Diacylglycerol kinase θ counteracts protein kinase C-mediated inactivation of the EGF receptor

The International Journal of Biochemistry & Cell Biology 44 (2012) 1791–1799

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Diacylglycerol kinase ␪ counteracts protein kinase C-mediated inactivation of the EGF receptor Jürgen van Baal a,1 , John de Widt a , Nullin Divecha b,∗∗ , Wim J. van Blitterswijk a,∗ a b

Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Paterson Institute for Cancer Research, Inositide Laboratory, The University of Manchester, Wilmslow Road, M20 4BX Manchester, UK

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Article history: Received 29 February 2012 Received in revised form 13 June 2012 Accepted 13 June 2012 Available online 23 June 2012 Keywords: Diacylglycerol kinase Epidermal growth factor receptor activity Protein kinase C Feedback regulation G protein-coupled receptor

a b s t r a c t Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC) signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGK␪ isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGK␪ physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGK␪ attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGK␪ downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGK␪ translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGK␪ attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Epidermal growth factor receptor (EGFR) signaling critically regulates cell proliferation and migration in normal and neoplastic cells (Yarden and Sliwkowski, 2001; Jorissen et al., 2003). It is known that EGFR number and activity are increased in many malignancies (Sebastian et al., 2006). However, the mechanisms that regulate EGFR signaling at the membrane level are incompletely understood. Much attention has focused on (direct) ligand-induced EGFR signaling and (indirect) EGFR transactivation by stimulation of G protein-coupled receptors (GPCR) (Jorissen et al., 2003; Gschwind et al., 2001; Schäfer et al., 2004), but less attention has

Abbreviations: DAG, diacylglycerol; DGK, diacylglycerol kinase; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; PLD, phospholipase D; PKC, protein kinase C; TPA, 2-Otetradecanoylphorbol-13-acetate. ∗ Corresponding author. Tel.: +31 756282239; fax: +31 205121940. ∗∗ Corresponding author. Tel.: +44 1619187159. E-mail addresses: [email protected] (N. Divecha), [email protected] (W.J. van Blitterswijk). 1 Present address: Wageningen University, Animal Nutrition Group, PO Box 338, 6700 AH Wageningen, The Netherlands. 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2012.06.021

been paid to regulation of negative feedback signaling (desensitization) of the EGFR. EGFR activity is inhibited in cells treated with 12-Otetradecanoylphorbol-13-acetate (TPA), which has been attributed to protein kinase C (PKC)-mediated phosphorylation of Thr654 at the juxtamembrane region of the EGFR (Davis and Czech, 1985; Lund et al., 1990; Welsh et al., 1991; Iwashita and Kobayashi, 1992). Physiologically, PKC can be activated by phospholipase C-mediated diacylglycerol (DAG) formation in response to stimulation of GPCR or (to a lesser extent) EGFR itself. In both cases, evidence suggests that this PKC pathway provides a negative feedback loop to attenuate EGFR signaling (Iwashita and Kobayashi, 1992; Chen et al., 1996; Grewal et al., 2001; Santiskulvong and Rozengurt, 2007). To add to this complexity, PKC itself is also subject to negative feedback regulation. PKC can interact with diacylglycerol kinase (DGK) (Yamaguchi et al., 2006; Luo et al., 2003; Van Baal et al., 2005), an enzyme that phosphorylates diacylglycerol (DAG) to phosphatidic acid, thereby removing the PKC activator. These two feedback principles may well be physiologically connected and, indeed, one paper suggests that the DGK␦ isotype regulates EGFR by modulating PKC signaling (Crotty et al., 2006). We previously identified DGK␪ (Houssa et al., 1997) as one of ten existing DGK isozymes (Van Blitterswijk and Houssa, 2000; Mérida et al., 2008) and reported on its structure–activity relationship (Los

