Negative regulation of RTKs Negative regulation of RTKs http://www.cell-research.com MINIREVIEW Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation Chanan RUBIN, Gal GUR, Yosef YARDEN* Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel ABSTRACT Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ulti- mately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cbl- mediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty. Keywords: endocytosis, growth factor, oncogene, signal transduction, ubiquitin, tyrosine kinase. INTRODUCTION a soluble ligand for the Drosophila EGF-receptor (DER). Receptor tyrosine kinases (RTKs) have evolved as pri- Cells in close proximity to midline cells experience high mary mediators of cell-to-cell communication in multi- levels of DER activation and express a specific set of cellular organisms. These membrane-spanning proteins markers, including orthodenticle. More distal cells exhibit mediate a variety of cellular responses ranging from cell lower DER activation and express other markers such as migration to survival, cell proliferation and differentiation. fasciclin III. Ligand binding to the cognate receptor triggers receptor It is becoming apparent that a large set of inhibitory dimerization and activation of the kinase domain, render- proteins act to attenuate the signal emanating from acti- ing the receptor catalytically active. Receptor trans- and vated receptors (Tab. 1). These negative regulators can auto-phosphorylation on tyrosine residues located within either exist prior to receptor activation or they are newly the cytoplasmic tail create docking sites for proteins con- synthesized following signaling initiation (see Fig. 1). taining phosphotyrosine-binding modules. In turn, the Indeed, receptor activation not only instigates multiple posi- recruited proteins initiate various signaling cascades [1]. tively acting pathways, such as the Ras-mitogen-activated Tuning of signaling amplitude and duration is crucial for protein kinase (MAPK); and the phosphatidylinositol 3- induction of the correct physiological outcome [2]. For kinase (PI3K)-Akt cascades, but also sets in motion example, the patterning of the Drosophila embryonic mechanisms that will ultimately terminate signaling. The ventral ectoderm relies on different levels of epidermal induction of negative feedback loops has been well growth factor receptor (EGFR) signaling for the in- characterized in insects. Examples of genes that are tran- duction of various cell fates [reviewed in [3]]. In this scriptionally induced following receptor activation include vein, ventral midline cells provide the source for Spitz, the intracellular membrane-bound protein Sprouty [4], the membrane spanning Kekkon proteins [5] and the antago- nistic ligand of the insect EGFR, namely Argos [6]. Similarly, mammalian RTK attenuators such as the pan-ErbB inhibitor *Correspondence: Yosef YARDEN Tel: +972-8-9343974; Fax: +972-8-9342488; RALT/Mig-6 [7] and the fibroblasts-derived growth factor E-mail: [email protected] receptor (FGFR) inhibitor, Sef [8], are also newly synthe- 66 Cell Research, 15(1):66-71, Jan 2005 | www.cell-research.com Chanan RUBIN et al Fig. 1 Activation-dependent mechanisms of signal attenuation. Growth factor binding to a receptor tyrosine kinase induces receptor auto-phosphorylation, followed by simultaneous activation of multiple positive signaling pathways. Cbl-mediated receptor ubiquitylation marks the onset of attenuation, starting with pre-existing molecules involved in endocytosis and cytoskeleton rearrangement (early attenuators), and culminating in transcription-dependent negative regulatory pathways (late attenuators). The list of up-regulated genes includes those encoding MAPK-specific phosphatases, various adaptors, transcription factors and secondary growth factors. DSPs, dual-specificity phosphatases; APs, clathrin-associated proteins. Tab. 1 Negative regulators of RTKs and their modes of action Protein name Domain structure Inhibitory targets Mechanism of action Ack1 TK, SH3, CRIB, UBA EGFR Unknown Argos EGF DER Ligand sequestration c-Cbl SH2, RF, UBA Several RTKs Mediates receptor ubiquitylation Echinoid Ig-like, FN3, TM EGFR Unknown [49] Kekkon LRR, Ig-like, TM DER