In response to epidermal growth factor (EGF), the EGF receptor is endocytosed and degraded. A substantial lag period exists between endocytosis and degradation, suggesting that endocytosis is more than a simple negative feedback. Phospholipase D (PLD), which has been implicated in vesicle formation in the Golgi, is activated in response to EGF and other growth factors. We report here that EGF receptor endocytosis is dependent upon PLD and the PLD1 regulators, protein kinase C ␣ and RalA. EGF-induced receptor degradation is accelerated by overexpression of either wild-type PLD1 or PLD2 and retarded by overexpression of catalytically inactive mutants of either PLD1 or PLD2. EGF-induced activation of mitogen-activated protein kinase, which is dependent upon receptor endocytosis, is also dependent upon PLD. These data suggest a role for PLD in signaling that facilitates receptor endocytosis.Phospholipase D (PLD) is a widely distributed enzyme that hydrolyzes phosphatidylcholine, a major phospholipid in the cell membrane, to form phosphatidic acid (PA) and choline. PLD activity, which can be detected in virtually all cell types as well as in most cellular organelles, is believed to play an important role in the regulation of cell physiology by extracellular signals, such as hormones, neurotransmitters, growth factors, and cytokines (8). Multiple PLD activities have been characterized in mammalian cells, and more recently, two mammalian PLD genes (PLD1 and PLD2) have been cloned (6,11,18,22,30). Recent studies indicate that PLD has many different functions in signal transduction, vesicle trafficking, and cytoskeletal dynamics (21). Vesicle budding in the Golgi network was shown to be mediated in part by Arf family GTPases (35). The discovery that Arf family GTPases regulate PLD activity (3, 5) suggested the possibility that PLD was also involved in vesicle transport. Consistent with this idea, PA formation by PLD-mediated hydrolysis of phosphatidylcholine has been reported to be required for the formation of Golgi vesicles (20) and for the transport of vesicles from the endoplasmic reticulum to the Golgi complex (1). PLD has also been reported to stimulate the release of secretory vesicles from the trans-Golgi network (4). It was therefore proposed that the role that Arf plays in vesicle budding in the Golgi network is to regulate PLD activity and PA production (15,33,34). However, there is controversy on this point (2, 40, 41), and it still is not clear how PLD and its primary metabolite, PA, might contribute to vesicle formation.PLD activity is elevated in response to many extracellular signals (8). Our laboratory has investigated the role PLD plays in the transduction of intracellular signals initiated by epidermal growth factor (EGF) (13,23,38). In response to EGF, the EGF receptor is internalized and then degraded (10). The internalization of the EGF receptor is a process that involves endocytic vesicles (10). Since PLD has been implicated in vesicle formation and membrane traffic as discussed above, we hypothesi...

A high percentage of patients with BRAF V600E mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAF V600E melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAF V600E inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance. Cancer Res; 72(4); 969-78. Ó2011 AACR.

3Y1 rat fibroblasts overexpressing the epidermal growth factor (EGF) receptor (EGFR cells) become transformed when treated with EGF.A common response to oncogenic and mitogenic stimuli is elevated phospholipase D (PLD) activity. RalA, a small GTPase that functions as a downstream effector molecule of Ras, exists in a complex with PLD1. In the EGFR cells, EGF induced a Ras-dependent activation of RalA. The activation of PLD by EGF in these cells was dependent upon both Ras and RalA. In contrast, EGF-induced activation of Erk1, Erk2, and Jun kinase was dependent on Ras but independent of RalA, indicating divergent pathways activated by EGF and mediated by Ras. The transformed phenotype induced by EGF in the EGFR cells was dependent upon both Ras and RalA. Importantly, overexpression of wild-type RalA or an activated RalA mutant increased PLD activity in the absence of EGF and transformed the EGFR cells. Although overexpression of PLD1 is generally toxic to cells, the EGFR cells not only tolerated PLD1 overexpression but also became transformed in the absence of EGF. These data demonstrate that either RalA or PLD1 can cooperate with EGF receptor to transform cells.Overexpression of a tyrosine kinase is a common genetic defect in a variety of human tumors (21). The epidermal growth factor (EGF) receptor, which has an intrinsic tyrosine kinase that is activated in response to EGF, is frequently overexpressed in human breast and ovarian cancer (35). However, overexpression of a tyrosine kinase such as the EGF receptor is not sufficient for a fully transformed or cancerous phenotype. We recently demonstrated that downregulation of protein kinase C ␦ (PKC ␦) transforms 3Y1 rat fibroblasts overexpressing either c-Src (28) or the EGF receptor (19). The EGF receptor-overexpressing cells (EGFR cells) could also be transformed when treated with EGF (19), suggesting that EGF could accomplish what PKC ␦ downregulation accomplished. Interestingly, downregulation of PKC ␦ also caused an increase in phospholipase D (PLD) activity (19,38), which is commonly elevated in response to oncogenic and mitogenic stimuli (11,41). Both EGF-induced increases in PLD activity and EGFinduced transformation were dependent upon the ␣ isoform of PKC (19), suggesting that PLD may be an important component of the mitogenic and oncogenic properties of the EGF receptor.We demonstrated previously (30) that PLD1 associates directly with the small GTPase RalA, a downstream target of Ras (13). RalA is required for PLD activation in response to v-Src and v-Ras (22). RalA has also been implicated in cell transformation (1, 39), indicating a possible role for PLD in mitogenic signaling. In this paper, we report that both RalA and PLD1 can cooperate with an overexpressed EGF receptor to transform cells. MATERIALS AND METHODSCells and cell culture conditions. Rat 3Y1 cells or rat 3Y1 cells expressing the EGF receptor were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (HyClone) as described previously (28, 29). The EGF...