Reciprocal regulation of the ubiquitin ligase Itch and the epidermal growth factor receptor signaling Bilal A. Azakir 1 , Annie Angers ⁎ Département de sciences biologiques, Université de Montréal, Montréal, QC, Canada H3C 3J7 abstract article info Article history: Received 26 November 2008 Received in revised form 18 March 2009 Accepted 24 March 2009 Available online 31 March 2009 Keywords: Cbl Endophilin c-Jun N-terminal kinase Akt Ubiquitin FAM Usp9x EGF-mediated stimulation of the EGF receptor activates a plethora of signaling cascades followed by receptor down regulation. Preventing down regulation leads to increased mitogenic signaling and potentially, cancer. Cbl and Endophilin are two key proteins required for EGF receptor down regulation and both become ubiquitylated and subject to proteasome-mediated degradation following EGF activation, providing a negative feedback loop for EGF receptor down regulation. The mechanism of this pathway is unknown. Here, we demonstrate that treatment of cells with EGF leads to JNK-dependent phosphorylation of the ubiquitin ligase Itch, stimulating Itch ligase activity. EGF-stimulated JNK activation causes an increased interaction between Itch and the de-ubiquitylating enzyme FAM, limiting the influence of Itch auto-ubiquitylation on its own degradation. Finally, JNK activation stimulates the association of Itch with its substrates. These effects combine to cause increased ubiquitylation of Itch substrates including Endophilin and Cbl, resulting in the proteasome-dependent down regulation of these key trafficking proteins. Thus, Itch is a key regulatory locus for EGF receptor degradation. © 2009 Elsevier Inc. All rights reserved. 1. Introduction Activation of EGF receptor triggers an intricate network of intracellular signaling cascade leading to diverse cell responses, including growth and proliferation [1–3]. Following EGF receptor activation, many signaling proteins are rapidly ubiquitylated, including the receptor itself, Eps15, Epsin and CIN85 [4,5]. The RING-domain ubiquitin ligase Cbl is the enzyme required for ligand- induced ubiquitylation of the EGF receptor and CIN85 [6,7], and another RING-domain ligase, Parkin, has been implicated in Eps15 ubiquitylation [8]. Cbl is directly recruited to activate EGF receptor through its SH2 domain, which accounts for the coupling of its ligase activity with receptor activation. Other ubiquitylation reactions triggered by receptor activation have been tentatively attributed to HECT-domain ubiquitin ligases [9]. However, it is not clear how these ubiquitylation reactions are coupled to receptor activation. Ubiquitin ligases play important regulatory roles in the cell as they catalyze the final step of the ubiquitylation reaction, and are thought to ensure the specificity of the process by selecting the correct substrate proteins. Itch is an ubiquitin ligase of the HECT family with several well characterized substrate interactions [10–16]. Cbl and Endophilin are two substrates of Itch that are involved in EGF receptor downregulation. Importantly, both substrates are ubiquitylated following treatment of cells with EGF [10,15]. Itch is also one of the few ubiquitin ligases that has been shown to be regulated by protein kinases. Phosphorylation of Itch has a major impact on its capacity to interact with and ubiquitylate JunB. The c-Jun N-terminal kinase (JNK) has been shown to phosphorylate Itch on at least three residues in its proline-rich domain, and leads to increased JunB ubiquitylation [17,18]. In contrast, the Src kinase Fyn phosphorylates a Tyr residue near the third WW domain that appears to reduce Itch interaction with JunB [19]. In several cell types, JNK is activated by treatment with EGF [3,20]. We thus examined if treatment of cells with EGF leads to Itch activation, and if this activation translates in an effect on Itch substrates. Particularly, two recognized Itch substrates, Endophilin and Cbl, are directly involved in EGF receptor internalization [21], raising the intriguing possibility that by increasing Itch activity, treatment with EGF could lead to Endophilin and Cbl degradation, thereby providing a negative feedback loop toward EGF receptor internalization. To determine if JNK-dependent activation of Itch is a potential mechanism through which ubiquitylation occurs after treatment with EGF, we have examined Itch phosphorylation and activity toward three different substrates: Endophilin, Cbl, and Itch itself, in response to treatment with EGF. We demonstrate that Itch activity increases after EGF treatment, and that this activation depends on JNK activity. Further, Endophilin is rapidly degraded after treatment with EGF, and Itch activity is directly Cellular Signalling 21 (2009) 1326–1336 Abbreviations: EGF, Epidermal growth factor; JNK, c-Jun N-terminal kinase; HECT, homologous to E6Ap C-terminus. ⁎ Corresponding author. Département de sciences biologiques, Université de Montréal, P.O. Box 6128, Station "Centre-ville", Montréal, QC, Canada H3C 3J7. Tel.: +1 514 343 7012; fax: +1 514 343 2293. E-mail address: annie.angers@umontreal.ca (A. Angers). 1 Present address: Neuromuscular Unit, Montreal Neurological Hospital, 3801 University Street, Room 736, Montreal, Qc, Canada H3A 2B4. 0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2009.03.020 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig