Protein phosphorylation, which is well known to function in recruitment of the ubiquitination machinery to the substrate [12
], may also act directly to regulate the activity of distinct ubiquitin ligases. The best-characterized circuitry involves the E3 ligase Cbl, which is responsible for the ubiquitination of several receptor tyrosine kinases (RTKs) [3
]. The mammalian Cbl protein family consists of the three homologs c-Cbl, Cbl-b, and Cbl-3, all of which associate with a wide variety of signaling proteins [13
]. Two highly conserved amino-terminal domains contribute strongly to E3 regulatory function. First, the amino-terminal tyrosine kinase binding (TKB) domain of Cbl recognizes phosphotyrosine residues and allows Cbl to interact directly with activated RTKs at the plasma membrane (Figure ). Second, the RING finger domain recruits ubiquitin-loaded E2s, whose interaction with Cbl results in the ubiquitination and subsequent degradation of the associated RTK. In the case of the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor receptor MET, the molecular mechanism of receptor ubiquitination has been investigated in detail. In both cases, Cbl binds directly to phosphotyrosine (pY)-sites on the activated receptor through its TKB [14
], as well as indirectly through its constitutive partner GRB2, which is recruited to receptors via other pY sites [7
]. Both direct and indirect interactions of Cbl with the EGFR or MET are required for full ubiquitination of these receptors (Figure ). Once bound, the ligase is phosphorylated and consequently activated [19
]. Two structural studies have now shed light on the mechanism of phosphorylation-induced activation of c-Cbl and Cbl-b [20
]. In the absence of substrate binding, the TKB and RING domains form a compact structure that masks the E2 binding site. Binding of the TKB to the substrate induces a first rotation of the linker region, allowing phosphorylation of tyrosine 371 (363 in Cbl-b). This phosphorylation event induces a complete rotation of the linker region that unmasks the RING E2 binding surface and activates the ligase [20
Figure 2 EGFR ubiquitination by Cbl. Upon EGF-dependent receptor activation, the GRB2-Cbl complex binds to the receptor through interactions of: i) the SH2 domain of GRB2 with pY1045 of EGFR, and ii) the TKB domain of Cbl (either c-Cbl or Cbl-b) with pY1068 or (more ...)
Another class of E3 ligases, the HECT NEDD4 family [22
], whose regulation has been extensively studied, also regulates endocytosis and sorting of numerous signaling receptors [3
]. These enzymes present a conserved modular organization with an amino-terminal C2 domain that is crucial for membrane localization, between two and four WW domains capable of recognizing substrates and adaptor proteins through PY motifs, and a carboxy-terminal catalytic HECT domain. In contrast to RING-based ligases in which the RING is an allosteric activator of the E2, HECT-containing E3s have intrinsic catalytic activity and directly ubiquitinate their targets. In humans, there are nine members of this family: NEDD4 (also known as NEDD4-1), NEDD4L (also known as NEDD4-2), ITCH (also known as AIP4), WWP1, WWP2, SMURF1, SMURF2, NEDL1 (also known as HECW1) and NEDL2 (also known as HECW2). Rsp5 is the unique, essential member of the NEDD4 family in Saccharomyces cerevisiae
. In normal conditions most of them appear to be in an inactive state because of an intramolecular inhibitory interaction between the carboxy-terminal HECT and the amino-terminal C2 domain (in the case of SMURF2, NEDD4 and WWP2 [23
]) or the WW domains (in the case of ITCH [24
]). Activation of this class of enzyme can occur in various ways that are briefly described below.
ITCH is the E3 ligase for the chemokine receptor CXCR4 [25
]. The ubiquitin moiety on CXCR4 serves as a signal on endosomes for entry into the degradative pathway and long-term attenuation or downregulation of signaling [25
]. ITCH can interact directly with CXCR4 through a non-canonical WW domain-mediated interaction involving serine residues within the carboxy-terminal tail of CXCR4. These serine residues are phosphorylated upon agonist activation, and are critical for mediating agonist-promoted binding of ITCH and the subsequent ubiquitination and degradation of CXCR4 [26
] (Figure ). Also in this case, the ligase appears to be regulated by phosphorylation. ITCH phosphorylation is activated by JNK1 [24
], and is thought to lead to conformational changes that disrupt the inhibitory intramolecular interactions between its WW and the HECT domains.
Figure 3 Activation of E3 ubiquitin ligases through recruitment to activated receptors. (a) Ubiquitination of CXCR4 by ITCH. ITCH activity is inhibited as a result of the intramolecular interaction between the WW domain and the carboxy-terminal catalytic HECT (more ...)
In the case of SMURF2, autoinhibition of the HECT domain by the C2 domain helps in maintaining the steady-state levels of this E3 ligase and can be relieved by adaptor-mediated substrate targeting [23
]. SMURF1 and SMURF2 bind to TGF-β family receptors through the inhibitory Smad proteins, SMAD6 and SMAD7, to induce their ubiquitin-dependent degradation. Wiesner et al.
] demonstrated that intramolecular interactions between the C2 and HECT inhibit SMURF2 catalytic activity interfering with ubiquitin thioester formation. This in cis
autoinhibition can be relieved by binding of the amino-terminal domain (NTD) of the adaptor protein SMAD7 to the E3 HECT domain (Figure ). The SMAD7 NTD further enhances the catalytic activity of the SMURF2 ligase by recruiting the E2 UbcH7 to the HECT domain [27
]. By releasing C2-mediated autoinhibition, stimulating E2 binding, and recruiting SMURF targets, SMAD7 functions at multiple levels to control E3 activity and ensure specificity in SMURF-catalyzed ubiquitination.
Recently, a role for a UBD present on the N-lobe of the HECT domain of NEDD4 and Rsp5 has been identified [28
]. The ability of the HECT domain to bind non-covalently to the distal ubiquitin at the growing end of the polyubiquitin chain on the substrate allows enzyme processivity [28
]. It is tempting to attribute an inhibitory role of the C2 binding for this critical feature of these enzymes. Accessibility of the UBD may be restored in response to upstream signaling events capable of inducing phosphorylation and/or ubiquitination of critical sites in the C2 or in the HECT domain, leading to full ligase activation. While this hypothesis needs to be experimentally verified, we notice that ubiquitination of NEDD4 is a critical event for the coupled monoubiquitination of EPS15 ([30
] and below).
In some cases, such as for the epithelial Na+
channel (ENaC), receptor:ligase interaction - and consequent receptor ubiquitination - is the default pathway, with phosphorylation negatively regulating ligase activity. NEDD4-2 binds constitutively to ENaC PPxY-containing motifs and catalyzes its ubiquitination, internalization and lysosomal targeting. This prevents Na+
overload in epithelial cells and is necessary for the maintenance of salt and fluid balance in the body. To increase ENaC abundance at the surface and enhance epithelial Na+
absorption, NEDD4-2 is phosphorylated by various kinases, including protein kinase A (PKA), serum- and glucocorticoid-inducible kinase (SGK), and IκB kinase (IKK)β (Figure ). Phosphorylation induces binding of 14-3-3 proteins, which prevents NEDD4-2 from binding to ENaC [31
Figure 4 Regulation of channels and transporters by ubiquitination. (a) ENaC ubiquitination by NEDD4-2. NEDD4-2 binds to PY motifs on the epithelial Na+ channel ENaC and catalyzes its ubiquitination. This induces ENaC endocytosis and lysosomal targeting, resulting (more ...)