPK activation in SCCHN cells (26). To determine the role of TACE in GRP-induced EGFR and MAPK activation, SCCHN cells (PCI-37A) were transfected with TACE small interfering RNA (siRNA) or negative control GFP duplex, followed by GRP treatment. As shown in Fig. 2a, GRP fails to induce EGFR and MAPK phosphorylation after suppression of TACE expression. However, in the GFP siRNA duplex-transfected cells, GRP retains the ability to induce EGFR and MAPK activation.

Fig. 2.

Fig. 2.

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TACE mediates GRP-induced EGFR activation. (a) Representative SCCHN cells (PCI-37A) were plated on 6-well plates, followed by TACE siRNA transfection or transfection with negative control GFP duplex. After serum starvation for 2 days, cells were treated with GRP (400 nM) for 10 min, followed by immunoblotting (IB) and immunoprecipitation (IP) as indicated. (b) An amphiregulin ELISA was performed on cell culture media according to the manufacturer’s instructions. TACE siRNA GRP treatment was compared to GFP siRNA GRP treatment. Cumulative results are shown from six independent experiments (P = 0.0011).

Our prior studies in SCCHN demonstrated that amphiregulin and TGF-α, but not heparin-binding-EGF or EGF, are released after treatment with GRP (27). To determine the role of TACE in GRP-mediated EGFR ligand release, we performed an amphiregulin ELISA after GRP stimulation in cell medium. As shown in Fig. 2b, suppression of TACE expression with siRNA abrogates GRP-induced amphiregulin release into the conditioned medium (P = 0.0011). In cell lysates, amphiregulin expression is higher in TACE siRNA transfected cell when compared with GFP siRNA-transfected cells (Fig. 10, which is published as supporting information on the PNAS web site). These results suggest that TACE is involved in GRP-induced EGFR transactivation.

c-Src Is Required for GRP Induced TACE Phosphorylation.

Phorbol-12-myristate-13-acetate (TPA), a well known shedding activator, has been reported to induce TACE phosphorylation on threonine residues (31, 32). EGF can induce TACE serine phosphorylation (33). To elucidate the mechanism by which GRP leads to TACE relocalization and subsequent amphiregulin release, we examined TACE serine and threonine phosphorylation after GRP treatment in SCCHN cells. GRP stimulates TACE phosphorylation as early as 2 min and reaches maximal level by 10 min after the addition of GRP, whereas GRP-induced EGFR and MAPK phosphorylation are first detectable at 5 min and peak at 10 min in PCI-37A cells (Fig. 11, which is published as supporting information on the PNAS web site), compatible with TACE acting upstream of EGFR and MAPK phosphorylation. Although phosphorylation was readily detected at both serine and threonine residues, we could not detect TACE phosphorylation on tyrosine residues (data not shown). The mechanism underlying GRP-induced TACE phosphorylation is unknown. ADAM15 has been reported to undergo Src family kinase-dependent phosphorylation, which contributed to the interaction between ADAM15 cytoplasmic domain and Src family proteins (34). Because c-Src translocates to the plasma membrane after GRP treatment, where c-Src associates with TACE, we hypothesized that GRP-induced Src family kinase activation could contribute to TACE phosphorylation. GRP-induced TACE and c-Src association and translocation is abrogated by treating cells with the Src family kinase inhibitor A-419259, indicating that the interaction between TACE and c-Src are phosphorylation-dependent (Fig. 12, which is published as supporting information on the PNAS web site).

To confirm the role of c-Src on GRP-induced TACE phosphorylation, SCCHN (PCI-37A) cells were transfected with c-Src siRNA, followed by GRP treatment. As shown in Fig. 3a, upon knockdown of c-Src expression, GRP fails to induce TACE phosphorylation. In addition, GRP fails to induce amphiregulin release into the medium in the presence of c-Src siRNA (Fig. 3b; P = 0.0011).

Fig. 3.

Fig. 3.

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Src mediates GRP-induced TACE phosphorylation in SCCHN. (a) PCI-37A cells were transfected with c-Src siRNA, followed by serum starvation for 2 days and treatment with GRP (400 nM) for 10 min. Cell lysates were subject to Western blotting for c-Src expression and TACE immunoprecipitation. IB, immunoblotting; IP, immunoprecipitation. (b) An amphiregulin ELISA was performed on cell culture media according to the manufacturer’s instructions. Cumulative results are shown from six independent experiments (P = 0.0011).