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et al., 2004), its regulation by interaction with RhoA (Houssa et al., 1999) and its translocation to the plasma membrane in response to GPCR agonists (Van Baal et al., 2005; Walker et al., 2001). We found that DGK␪ negatively regulates PKC␧/␩ activity in A431 carcinoma cells (Van Baal et al., 2005). DGK␪ translocated from the cytosol to the plasma membrane upon activation of these PKC isotypes in response to GPCR stimulation or by their direct activators TPA or membrane-permeable DAG (Van Baal et al., 2005). Since these cells express an extremely high number of EGFR, and we could block DGK␪ translocation by the EGFR kinase inhibitor AG1478 (see Section 3), we hypothesized that both the EGFR and PKC signaling are required for translocation of DGK␪ to the membrane and that, conversely, DGK␪ at the plasma membrane may regulate PKC and EGFR activity. Here, we provide evidence that this is indeed the case, and that the three kinases thereby (inter)act in one ternary signaling complex. Our data thus suggest that DGK␪ can indirectly enhance EGFR activity. 2. Materials and methods 2.1. Reagents and antibodies Dulbecco’s modified Eagle’s medium (DMEM) and Geneticin (G418) were purchased from Life Technologies. Ro31-8220 and tyrphostin AG1478 were obtained from Calbiochem. [␥-32 P]ATP was from Amersham Pharmacia Biotech. Tetramethylrhodamineconjugated epidermal growth factor (EGF-rh) was purchased from molecular probes. All other chemicals, including human recombinant EGF, uridine-5 -triphosphate (UTP), bradykinin (BK), and 12-O-tetradecanoylphorbol-13-acetate (TPA) were from Sigma. Affinity-purified rabbit polyclonal antibodies directed against ErbB3 were from Santa Cruz Biotechnology. Rabbit anti-human ErbB1 (EGFR) and mouse anti-human ErbB2 were from Neomarkers. Affinity-purified rabbit phospho-specific antibodies directed against the EGFR sites Y845 and Y1068 were from Cell Signaling Technology. Mouse monoclonal anti-human EGFR and anti-phospho-Thr654 were obtained from Nanotools. Mouse monoclonal anti-phosphotyrosine (4G10) was from Upstate, monoclonal anti-DGK␪ from BD Biosciences, and monoclonal anti-actin from Sigma. 2.2. Cells and plasmids Human A431 epidermoid carcinoma, HEK293 embryonic kidney and Phoenix packaging cells were routinely grown in DMEM supplemented with 8% heat-inactivated fetal bovine serum, penicillin (100 units/ml), streptomycin (100 units/ml) and 2 mM l-glutamine (complete medium). pMT2-EGFR vector was a gift of W. Moolenaar (The Netherlands Cancer Institute, Amsterdam). Human wild-type DGK␨ (in pBABE vector) (Los et al., 2006), DGK␪ and catalytically inactive DGK␪ (kinase-dead G648D mutant; kdDGK␪, in pcDNA3 vector) (Los et al., 2004), were described previously. NH2 -terminally FLAG-tagged DGK␪ and FLAG-kdDGK␪ were generated by PCR and inserted into the EcoR1 restriction site of pBABE. To suppress endogenous DGK␪ expression, we used the retroviral derivative of the mammalian expression vector pSUPER (Brummelkamp et al., 2002) to introduce synthetic shortinterfering RNAs (siRNAs) into A431 cells. SiRNA primers were: GATCCCCTGC GAC GTC TGC AAT TTC A TTCAAGA G ATG AAA TTG CAG ACG TCG CAT TTTTGGAA (sense). AGCTTTCCAAAAATG CGA CGT CTG CAA TTT CAT CTCTTGAAT GAA ATT GCA GAC GTC GCA GGG (antisense). Underlined bases target the codons of the amino acid residues CDVNF in the Cysteine-rich domain 1 (CRD1) of human DGK␪ (Houssa et al., 1997). After annealing of both primers, BglII and HindIII sites were used for cloning of the insert in pSUPER.

