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

Abstract

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 DGKθ 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, DGKθ physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGKθ attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGKθ downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGKθ translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGKθ attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling.

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.