PI3-K Acts as an Intermediary and Is Required for GRP-Induced TACE Phosphorylation.

Because c-Src is a tyrosine kinase and phosphorylates substrates exclusively on tyrosine residues and TACE is phosphorylated on serine/threonine residues, we reasoned that a serine/threonine kinase likely acts as an intermediary between c-Src and TACE phosphorylation. Src family kinases have been reported to activate PI3-K by interaction between Src homology 3 domains and the p85 regulatory subunit of PI3-K with subsequent phosphorylation of Y688 and release of the inhibitory of activity of p85 on the p110 catalytic subunit (35, 36). Furthermore, Src family kinases can inhibit PTEN activity, also leading to increased signaling through the PI3-K pathway (37). To examine whether the PI3-K pathway was involved in GRP-induced EGFR signaling by acting downstream of Src family kinases, SCCHN cells (PCI-37A) were treated with the Src family kinase inhibitor A-419259 (100 nM), followed by GRP treatment. Using phospho-Akt as a surrogate marker for activation of the PI3-K pathway, we found that GRP-induced PI3-K activity is suppressed by Src family kinase blockade (Fig. 4a). In addition to Src kinase inhibitors, c-Src siRNA also was used and similar results were observed (Fig. 13, which is published as supporting information on the PNAS web site).

Fig. 4.

Fig. 4.

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PI3-K acts as an intermediary and is required for GRP-induced TACE phosphorylation. (a) PCI-37A cells were serum-starved for 2 days. After pretreatment with the Src family kinase inhibitor PD0180970 (500 nM) for 2 h, cells were treated with GRP or EGF for 10 min. Cell lysates were subject to immunoblotting with phospho-Akt (Ser-473) and total Akt. (b) PCI-37A cells were transfected with p85α siRNA, followed by serum starvation for 2 days and treatment with GRP (400 nM) for 10 min. Cell lysates were subject to Western blotting for p85α expression and TACE immunoprecipitation. IB, immunoblotting; IP, immunoprecipitation. (c) An amphiregulin ELISA was performed on cell culture media according to the manufacturer’s instructions. Cumulative results are shown from four independent experiments (P = 0.014).

Because PI3-K activation by GRP occurred via a c-Src dependent manner, we hypothesized that PI3-K could potentially acts as an intermediate between TACE and c-Src. To test this hypothesis, we transfected SCCHN cells with p85 siRNA. Upon knockdown of p85 expression, GRP failed to induce TACE and c-Src association, indicating that the interaction is not direct (data not shown). It is likely that PI3-K acts as an intermediate between c-Src and TACE. Because PI3-K mediates c-Src and TACE association, we hypothesized that PI3-K activity is required for GRP-induced TACE phosphorylation on the cytoplasmic domain. As shown in Fig. 4b, GRP fails to induce TACE phosphorylation in p85α siRNA-transfected cells, whereas GRP retains the ability to stimulate TACE phosphorylation in control siRNA-transfected SCCHN cells. To confirm that PI3-K acts downstream of c-Src kinase and contributes to GRP-induced EGFR ligand release, the role of p85α on GRP-induced EGFR ligand release was examined. p85α siRNA abrogates GRP-induced amphiregulin release (P = 0.014; Fig. 4c). Similar results were observed in PCI-15B cells (data not shown). In addition to p85 siRNA, two PI3-K inhibitors, Wortmannin or LY294002, were used. Both compounds inhibited PI3-K in a dose-dependent manner without affecting Src activity (Figs. 14 a and b, which is published as supporting information on the PNAS web site). Also, both compounds abrogated GRP-induced TACE phosphorylation, EGFR ligand release and EGFR/MAPK phosphorylation (Figs. 15–17, which is published as supporting information on the PNAS web site). Thus, PI3-K is required for GRP-induced TACE phosphorylation and EGFR ligand release in SCCHN cells.

PDK1 Phosphorylates TACE in Response to GRP.