For mock transfection, a plasmid with scrambled siRNA was used. A431 and HEK293 cells were transfected with DGK␪ plasmids using Fugene according to manufacturer’s instructions (Roche). For stable DGK␪ (over)expression, Phoenix packaging cells were transfected with DGK␪-pBABE using Fugene. A431 cells were transduced with retroviral supernatants and selected with puromycin (2 ␮g/ml). 2.3. Reverse transcriptase PCR Total RNA was extracted from exponentially growing A431 or HEK293 cells using Trizol (Invitrogen). First-strand synthesis was performed from 3 ␮g of total RNA using 3 ␮g of random p(N6) primers (Roche), 0.5 mM dNTPs (Roche), 1× first strand buffer (Roche), 10 mM dithiothreitol, and 200 units of Superscript II-RT (Invitrogen) in a volume of 20 ␮L at 42 ◦ C for 1 h. A 5% fraction of the resulting single-stranded cDNA sample was amplified by PCR using Taq DNA polymerase (Invitrogen) in a final volume of 50 ␮l. For EGFR/ErbB1 the forward primer 5 -CCACCTGTGCCATCCAAACT-3 and the reverse primer 5 -CCTTATACACCGTGCCGAAC-3 were used. For ErbB2, we used forward primer 5 -GCCTTGCCCCATCAACTG3 and the reverse primer 5 -CAAGCACCTTCACCTTCCTC-3 . For ErbB3, forward primer 5 -CCTGCCATGAGAACTGCAC-3 and reverse primer 5 -ACTCTGCCGTCCACTCTTGT-3 . For ErbB4, forward primer 5 -CAACATCCCACCTCCCATCTATAC-3 and reverse primer 5 GGTGCCATTACAGCAGGAGT-3 . PCR was performed as follows: 1 min at 94 ◦ C, 30 cycles of (94 ◦ C for 30 s, 58 ◦ C for 30 s, and 72 ◦ C for 45 s), followed by 72 ◦ C for 10 min. PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide and visualized under UV light. 2.4. Cell imaging Imaging of cells was performed as described previously (Van Baal et al., 2005). Briefly, cells expressing GFP-tagged DGK␪ (Van Baal et al., 2005) were seeded onto glass coverslips, allowed to attach for 48 h. Before live cell imaging, cells were transferred to serum-free Dulbecco’s modified Eagle’s medium (supplemented with 20 mM HEPES buffer, pH 7.4) for 2 h and then placed inside a prewarmed (37 ◦ C) chamber. Images were taken on a Leica inverted confocal laser microscope. GFP fluorescence was excited with an argon laser emitting at 488 nm, and images were obtained with a ×63 numerical aperture 1.4 oil immersion lens and Leica software. 2.5. DGK activity in vivo A431 cells stably expressing FLAG-tagged wild-type or kinaseinactive G648D-DGK␪ or HA-tagged DGK␨ (Los et al., 2006) were plated on 60 mm dishes (Falcon) in complete medium DMEM at 80% confluency. After 16 h, cells were rinsed twice with phosphate-free DMEM (without serum) and incubated in 2 ml of phosphate-free DMEM for 1 h at 37 ◦ C. Cells were then metabolically labeled with 1.0 mCi/ml [32 P]orthophosphate for 2 h. The 32 P-labelled cells were stimulated with UTP (100 ␮M) or TPA (300 nM) at 37 ◦ C. Reactions were stopped by quickly aspirating the medium and freezing the cells with liquid nitrogen. Lipids were extracted, radioactive PA was separated by TLC, visualized and quantitated by phosphorimaging (Van Baal et al., 2005). 2.6. Western blotting and immunoprecipitation Cell lysates were cleared by centrifugation and separated using the NUPAGE Pre-Cast SDS-PAGE Gel System (4–12% gels; Invitrogen). Separated proteins were blotted to nitrocellulose membranes, probed with primary antibodies, then with swine anti-rabbit or rabbit anti-mouse horseradish peroxidase-conjugated secondary

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antibodies (DAKO) and eventually developed by ECL Western Blotting Detection Reagents (Amersham Biosciences). All presented blots are representative of at least three separate experiments yielding similar results. Immunoprecipitates of FLAG-DGK␪ from cell lysates using monoclonal M2 (anti-FLAG) antibody (final concentration 1 ␮g/ml; Sigma) were subjected to SDS-PAGE and subsequent immunoblotting with relevant antibodies. As a control, FLAG-peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (4 ␮g/ml, pH 7.5) was used to compete with specific immunoprecipitation. 3. Results 3.1. EGF receptor activity is required for hormone-induced DGK translocation We previously reported that stimulation of A431 cells by UTP, ATP, bradykinin or thrombin induced the PKC-mediated translocation of green fluorescent protein (GFP)-tagged DGK␪ from the cytosol to the plasma membrane (Van Baal et al., 2005). A431 cells are known to express an extremely high number of EGFR, that leads to basal activity in the absence of EGFR ligand (Sako et al., 2000). To our surprise, preincubation of A431 cells with AG1478 completely blocked the UTP-induced translocation of DGK␪-GFP (Fig. 1A). AG1478 likewise inhibited ATP- and bradykinin-induced DGK␪-GFP translocation (data not shown). Our previous work has shown that PKC is a direct mediator of this translocation (Van Baal et al., 2005). These data therefore suggest that EGFR activity is required for DGK␪ translocation to the plasma membrane in response to GPCR stimulation and subsequent PKC activation. We next tested this concerted action of activated PKC and EGFR in the regulation of DGK␪ translocation in another cell type, HEK293. Stimulation of these cells with either phorbol ester (TPA, strong activator of PKC) or EGF alone did not induce translocation of transfected DGK␪-GFP to the plasma membrane (Fig. 1B). However together, TPA and EGF induced DGK␪-GFP translocation in about 43% of the cells (Fig. 1B). This co-stimulatory effect was completely abolished by pretreatment with PKC inhibitors staurosporine (1 ␮M; 15 min) or Ro31-8220 (5 ␮M; 15 min) or with the EGFR inhibitor, AG1478 (5 ␮M; 30 min) (data not shown). DGK␪GFP translocation in HEK293 cells was much more pronounced when the human EGFR (ErbB1/HER1) was overexpressed (HEK293EGFR cells). In this case, TPA readily induced translocation of DGK␪ even without co-stimulation with EGF in about 40% of the cells (Fig. 2), similar to A431 cells (Van Baal et al., 2005). When these HEK293-EGFR cells were stimulated with TPA and EGF together, DGK␪ translocation was observed in up to 70% of the cells. The translocation induced by TPA (with or without EGF) was completely prevented by AG1478 (Fig. 2). From these data, we conclude that EGFR tyrosine kinase activity is required for the PKC-mediated DGK␪ translocation from the cytosol to the plasma membrane. 3.2. Colocalization of DGK with EGFR at the plasma membrane and in endosomes Since EGFR activity was required for hormone-induced, PKCmediated DGK␪ translocation to the plasma membrane, we investigated if DGK␪ was recruited towards the EGFR. For this purpose, we used rhodamine-conjugated EGF to activate and trace EGFRs in A431 cells expressing DGK␪-GFP. Rhodamine-EGF binding to the EGFR stained the cell surface within 2 min (Fig. 3A), and significant internalization of the EGFR was seen after 10 min, resulting in rhodamine-EGF-stained vesicles, without any sign of DGK␪-GFP translocation. However, upon co-stimulation with TPA (to activate PKC), translocation of DGK␪ to the plasma membrane was observed

