The International Journal of Biochemistry & Cell Biology
Diacylglycerol kinase θ counteracts protein kinase C-mediated inactivation of the EGF receptor
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 been paid to regulation of negative feedback signaling (desensitization) of the EGFR.
EGFR activity is inhibited in cells treated with 12-O-tetradecanoylphorbol-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 DGKδ isotype regulates EGFR by modulating PKC signaling (Crotty et al., 2006).
We previously identified DGKθ (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 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 DGKθ negatively regulates PKCɛ/η activity in A431 carcinoma cells (Van Baal et al., 2005). DGKθ 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 DGKθ translocation by the EGFR kinase inhibitor AG1478 (see Section 3), we hypothesized that both the EGFR and PKC signaling are required for translocation of DGKθ to the membrane and that, conversely, DGKθ 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 DGKθ can indirectly enhance EGFR activity.
Section snippets
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. [γ-32P]ATP was from Amersham Pharmacia Biotech. Tetramethylrhodamine-conjugated 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
EGF receptor activity is required for hormone-induced DGKθ 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 DGKθ 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
Discussion
In this paper we describe a new regulatory function of the DGKθ 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,
Acknowledgement
This work was funded by the Dutch Cancer Society.
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Present address: Wageningen University, Animal Nutrition Group, PO Box 338, 6700 AH Wageningen, The Netherlands.