Although PI3-K contains serine/threonine protein kinase activity, the only identified PI3-K protein substrate is PI3-K itself. Thus, we reasoned that an alternative intermediary kinase must directly phosphorylate TACE in response to GRP stimulation. There are a number of possible candidates because the three phosphorylated phosphatidylinositols produced by PI3-K activate, both directly and indirectly (38–40). Of particular interest, PDK1-specific docking with these substrates was reportedly required for efficient phosphorylation, which was localized to the hydrophobic Phe-Xaa-Xaa-Phe (PXXP) domain on the substrates (41, 42). Because TACE contains several PXXP domains on the cytoplasmic domain, we hypothesized that upon PI3-K activation by GRP, PDK1 translocates to the membrane, where it recognizes the TACE cytoplasmic PXXP domain and regulates TACE phosphorylation. To test whether PDK1 kinase can phosphorylate full-length TACE, SCCHN (37A) cells were serum-starved, followed by an in vitro kinase assay with recombinant PDK1 enzyme. As shown in Fig. 5a, PDK1 induces TACE phosphorylation. Similar results were observed in PCI-15B cells (data not shown). To confirm that PDK1 is able to phosophorylate TACE intracellularly, we purified a GST fusion protein linked to the cytoplasmic domain of TACE (GST-TACEc). As shown in Fig. 5b, PDK1 kinase can phosphorylate TACE on the cytoplasmic domain in vitro, implicating PDK1 as one effector mediating GRP-induced TACE phosphorylation. In contrast, the PKC active enzyme failed to induce GST-TACEc phosphorylation (data not shown). To investigate the requirement of PDK1 in GRP-induced TACE phosphorylation, PDK1 siRNA was used to knockdown endogenous PDK1 expression. Upon PDK1 knockdown, GRP fails to induce TACE phosphorylation (Fig. 5c). In addition, PDK1 knockdown abrogated GRP-induced TACE translocation to the cell membrane (Fig. 18, which is published as supporting information on the PNAS web site). These results demonstrate that PDK1 can mediate GRP-induced TACE phosphorylation and translocation.

Fig. 5.

Fig. 5.

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PDK1 kinase phosphorylates TACE upon GRP treatment in SCCHN. (a) SCCHN cells (PCI-37A) were serum-starved for 2 days, followed by TACE immunoprecipitation. PDK1 enzyme was used to incubate with the cell pellets, followed by in vitro kinase assay. The same membrane was used to probe for total TACE. IB, immunoblotting; IP, immunoprecipitation. (b) Active PDK1 kinase was incubated with recombinant GST or GST-TACEc, followed by in vitro kinase assay. The same membrane was subject to immunoblotting with anti-GST antibody to ensure equal loading. (c) SCCHN cells (PCI-37A) were transfected with PDK1 siRNA or negative control (GFP) siRNA, followed by immunoblotting with PDK1 antibody, or immunoprecipitation with TACE, followed by immunoblotting with phosphothreonine or serine antibody.

Targeting of PDK1 Enhances the Cytotoxic and Antiinvasive Effects of an EGFR Inhibitor.

EGFR inhibitors, including the tyrosine kinase inhibitor erlotinib (OSI-774/Tarceva), have resulted in limited clinical responses in cancer patients whose tumors do not contain activating EGFR mutations (5). Because GRP stimulates SCCHN growth through transactivation of EGFR, we hypothesized that the cytotoxic and antiinvasive effects of an EGFR inhibitor could be enhanced by simultaneous targeting of PDK1. Erlotinib treatment (1 μM) resulted in an ≈50% growth inhibition at 24 h. The cytotoxic effects of erlotinib were increased significantly when combined with PDK1 siRNA (Fig. 6a; P = 0.0011). In addition, PDK1 siRNA significantly reduced SCCHN cell invasion ability when combined with erlotinib (Fig. 6b; P < 0.001). These results indicate that the cytotoxic and antiinvasive effects of EGFR inhibitors can be enhanced by PDK1 blockade.

Fig. 6.

Fig. 6.

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Enhanced antitumor effects by combined targeting of PDK1 and EGFR. SCCHN (PCI-37A) cells were plated on 24-well plates, followed by transfection with PDK1 siRNA or GFP duplex siRNA. Twenty-four hours after transfection, erlotinib (1 μM) was added to the well. (a) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed after 24 h of erlotinib treatment. Results are from six independent experiments (P = 0.0011). (b) Cells were plated in a matrigel invasion chamber in duplicate, followed by transfection with PDK1 siRNA or GFP duplex siRNA. Twenty-four hours after transfection, erlotinib (1 μM) was added for 24 h. Invading cells in 10 representative fields were counted by using light microscopy at ×400 magnification.