Fig. 1. DGK␪ translocation to the plasma membrane in response to UTP or phorbol ester stimulation requires EGFR activity. Cells were stably transfected with GFPtagged DGK␪. (A) A431 cells were pretreated with or without AG1478 (5 ␮M, 2 h) and then stimulated with UTP (100 ␮M, 15 min). AG1478 prevents DGK␪-GFP translocation to the plasma membrane. (B) HEK293 cells were stimulated with EGF (40 ng/ml) or TPA (300 nM), or both agonists for 15 min at 37 ◦ C. Stimulation with TPA or EGF alone is not sufficient to induce translocation of DGK␪-GFP, visualized by confocal laser scanning microscopy. Bars: 10 ␮m.

within 5 min, and increasing co-localization with EGFR was seen in time. From 15 to 30 min, next to plasma membrane staining, DGK␪-GFP also increasingly appeared in intracellular vesicle-like structures, again co-localizing with EGFR (Fig. 3A). We calculated that, at 25 min, 83 ± 5% (N = 300 cells) of GFP-stained vesicles in A431 cells contained rhodamine-EGF. Of note, the presence of DGK␪-GFP in vesicles was not detected when cells were pretreated with AG1478 (30 min, 5 ␮M; data not shown). Also in HEK293 cells, we observed this AG1478-sensitive translocation of DGK␪-GFP and co-localization with rhodamine-EGF-stained EGFR in endosomal

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Fig. 2. Overexpression of EGFR in HEK293 cells enables PKC-mediated DGK␪ translocation. HEK293 cells were transfected with both EGFR and DGK␪-GFP and then stimulated with TPA (300 nM) for the indicated time periods. TPA readily induced translocation of DGK␪ to the plasma membrane. This translocation was blocked by preincubation (30 min) with AG1478 (5 ␮M). Bars: 10 ␮m.

vesicles (Fig. 3B). These findings indicate that not only the PKCmediated translocation of DGK␪ towards the plasma membrane but also its final redistribution to EGFR-containing endosomes depends on the activity of the EGFR.

3.3. DGK physically interacts with the EGFR, becomes phosphorylated on tyrosine and is active EGFR is one of the four members, designated ErbB1 (EGFR), ErbB2, ErbB3 and ErbB4, of the ErbB family of receptor tyrosine kinases (Linggi and Carpenter, 2006). Reverse transcription PCR revealed that A431 cells express ErbB1, ErbB2 and ErbB3, whereas HEK293 cells express all four ErbB family members (Fig. 4A). To determine possible interaction of DGK␪ with any of the expressed ErbB family members in A431 cells, we tried immunoprecipitation of endogenous DGK␪ but, due to its low expression, we could hardly detect the protein on a Western blot. We therefore transfected A431 cells with FLAG-tagged DGK␪ (FLAG-DGK␪). The Western blot of specifically immunoprecipitated FLAG-DGK␪ revealed a significant amount of EGFR (ErbB1) binding, but not of other ErbB family members (Fig. 4B). This physical interaction seems constitutive, as it was not significantly affected by EGF or TPA stimulation. We next investigated whether DGK␪ could likewise interact with EGFR in HEK293 cells. Since these cells express a relatively low amount of EGFR in comparison with A431 cells, co-transfection of EGFR was required to enhance sensitivity. Fig. 5A shows that, similar to A431 cells, the EGFR in HEK293 cells co-immunoprecipitated with FLAG-DGK␪, while the interaction in this case was slightly more pronounced when the cells were stimulated with TPA or EGF, or both. Interestingly, this physical interaction between DGK␪ and the EGFR was dependent on the receptor activity, since AG1478 prevented the interaction. Binding of DGK␪ to active EGFR resulted in phosphorylation of DGK␪ on tyrosine, as detected with the antiphospho-tyrosine-specific monoclonal antibody 4G10 (Fig. 5B). Using this antibody, we confirmed EGF-induced activation of EGFR that was co-immunoprecipitated with FLAG-DGK␪, by the strong overall EGFR tyrosine phosphorylation. EGF-induced tyrosine phosphorylation of both EGFR and DGK␪ was blocked by AG1478 or by