Inhibits growth factor binding LRIG1 LRR, Ig-like, TM ErbB family members Enhances receptor ubiquitylation Nedd4 C2, WW, HECT IGF1R, VEGFR Mediates receptor ubiquitylation [50,51] Sef None identified Multiple RTKs Retains Erk in the cytoplasm [8,52] Senseless Zinc fingers DER Represses pointed- mediated transcription [53] Socs 1 and 3 SH2, SOCS box Insulin and cytokine receptors Mediate IRS1/2 ubiquitylation and block access of substrates [54] Spred EVH-1, CRD RTKs Inhibits Ras-mediated Raf activation [55] Sprouty CRD Several RTKs Unknown Ralt/Mig6 None identified EGFR, ErbB-2 Unknown [7] sized in response to growth factors. hypomorphic mutations of EGFR/LET-23 in C. elegans identified SLI-1 as a negative regulator of the EGFR sig- REGULATION OF RECEPTOR ENDOCYTOSIS naling pathway [9]. In line with genetic evidence indicat- Of the negative regulators, which are independent of ing that SLI-1 acts downstream to the receptor and up- new transcription, the E3 ubiquitin ligase c-Cbl/Sli-1 is stream of Ras, c-Cbl, a mammalian ortholog of SLI-1, relatively well understood. Screens for suppressors of was shown to be rapidly phosphorylated and to complex www.cell-research.com | Cell Research, 15(1):66-71, Jan 2005 67 Negative regulation of RTKs Negative regulation of RTKs http://www.cell-research.com Fig. 2 Modulators of ErbB signaling. The domain structures of LRIG-1, Cbl and Sprouty proteins are schematically presented. Arrows indicate direct or indirect interactions. Thus, the extracellular domains of ErbB and LRIG proteins physically interact in a ligand-independent manner, and ErbB increases tyrosine phosphorylation of Sprouty. Cbl proteins bind in a phosphorylation- dependent manner to ErbB and Sprouty proteins, but the interaction with LRIG-1 is phosphorylation-independent. All three targets of Cbl undergo ubiquitylation, which dictates degradation by lysosomes or proteasomes. CRD, cysteine-rich domain; Ig, immunoglo- bulin-like domain; LRD, leucine-rich region; PRR, proline-rich domain; RF, RING finger. with the EGFR following EGF stimulation [10]. The considered as a signal termination mechanism. Emerging domain structure of c-Cbl consists of a tyrosine kinase data indicate that the specificity, kinetics and magnitude binding (TKB) domain through which it binds tyrosine- of receptor’s responses may be regulated by the locationof phosphorylated targets, a RING finger domain, which binds the activated receptor in the endocytotic pathway [15]. E2-conjugating enzymes, and a C-terminal ubiquitin-asso- Ligand binding initiates receptor auto-phosphorylation, ciated (UBA) domain. Additional studies demonstrated that which is followed by the recruitment of the Cbl TKB domain c-Cbl facilitates ligand-induced ubiquitylation of the EGFR to a specific phosphotyrosine, such as tyrosine 1045 of [11,12], as well as several other RTKs (reviewed in [13]) the EGFR [11]. This proximity facilitates Cbl-mediated re- by means of RING finger-dependant recruitment of E2 ceptor mono-ubiquitylation [16,17], which commences at ubiquitin-conjugating enzymes to the receptor’s vicinity. the plasma membrane [18,19]. It is believed that mono- Receptor ubiquitylation results in accelerated removal from ubiquitylation of the receptor on multiple lysine residues the cell surface and to subsequent receptor degradation in robustly generates docking sites for endocytotic adaptor the lysosomal compartment, thereby terminating RTK proteins possessing ubiquitin-binding domains. Adaptors, signaling. such as Eps15, may recruit receptors to clathrin-coated Recent studies have linked RTK ubiquitylation to the pits as they comprise both ubiquitin interacting motifs process of receptor endocytosis [14]. This phenomenon (UIMs) [20] and DPF motifs that couple to clathrin adap- entails the internalization of the receptor from the plasma tors [AP2; [21]]. Recruited receptors are thus linked to membrane and its routing through several intracellular AP2 complexes that drive the assembly of clathrin-coated compartments. Multiple sorting steps, along the endocy- vesicles (CCVs). The CCVs shed clathrin and fuse with totic course, ultimately determine whether the receptor internal vesicles to form early endosomes that proceed will be destined for