Discussion

The integration of EGFR and GPCR signaling pathways has been shown to contribute to carcinogenesis in a variety of cancer models (8, 16, 21, 24, 43, 44). The precise mechanism of EGFR activation by GPCRs is incompletely understood. The results of the present study suggest that after GPCR stimulation, c-Src is activated, leading to downstream induction of PI3-K. After PI3-K activation, PDK1 could phosphorylate TACE on threonine and serine residues, leading to translocation of TACE, EGFR proligand (e.g., amphiregulin) cleavage, and subsequent EGFR and MAPK phosphorylation. This model indicates a pivotal role of GPCR ligand-induced amphiregulin release in mitogenic signaling, which is consistent with the previous finding that GPCR ligands induced amphiregulin release, leading to EGFR transactivation proliferation, motility, and invasion in head and neck cancer cells (20, 26).

The mechanisms underlying TACE phosphorylation in the context of GPCR ligand stimulation of EGFR have not been identified previously. Phosphorylation of TACE may result in its translocation to its targets or the formation of a functional complex of Src and TACE on the membrane. We previously reported that Src family kinases contribute to GRP-induced EGFR phosphorylation in SCCHN (15). Both c-Src and TACE are located in punctuate foci in the cytosol compatible with the association of c-Src and TACE with intracellular membranes through myristoylation of the Src N-terminal domain and TACE transmembrane domain, respectively. In the present study, we demonstrated by coimmunoprecipitation and confocal microscopy in SCCHN cells that TACE associates with c-Src in the cytoplasm in the absence of treatment with exogenous GRP. The interaction between TACE and c-Src could result from a GRP/gastrin-releasing peptide receptor autocrine pathway in this tumor system (28). Upon GRP treatment, the association significantly increased, followed by TACE and c-Src translocation to the membrane. Translocation of TACE to the plasma membrane likely plays an important role in TACE function by placing it in the proximity of its target, proamphiregulin. However, it remains to be determined whether TACE directly or indirectly mediates amphiregulin release.

The mechanism by which GRP induces TACE association with c-Src is unknown but could involve the proline-rich sequence in the cytoplasmic domain of TACE and the Src homology 3 domain of c-Src. Alternatively, because p85 binds TACE and c-Src (data not shown), p85 could be an intermediary between TACE and c-Src. Several observations suggest that the interaction between TACE and c-Src is of physiological significance. First, the transactivation of EGFR and the activation of downstream MAPK in response to GRP require active c-Src. Second, stimulation of SCCHN cells with GRP resulted in increased TACE and c-Src association in the cytoplasm. Third, TACE translocated with c-Src to the plasma membrane where cleavage of amphiregulin by TACE occurs. Finally, GRP stimulation of SCCHN cells induced TACE phosphorylation in a c-Src-dependent fashion.

GRP-induced phosphorylation of TACE occurs on serine and threonine but not on tyrosine residues. Therefore, we reasoned that additional signaling molecules are required to regulate TACE phosphorylation. PDK1 has been reported to facilitate the activation of several AGC protein kinases, including PKA, PKG, and PKC (38–40). Here we show that in addition to AGC protein kinases, PDK1 also can phosphorylate TACE. PDK1 is mainly cytoplasmic with some localization on the plasma membrane under basal condition (45, 46). Unlike other kinases, PDK1 exists as a constitutively active kinase, even in the absence of exogenous stimulation. Furthermore, phosphorylation of PDK1 appears to be resistant to agonist stimulation of PI3-K (39, 40, 47). Consistent with these previous findings, upon GRP treatment, we did not detect increased PDK1 phosphorylation by in vitro kinase assay. However, we observed that PDK1 siRNA abrogated GRP-induced TACE translocation to the membrane. We propose the following model of PDK1-induced TACE phosphorylation: (i) GRP induces c-Src activation and downstream PI3-K activation, giving rise to PIP2 and PIP3 production; (ii) these lipid molecules elicit the translocation of PDK1 and TACE from the cytoplasm to the plasma membrane; and (iii) TACE undergoes a conformational change, which can serve as a substrate for PDK1, through recognition of multiple PXXP motifs on the TACE cytoplasmic domain by PDK1. Although it was previously reported that the cytoplasmic domain of TACE is not required for the cleavage of some transmembrane molecules (48), we show here that phosphorylation of the TACE cytoplasmic domain may contribute to EGFR ligand release. It is possible that the requirement of the cytoplasmic domain of TACE for activation may depend on the precise model under investigation.

Elucidation of the critical mediators of GPCR/EGFR crosstalk has important clinical implications. PDK1, as a kinase at the hub of many signaling pathways, has been reported to bring key signaling molecules into proximity, activate cell signaling through translocation, and nucleate receptor-induced signaling complex for downstream molecule activation such as NF-κB (49). Inhibition of PDK1 and Akt with the broad-spectrum kinase inhibitor, staurosporine, promotes apoptosis in a variety of cancer cells (50). However, because of nonselectivity, this compound is too toxic for clinical development (51). 7-hydroxystaurosporine (UCN-01) inhibits PDK1 nonselectively and has been reported to abrogate tumor cell growth and promote apoptosis (52, 53). PDK1 antisense oligonucleotides have shown to reduce glioblastoma cell proliferation and survival (54).

Although PDK1 and PI3-K are known downstream components of EGFR-signaling pathways, our evidence suggests that they can also act upstream of EGFR by mediating EGFR-ligand release. The results of the present study suggest that the combination of PDK1 targeting with EGFR blockade may enhance the therapeutic effects of EGFR inhibitors. PDK1 targeting may block both EGFR-dependent and EGFR-independent pathways that contribute to cancer cell proliferation and invasion. PDK1 appears to play a critical role in EGFR activation by GPCRs, where it may serve as a target for cancer therapy.

Materials and Methods

Cell Culture.

All SCCHN cell lines (PCI-37A and PCI-15B) were of human origin (55–57). Cells were maintained in DMEM with 10% heat-inactivated FCS (Invitrogen) at 37°C with 5% CO2.

Western Blotting and Immunoprecipitation.

Twenty-five micrograms of protein was resolved in an 8% SDS/PAGE. For immunoprecipitation, 200 μg of total protein was incubated for 2 h at 4°C with 3 μg of anti-TACE antibody (QED Biosciences, San Diego), anti-EGFR antibody (Upstate Biotechnology), or 2 μg of anti-c-Src antibody (Santa Cruz Biotechnology) with gentle agitation, followed by protocols reported in ref. 15.

Supporting Information.

Chemicals and reagents, siRNA transfection, ELISAs, cloning of the human TACE cytoplasmic domain (TACEc) and its expression as GST-TACEc fusion protein, immunofluorescence microscopic analysis, in vitro kinase assay, in vitro cytotoxicity assay, matrigel invasion assay, and statistics are published as Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

Supplementary Material

Supporting Information
pnas_0509719103_index.html (15.3KB, html)

Acknowledgments

This work was supported by National Institutes of Health Specialized Programs of Research Excellence Grants P50CA90440 and R01 CA098372 (to J.R.G.).