co-stimulation with TPA, to activate PKC (Fig. 5B). Collectively, the data indicate that DGK␪ interacts with, and is phosphorylated by the EGFR. Since DGK is essentially active when it is membrane-bound and its substrate DAG is available (Van Blitterswijk and Houssa, 2000; Mérida et al., 2008), the translocation of DGK␪ from the cytosol to the plasma membrane is expected to be accompanied by phosphorylation of DAG to produce phosphatidic acid. Stimulation of GPCRs receptors makes DAG available through the hydrolysis of PIP2 . To measure DGK␪ activity in response to receptor stimulation, we transduced A431 cells with FLAG-DGK␪ to amplify the production of phosphatidic acid. After labeling the ATP pool in the cells with ortho-[32 P]phosphate, we stimulated G protein-coupled purinergic GPCRs with UTP and measured [32 P]phosphatidic acid synthesis in time. Fig. 6 shows that UTP transiently elevates phosphatidic acid formation with an optimum (∼2-fold; P < 0.05) around 9 min. To demonstrate that this effect was specific for DGK␪, we found no phosphatidic acid synthesis when cells were transfected with an enzymatically inactive (G648D)-DGK␪ mutant, or with DGK␨, a different isoform expressed in these cells (Supplementary Fig. 1). Stimulation of cells with TPA (300 nM) did not result in phosphatidic acid formation, irrespective of the expressed construct (data not shown). Remarkably, the time dependence of UTP-induced phosphatidic acid production closely resembles that of our previously reported PKC-mediated translocation of DGK␪ to the plasma membrane in these A431 cells (Van Baal et al., 2005).

3.4. Negative regulation of EGFR by PKC-mediated phosphorylation at Thr654 is counteracted by DGK The above in vivo enzymatic activity data suggest that DGK␪ temporarily lowers DAG levels in response to GPCR stimulation. Since DAG activates PKC, and the PKC␧ and PKC␩ isotypes were previously found to mediate DGK␪ translocation to the plasma membrane in A431 cells (Van Baal et al., 2005), it is conceivable that DGK␪ attenuates PKC-mediated signals in A431 cells in a negative feedback loop. Since DAG-sensitive PKC phosphorylates the EGFR at Thr654, which attenuates the EGFR (see Section 1),

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Fig. 3. Co-localization of DGK␪-GFP and EGFR at the plasma membrane and subsequently in endocytic vesicles upon stimulation with TPA and rhodamine-EGF. A431 cells (indicated) (A) or HEK293 cells (indicated) (B) were transfected with DGK␪-GFP and, in case of HEK293, cotransfected with EGFR. Cells were stimulated with TPA (300 nM) for the indicated time periods. To visualize and activate EGFRs, cells were pretreated with rhodamine-conjugated EGF (EGF-Rh; 60 ng/ml) for 2 min. DGK␪-GFP undergoes time-dependent translocation from the cytosol to the plasma membrane and eventually also appears in EGF-Rh containing vesicles (arrows). The magnified lower photograph in panel (B) represents an optimal visualization of intracellular vesicles containing DGK␪-GFP (arrows) in HEK293 cells that overexpress the EGFR. Bars: 10 ␮m.

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Fig. 4. EGFR (ErbB1) but not other ErbB family members co-immunoprecipitate with DGK␪ in A431 cells. (A) Expression of ErbB isoforms in A431 and HEK293 cells. Total RNA from A431 or HEK293 cells was reverse-transcribed into cDNA and PCR was performed using specific primers. PCR products were separated on 1.5% (w/v) agarose gel and visualized by ethidium bromide staining. The figure is representative for three independent experiments. (B) DGK␪ specifically interacts with ErbB1 (EGFR). A431 cells expressing FLAG-tagged DGK␪ were stimulated with EGF (40 ng/ml) or TPA (300 nM) for 15 min at 37 ◦ C. Cell lysates (L) were subjected to immunoprecipation (IP) with anti-FLAG monoclonal antibodies, followed by immunoblotting (IB) using specific antibodies against the various ErbB receptors. Where indicated, an excess of FLAG-peptide was added to lysate to demonstrate the specificity of the immunoprecipitation.

