E3 families specifically involved in the internalization step of endocytosis

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Ubiquitin ligases are proteins or protein complexes that binds to both E2 and the substrate. Interaction with the substrate may be direct or may involve other proteins. E3s are heterogeneous, but may be classified into two major groups — HECT domain and RING finger-containing E3s — and several minors groups, such as U box-containing proteins, also termed E4, involved in elongating polyubiquitin chains [9], or a subset of the PHD-containing proteins with divergent RING domains [14]. The E3s involved in endocytosis include RING finger E3s, HECT domain proteins, and PHD-containing proteins.

Most of E3 ubiquitin ligases are RING finger-containing enzymes. These E3s serve as a scaffold responsible for optimal positioning of E2 and the substrate for the efficient transfer of activated ubiquitin from E2 to the substrate. The RING finger domain has been defined as a pattern of conserved Cys and His residues forming a cross-brace structure that probably binds two Zn cations. The RING finger family of E3 enzymes is composed of two distinct groups: single and multisubunit proteins. Monomers (or homodimers) contain both the RING finger domain and the substrate-binding/recognition site in the same molecule [9]. This is the case for c-Cbl, a RING finger ligase involved in targeting activated receptor tyrosine kinases [15]. A multisubunit RING E3, SCF^HOS, was also described to be involved in endocytosis [16].

The HECT domain superfamily is the only  family of E3 enzymes known to catalyze substrate ubiquitylation directly [17, 18]. HECT domain proteins contain a 350 amino acid sequence homologous to the COOH-terminal domain of the prototype member of the E6-AP family (E6-associated protein). This domain contains a conserved Cys residue, to which the activated ubiquitin moiety is transfered from E2 [17, 18]. The NH₂-terminal domain, which varies between HECT domain proteins, is probably devoted to specific substrate recognition, as is the case for members of the Nedd4 family. The N-terminus of members of the Nedd4 family harbors a C2 domain, followed by two to four WW domains (Fig. 1) [19]. The C2 domain is a 120-amino acid sequence that has been shown to bind phospholipids and membrane proteins in a Ca⁺⁺-dependent manner in several proteins, including Nedd4 [20, 21], whereas Rsp5p C2 domain was shown to  bind phosphoinositides in a Ca⁺⁺-independent manner in vitro [6]. WW domains are 40-amino acid protein:protein interaction modules that bind Pro-rich ligands. Members of the Nedd4 family have two to four WW domains, suggesting that they may interact with several proteins simultaneously. Based on their binding specificity, WW domains can be classified into two major and three minor groups [22]. Group I WW domains bind PPXY motifs, whereas Group II WW domains bind PPLP motifs. Two of the three minor groups, Group III and V, bind proline-rich sequences, whereas Group IV WW domains interact with short sequences containing phosphorylated serine and threonine residues followed by proline. The binding of Group IV WW domains to their ligands has been shown to be phosphorylation-dependent [23].

One major function of Nedd4/Rsp5p family members is regulation of the stability of several yeast and animal cell-surface transmembrane proteins by ubiquitylation, which controls subsequent internalization. For example, Nedd4-2 targets the kidney epithelial Na+ channel [24], whereas the degradation of several receptors and transporters in yeast is mediated by the Rsp5p ligase, the only member of this family in S. cerevisiae [21].

The third family of E3s involved in the internalization step of endocytosis is that of PHD-containing E3 ligases. The plant homeodomain (PHD) motif encodes a specialized form of Zn finger [25]. This motif consists of seven Cys and one His residue in the order 4 Cys, His, 3 Cys, and is similar in both sequence and structure to RING finger domains. A PHD domain is present in a membrane-bound protein known to be involved in ubiquitin-dependent ER degradation in yeast. This protein was demonstrated to have ubiquitin ligase activity in vitro [26]. PHD motifs have more recently been identified in several viral and human proteins playing key roles as E3s in the downregulation of cell surface proteins (reviewed in [14]).

A key issue in the ubiquitin field is the way in which the system achieves its high specificity and selectivity, an E3-dependent property. In the case of cell-surface proteins, ubiquitylation is often specifically triggered in response to extracellular stimuli, such as ligand binding or nutrient modification. For E3s involved in the endocytic pathway, and for other E3s, ubiquitylation may require the involvement of other modifying enzymes and additional proteins. Many substrates are not recognized constitutively and are not recognized directly by E3s. In some instances, E3s must be activated or deactivated by posttranslational modification. In other cases, the substrate undergoes modifications rendering its recognition possible. Thus, in addition to E3s themselves, modifying proteins such as kinases and other proteins also play an important role in the recognition process.