Abbreviations

ADAM

a disintegrin and metalloprotease

EGFR

EGF receptor

GPCR

G protein-coupled receptor

GRP

gastrin-releasing peptide

MAPK

mitogen-activated protein kinase

PDK1

phosphoinositide-dependent kinase 1

PI3-K

phosphatidylinositol 3-kinase

SCCHN

squamous cell carcinoma of head and neck

siRNA

small interfering RNA

TACE

TNF-α converting enzyme

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

References

  • 1.Lynch T. J., Bell D. W., Sordella R., Gurubhagavatula S., Okimoto R. A., Brannigan B. W., Harris P. L., Haserlat S. M., Supko J. G., Haluska F. G., et al. N. Engl. J. Med. 2004;350:2129–2139. doi: 10.1056/NEJMoa040938. [DOI] [PubMed] [Google Scholar]
  • 2.Paez J. G., Janne P. A., Lee J. C., Tracy S., Greulich H., Gabriel S., Herman P., Kaye F. J., Lindeman N., Boggon T. J., et al. Science. 2004;304:1497–1500. doi: 10.1126/science.1099314. [DOI] [PubMed] [Google Scholar]
  • 3.Baselga J. Ann. Oncol. 2000;11(Suppl. 3):187–190. doi: 10.1093/annonc/11.suppl_3.187. [DOI] [PubMed] [Google Scholar]
  • 4.Garber K. J. Natl. Cancer Inst. 2000;92:967–969. doi: 10.1093/jnci/92.12.967. [DOI] [PubMed] [Google Scholar]
  • 5.Arteaga C. L., Baselga J. Cancer Cell. 2004;5:525–531. doi: 10.1016/j.ccr.2004.05.028. [DOI] [PubMed] [Google Scholar]
  • 6.Daub H., Weiss F. U., Wallasch C., Ullrich A. Nature. 1996;379:557–560. doi: 10.1038/379557a0. [DOI] [PubMed] [Google Scholar]
  • 7.Daub H., Wallasch C., Lankenau A., Herrlich A., Ullrich A. EMBO J. 1997;16:7032–7044. doi: 10.1093/emboj/16.23.7032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Prenzel N., Zwick E., Daub H., Leserer M., Abraham R., Wallasch C., Ullrich A. Nature. 1999;402:884–888. doi: 10.1038/47260. [DOI] [PubMed] [Google Scholar]
  • 9.Zwick E., Daub H., Aoki N., Yamaguchi-Aoki Y., Tinhofer I., Maly K., Ullrich A. J. Biol. Chem. 1997;272:24767–24770. doi: 10.1074/jbc.272.40.24767. [DOI] [PubMed] [Google Scholar]
  • 10.Yarden Y., Sliwkowski M. X. Nat. Rev. Mol. Cell Biol. 2001;2:127–137. doi: 10.1038/35052073. [DOI] [PubMed] [Google Scholar]
  • 11.Gschwind A., Zwick E., Prenzel N., Leserer M., Ullrich A. Oncogene. 2001;20:1594–1600. doi: 10.1038/sj.onc.1204192. [DOI] [PubMed] [Google Scholar]
  • 12.Pai R., Soreghan B., Szabo I. L., Pavelka M., Baatar D., Tarnawski A. S. Nat. Med. 2002;8:289–293. doi: 10.1038/nm0302-289. [DOI] [PubMed] [Google Scholar]
  • 13.Luttrell L. M., Hawes B. E., van Biesen T., Luttrell D. K., Lansing T. J., Lefkowitz R. J. J. Biol. Chem. 1996;271:19443–19450. doi: 10.1074/jbc.271.32.19443. [DOI] [PubMed] [Google Scholar]
  • 14.Shah B. H., Farshori M. P., Jambusaria A., Catt K. J. J. Biol. Chem. 2003;278:19118–19126. doi: 10.1074/jbc.M212932200. [DOI] [PubMed] [Google Scholar]
  • 15.Zhang Q., Thomas S. M., Xi S., Smithgall T. E., Siegfried J. M., Kamens J., Gooding W. E., Grandis J. R. Cancer Res. 2004;64:6166–6173. doi: 10.1158/0008-5472.CAN-04-0504. [DOI] [PubMed] [Google Scholar]
  • 16.Thomas S. M., Grandis J. R., Wentzel A. L., Gooding W. E., Lui V. W., Siegfried J. M. Neoplasia. 2005;7:426–431. doi: 10.1593/neo.04454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Hawes B. E., van Biesen T., Koch W. J., Luttrell L. M., Lefkowitz R. J. J. Biol. Chem. 1995;270:17148–17153. doi: 10.1074/jbc.270.29.17148. [DOI] [PubMed] [Google Scholar]