Fig. 5. DGK␪ binds to active EGFR in HEK293 cells and becomes phosphorylated on tyrosine. HEK293 cells were cotransfected with FLAG-DGK␪ and with empty vector or EGFR, as indicated. Two days after transfection, cells were left untreated or were pretreated with AG1478 (5 ␮M, 2 h) and stimulated with EGF (40 ng/ml) with or without TPA (300 nM) for 15 min. Lysates were subjected to immunoprecipitation (IP) using monoclonal anti-FLAG antibody followed by immunoblotting (IB) with the indicated antibodies.

We next investigated the effect of DGK␪ on the PKC-mediated EGFR phosphorylation at Thr654. To this end, we transfected A431 cells with FLAG-tagged DGK␪ and stimulated PKC directly with TPA or indirectly by the GPCR agonist bradykinin. Expression of

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PA formation

and since we found DGK␪ colocalization and interaction with the EGFR, we investigated if DGK␪ would regulate this PKC-mediated phosphorylation of the EGFR. We first confirmed that stimulation of A431 cells with various hormones (bradykinin, UTP), growth factor (EGF) or a direct activator of PKC (TPA) induced the PKC-mediated EGFR-Thr654 phosphorylation (Fig. 7A). In all cases, preincubation of cells with the PKC inhibitor Ro31-8220 blocked this phosphorylation completely. Another PKC inhibitor chelerythrine chloride (5 ␮M; 15 min preincubation) blocked Thr654 phosphorylation in a similar fashion (data not shown). Ro31-8220 pretreatment furthermore enhanced EGFR phosphorylation at Tyr1068, a prominent EGFR autophosphorylation site, and at Tyr845, a site phosphorylated by Src (Jorissen et al., 2003; Sebastian et al., 2006) (Fig. 7A). Phosphorylation at these two sites reflects the degree of EGFR activation (Jorissen et al., 2003; Sebastian et al., 2006). The data confirm that PKC-mediated Thr654 phosphorylation by the various stimuli suppresses activation of the EGFR.

(% of control)

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wtDGKθ kdDGKθ wtDGKζ

150 125 100 75 0 3

6

9

12

15

UTP stimulation time (min) Fig. 6. Transient activity of DGK␪ in response to UTP stimulation. A431 cells stably transfected with either wild-type DGK␪ (closed circles), kinase-inactive (kd) DGK␪ (open circles) or wild-type DGK␨ (squares) were radiolabeled for 2 h with ortho[32 P]phosphate and then stimulated with UTP (100 ␮M) for the indicated periods of time. Lipids were extracted, separated by TLC and visualized and counted by phosphoimaging. Phosphatidic acid (PA) formation is expressed as percentage of controls (N = 4).

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Fig. 7. Agonist-stimulated, PKC-mediated phosphorylation of EGFR at Thr654 and concomitant suppression of Tyr1068 and Tyr845 phosphorylation. (A) A431 cells were preincubated without (control; 0.1% DMSO vehicle) or with 1 ␮M Ro31-8220 for 30 min, and then stimulated with bradykinin (BK; 1 ␮M), UTP (100 ␮M), EGF (40 ng/ml) or TPA (300 nM) for 10 min. Cell lysates were subjected to Western blotting analysis using specific antibodies against the indicated phosphorylated EGFR sites or total EGFR. (B) DGK␪ overexpression inhibits bradykinin-induced, but not TPA-induced Thr654 phosphorylation. A431 cells stably transduced with empty vector (control) or FLAG-DGK␪ were stimulated with bradykinin (BK; 1 ␮M) or TPA (300 nM) for the indicated time periods. Cell lysates were analyzed by immunoblotting.

DGK␪ decreased the bradykinin-induced Thr654 phosphorylation, whereas it had no effect on TPA-induced Thr654 phosphorylation (Fig. 7B). This makes sense, since TPA-activated PKC (unlike GPCRinduced, DAG-activated PKC) cannot be inactivated by active (i.e. DAG-consuming) DGK. To investigate if DGK␪ plays a regulatory role in the inactivation of the EGFR following PKC-mediated Thr654 phosphorylation, DGK␪-transfected A431 cells or (empty vector) control cells were stimulated with bradykinin (to generate DAG and activate PKC) prior to EGF stimulation. Fig. 8 shows that, in (vector) control cells and in the absence of bradykinin, EGF induced only weak Thr654 phosphorylation. Short (5 min) prestimulation with bradykinin greatly enhanced this phosphorylation, which then gradually decreased in time. Upon DGK␪ expression, the EGFand bradykinin-induced Thr654 phosphorylation was dramatically reduced. Similar results were obtained when using UTP prestimulation (Supplementary Fig. 2). Furthermore, while in vector-control cells, bradykinin significantly attenuated the EGFinduced phosphorylations at Tyr1068 and Tyr845, this attenuation was abrogated when DGK␪ was expressed. These data indicate that DGK␪ decreases Thr654 phosphorylation and thus counteracts the inactivation of the EGFR by PKC. While the above effects were most clear when DGK␪ was overexpressed, we wished to confirm these results for endogenous DGK␪, which is expressed at only very low levels and barely detectable on a Western blot. RT-PCR analysis revealed that A431 cells express DGK␪, DGK␣, DGK␦, DGK␧, DGK␩ and two splicing variants of DGK␨ (Supplementary Fig. 1). DGK␤, DGK␥, and DGK␫ were not expressed. We downregulated the DGK␪ isozyme by siRNA, as confirmed by Western blotting (Fig. 9, lower panel). The

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Fig. 8. DGK␪ attenuates Thr654 phosphorylation in response to bradykinin and consequently increases phosphorylation of the activated EGFR at Tyr845 and Tyr1068. A431 cells stably transduced with FLAG-DGK␪ or empty vector were preincubated without (control) or with bradykinin (BK; 1 ␮M) for 5 min, and then stimulated with EGF (40 ng/ml) for the indicated time periods. Cell lysates were analyzed by immunoblotting using phospho-specific antibodies against the indicated EGFR phosphorylation sites or total EGFR. For all stimulated samples, vector and DGK␪ blots were run and probed at the same time. To this end, one blot corresponding to DGK␪- and one for the empty vector-stimulated cells were placed together in one container to which one of the indicated antibodies was added. Exposure times following addition of ECL detection reagent were the same for each antibody.

Fig. 9. Effect of DGK␪ silencing on agonist-induced EGFR-Thr654 phosphorylation. A431 cells stably transduced with pRetroSuper vector encoding either scrambled (control) or DGK␪–siRNA were stimulated with Bradykinin (BK; 1 ␮M) or UTP (100 ␮M) for indicated periods of time. For comparison, cells were also stimulated with EGF (40 ng/ml) or TPA (300 nM) for 5 min. Cell lysates were subjected to Western blot analysis using antibodies against phospho-Thr654 EGFR, total EGFR or ␤-actin. Control and DGK␪–siRNA blots were run and probed at the same time, with similar exposure times, in the same way as described in the legend of Fig. 8. Lower panel: Depletion of endogenous DGK␪ by siRNA, demonstrated by Western blotting using a monoclonal antibody against DGK␪.

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DGK␪-depleted A431 cells, indeed, showed enhanced Thr654 phosphorylation in response to stimulation with EGF, bradykinin or UTP, but not to TPA (Fig. 9, upper panel). These data confirm our above notion that DGK␪ expression (and activity) affects only hormoneinduced elevation of DAG levels and subsequent PKC activation, not phorbol ester-activated PKC. We conclude that DGK␪, endogenous or overexpressed, suppresses PKC-mediated Thr654 phosphorylation, thereby attenuating the negative feedback regulation of EGFR activity.

4. Discussion In this paper we describe a new regulatory function of the DGK␪ isotype in growth factor receptor signaling. It was already known that activation of PKC by phorbol ester or GPCR stimulation leads to phosphorylation of the EGFR at residue Thr654, thereby inactivating/desensitizing this growth factor receptor in a negative feedback loop (Davis and Czech, 1985; Lund et al., 1990; Welsh et al., 1991; Iwashita and Kobayashi, 1992; Chen et al., 1996; Grewal et al., 2001; Santiskulvong and Rozengurt, 2007). Our present work adds a new level of complexity in this EGFR regulation, that is the attenuation and fine-tuning of the PKC-mediated Thr654 phosphorylation by DGK␪. We showed by co-immunoprecipitation that DGK␪ interacts with, and is phosphorylated by the EGFR. DGK␪ is recruited to the plasma membrane by active EGFR and PKC signaling, where it eliminates the PKC activator DAG through phosphorylation to phosphatidic acid. Using confocal microscopy, we demonstrated DGK␪ translocation to the plasma membrane and co-localization with the EGFR. Inhibition of EGFR kinase activity by AG1478 fully blocked translocation and interaction of DGK␪ with the receptor. Our previous work (Van Baal et al., 2005) has suggested that, in A431 cells, activation of PKC␧/␩ isotypes induced the translocation of DGK␪ to the plasma membrane. In the present paper, we concluded that this translocation required the activities of both PKC and the highly expressed EGFR. Since DGK␪ specifically bound to, and was phosphorylated by the PKC␧ and PKC␩ isotypes (Van Baal et al., 2005), and it also bound to the EGFR (this study), it is conceivable that DGK␪, PKC␧/␩ and the EGFR interact in one ternary signaling complex, fine-tuning EGFR activity by feedback loops. Desensitization of the EGFR through PKC-mediated Thr654 phosphorylation occurs in the direct, EGF-activated pathway as well as indirectly, through GPCR-stimulated PKC signaling (Fig. 7A). Importantly, in both pathways DGK␪ counteracts this process, as found upon DGK␪ overexpression (Figs. 7B and 8, Supplementary Fig. 2) as well as siRNA-induced downregulation of endogenous DGK␪ (Fig. 9). EGFR activation results in autophosphorylation of a number of Tyr-residues of the receptor (Sebastian et al., 2006), two of which (pTyr1173 and pTyr992) recruit and bind phospholipase C-␥ (Chattopadhyay et al., 1999), resulting in the hydrolysis of PIP2, DAG formation and activation of PKC. Our data suggest that this latter step is attenuated by DGK␪, which converts DAG to phosphatidic acid. It would be interesting to investigate the temporal sequence of events of PIP2-to-DAG-to-phosphatidic acid formation in more detail, in relation to possible physical association of PLC-␥ with the above-proposed EGFR-PKC-DGK␪ complex. EGF stimulation of cells also activates other lipid-related enzymes (Jorissen et al., 2003). One of these enzymes, phospholipase D (PLD) also produces phosphatidic acid, like DGK does. The PLD2 isotype has also been shown to be associated with, phosphorylated and activated by the EGFR (Slaaby et al., 1998). However, PLD acts on a different substrate, phosphatidylcholine, and seems therefore unrelated to PKC-mediated receptor desensitization. Moreover, n-butanol, which inhibits PLD-mediated phosphatidic acid formation did not affect DGK␪ translocation induction by any agonist (Van Baal et al., 2005). Phosphoinositide kinases also bind