  • 18.Touhara K., Hawes B. E., van Biesen T., Lefkowitz R. J. Proc. Natl. Acad. Sci. USA. 1995;92:9284–9287. doi: 10.1073/pnas.92.20.9284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lopez-Ilasaca M., Crespo P., Pellici P. G., Gutkind J. S., Wetzker R. Science. 1997;275:394–397. doi: 10.1126/science.275.5298.394. [DOI] [PubMed] [Google Scholar]
  • 20.Schafer B., Marg B., Gschwind A., Ullrich A. J. Biol. Chem. 2004;279:47929–47938. doi: 10.1074/jbc.M400129200. [DOI] [PubMed] [Google Scholar]
  • 21.Schafer B., Gschwind A., Ullrich A. Oncogene. 2004;23:991–999. doi: 10.1038/sj.onc.1207278. [DOI] [PubMed] [Google Scholar]
  • 22.Le Gall S. M., Auger R., Dreux C., Mauduit P. J. Biol. Chem. 2003;278:45255–45268. doi: 10.1074/jbc.M307745200. [DOI] [PubMed] [Google Scholar]
  • 23.Yan Y., Shirakabe K., Werb Z. J. Cell Biol. 2002;158:221–226. doi: 10.1083/jcb.200112026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Razandi M., Pedram A., Park S. T., Levin E. R. J. Biol. Chem. 2003;278:2701–2712. doi: 10.1074/jbc.M205692200. [DOI] [PubMed] [Google Scholar]
  • 25.Roelle S., Grosse R., Aigner A., Krell H. W., Czubayko F., Gudermann T. J. Biol. Chem. 2003;278:47307–47318. doi: 10.1074/jbc.M304377200. [DOI] [PubMed] [Google Scholar]
  • 26.Gschwind A., Hart S., Fischer O. M., Ullrich A. EMBO J. 2003;22:2411–2421. doi: 10.1093/emboj/cdg231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Lui V. W., Thomas S. M., Zhang Q., Wentzel A. L., Siegfried J. M., Li J. Y., Grandis J. R. Oncogene. 2003;22:6183–6193. doi: 10.1038/sj.onc.1206720. [DOI] [PubMed] [Google Scholar]
  • 28.Lango M. N., Dyer K. F., Lui V. W., Gooding W. E., Gubish C., Siegfried J. M., Grandis J. R. J. Natl. Cancer Inst. 2002;94:375–383. doi: 10.1093/jnci/94.5.375. [DOI] [PubMed] [Google Scholar]
  • 29.Feng S., Chen J. K., Yu H., Simon J. A., Schreiber S. L. Science. 1994;266:1241–1247. doi: 10.1126/science.7526465. [DOI] [PubMed] [Google Scholar]
  • 30.Lautrette A., Li S., Alili R., Sunnarborg S. W., Burtin M., Lee D. C., Friedlander G., Terzi F. Nat. Med. 2005;11:867–874. doi: 10.1038/nm1275. [DOI] [PubMed] [Google Scholar]
  • 31.Black R. A., Rauch C. T., Kozlosky C. J., Peschon J. J., Slack J. L., Wolfson M. F., Castner B. J., Stocking K. L., Reddy P., Srinivasan S., et al. Nature. 1997;385:729–733. doi: 10.1038/385729a0. [DOI] [PubMed] [Google Scholar]
  • 32.Diaz-Rodriguez E., Montero J. C., Esparis-Ogando A., Yuste L., Pandiella A. Mol. Biol. Cell. 2002;13:2031–2044. doi: 10.1091/mbc.01-11-0561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Fan H., Turck C. W., Derynck R. J. Biol. Chem. 2003;278:18617–18627. doi: 10.1074/jbc.M300331200. [DOI] [PubMed] [Google Scholar]
  • 34.Poghosyan Z., Robbins S. M., Houslay M. D., Webster A., Murphy G., Edwards D. R. J. Biol. Chem. 2002;277:4999–5007. doi: 10.1074/jbc.M107430200. [DOI] [PubMed] [Google Scholar]
  • 35.Pleiman C. M., Hertz W. M., Cambier J. C. Science. 1994;263:1609–1612. doi: 10.1126/science.8128248. [DOI] [PubMed] [Google Scholar]
  • 36.Cuevas B. D., Lu Y., Mao M., Zhang J., LaPushin R., Siminovitch K., Mills G. B. J. Biol. Chem. 2001;276:27455–27461. doi: 10.1074/jbc.M100556200. [DOI] [PubMed] [Google Scholar]
  • 37.Lu Y., Yu Q., Liu J. H., Zhang J., Wang H., Koul D., McMurray J. S., Fang X., Yung W. K., Siminovitch K. A., et al. J. Biol. Chem. 2003;278:40057–40066. doi: 10.1074/jbc.M303621200. [DOI] [PubMed] [Google Scholar]
  • 38.Stokoe D., Stephens L. R., Copeland T., Gaffney P. R., Reese C. B., Painter G. F., Holmes A. B., McCormick F., Hawkins P. T. Science. 1997;277:567–570. doi: 10.1126/science.277.5325.567. [DOI] [PubMed] [Google Scholar]
  • 39.Alessi D. R., Deak M., Casamayor A., Caudwell F. B., Morrice N., Norman D. G., Gaffney P., Reese C. B., MacDougall C. N., Harbison D., et al. Curr. Biol. 1997;7:776–789. doi: 10.1016/s0960-9822(06)00336-8. [DOI] [PubMed] [Google Scholar]
  • 40.Pullen N., Dennis P. B., Andjelkovic M., Dufner A., Kozma S. C., Hemmings B. A., Thomas G. Science. 1998;279:707–710. doi: 10.1126/science.279.5351.707. [DOI] [PubMed] [Google Scholar]
  • 41.Biondi R. M. Trends Biochem. Sci. 2004;29:136–142. doi: 10.1016/j.tibs.2004.01.005. [DOI] [PubMed] [Google Scholar]
  • 42.Biondi R. M., Nebreda A. R. Biochem. J. 2003;372:1–13. doi: 10.1042/BJ20021641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Lemjabbar H., Li D., Gallup M., Sidhu S., Drori E., Basbaum C. J. Biol. Chem. 2003;278:26202–26207. doi: 10.1074/jbc.M207018200. [DOI] [PubMed] [Google Scholar]
  • 44.McCole D. F., Keely S. J., Coffey R. J., Barrett K. E. J. Biol. Chem. 2002;277:42603–42612. doi: 10.1074/jbc.M206487200. [DOI] [PubMed] [Google Scholar]
  • 45.Anderson K. E., Coadwell J., Stephens L. R., Hawkins P. T. Curr. Biol. 1998;8:684–691. doi: 10.1016/s0960-9822(98)70274-x. [DOI] [PubMed] [Google Scholar]
  • 46.Currie R. A., Walker K. S., Gray A., Deak M., Casamayor A., Downes C. P., Cohen P., Alessi D. R., Lucocq J. Biochem. J. 1999;337:575–583. [PMC free article] [PubMed] [Google Scholar]
  • 47.Vanhaesebroeck B., Alessi D. R. Biochem. J. 2000;346:561–576. [PMC free article] [PubMed] [Google Scholar]
  • 48.Reddy P., Slack J. L., Davis R., Cerretti D. P., Kozlosky C. J., Blanton R. A., Shows D., Peschon J. J., Black R. A. J. Biol. Chem. 2000;275:14608–14614. doi: 10.1074/jbc.275.19.14608. [DOI] [PubMed] [Google Scholar]
  • 49.Lee K. Y., D’Acquisto F., Hayden M. S., Shim J. H., Ghosh S. Science. 2005;308:114–118. doi: 10.1126/science.1107107. [DOI] [PubMed] [Google Scholar]
  • 50.Hill M. M., Andjelkovic M., Brazil D. P., Ferrari S., Fabbro D., Hemmings B. A. J. Biol. Chem. 2001;276:25643–25646. doi: 10.1074/jbc.C100174200. [DOI] [PubMed] [Google Scholar]
  • 51.Harris T. K. IUBMB Life. 2003;55:117–126. doi: 10.1080/1521654031000115951. [DOI] [PubMed] [Google Scholar]
  • 52.Sato S., Fujita N., Tsuruo T. Oncogene. 2002;21:1727–1738. doi: 10.1038/sj.onc.1205225. [DOI] [PubMed] [Google Scholar]
  • 53.Senderowicz A. M. Cancer Biol. Ther. 2003;2:S84–95. [PubMed] [Google Scholar]
  • 54.Flynn P., Wongdagger M., Zavar M., Dean N. M., Stokoe D. Curr. Biol. 2000;10:1439–1442. doi: 10.1016/s0960-9822(00)00801-0. [DOI] [PubMed] [Google Scholar]
  • 55.Heo D. S., Snyderman C., Gollin S. M., Pan S., Walker E., Deka R., Barnes E. L., Johnson J. T., Herberman R. B., Whiteside T. L. Cancer Res. 1989;49:5167–5175. [PubMed] [Google Scholar]
  • 56.Sacks P. G., Parnes S. M., Gallick G. E., Mansouri Z., Lichtner R., Satya-Prakash K. L., Pathak S., Parsons D. F. Cancer Res. 1988;48:2858–2866. [PubMed] [Google Scholar]
  • 57.Riser B. L., Mitra R., Perry D., Dixit V., Varani J. Cancer Res. 1989;49:6123–6129. [PubMed] [Google Scholar]

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