to, and are activated by the EGFR. PI3-kinase is a well known example (Sebastian et al., 2006), but also PI 4-kinase and PI(4)P 5-kinase have been detected in immunoprecipitates of the activated EGFR (Cochet et al., 1991). It is not known if these enzymes contribute to EGFR activity regulation. Activated EGFRs are known to be ubiquitinated and endocytosed and are then either returned back to the cell surface or degraded in lysosomes (Sebastian et al., 2006). Sorting to lysosomes downregulates the receptor and serves another way of negative-feedback regulation of receptor signaling. At the same time, internalized EGFR continues to signal from endosomes, and this endosomal signaling is thought to play a role in determining intensity, duration and specificity of signaling processes (Sebastian et al., 2006; Sorkin and von Zastrow, 2002). We showed that DGK␪, after initial translocation from the cytosol to the activated EGFR at the plasma membrane, moved into endosomal vesicles, presumably in a complex with the EGFR. This suggests that, also in endosomes, DGK␪ continues to attenuate Thr654 phosphorylation, thus enhancing EGFR activity. In CHO cells, PKC-mediated EGFR-Thr654 phosphorylation inhibited ubiquitination and redirected receptor trafficking from lysosomal degradation to a recycling endosomal pathway (Bao et al., 2000). In A431 cells, we confirmed that (GPCRactivated) PKC inhibited EGF-induced EGFR ubiquitination (data not shown), but we found no significant changes in EGFR on Western blots within the time scale of our experiments. The activity of the EGFR was estimated here by the phosphorylation status of two sites, Tyr1068 and Tyr845 in the catalytic domain of the receptor. Tyr1068 is a major autophosphorylation site that, in its phosphorylated state, recruits the adaptor protein Grb2, the main step in EGF-dependent induction of the Ras-MAPkinase/ERK and Akt/PKB pathways (Sebastian et al., 2006). Tyr845, a highly conserved residue in tyrosine kinases, is phosphorylated by Src, which is important for EGFR function and tumor progression (Biscardi et al., 1999). EGFR-pTyr845 binds the transcription factor STAT5b (overexpressed in several tumors), which mediates EGF-induced DNA synthesis and confers tamoxifen resistance in breast tumors (Kloth et al., 2003; Fox et al., 2008). It also regulates the cytochrome C oxidase subunit II-related trafficking of EGFR to mitochondria, which contributes to cell survival (Boerner et al., 2004). Clearly, enhanced phosphorylation of these two EGFR sites, Tyr1068 and Tyr845 (and possibly other (auto)phosphorylation sites as well) under the influence of DGK␪ activity is an important new EGFR regulatory principle, which also implies that intervention at the level of DGK might be considered as an alternative or additional therapeutic approach to treat EGFR-dependent cancer. Acknowledgement This work was funded by the Dutch Cancer Society. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.biocel.2012.06.021. References Bao J, Alroy I, Waterman H, Schejter ED, Brodie C, Gruenberg J, Yarden Y. Threonine phosphorylation diverts internalized epidermal growth factor receptors from a degradative pathway to the recycling endosome. Journal of Biological Chemistry 2000;275:26178–86. Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, Parsons SJ. c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. Journal of Biological Chemistry 1999;274:8335–43. Boerner JL, Demory ML, Silva C, Parsons SJ. Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial

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