The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP Cterminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of b-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pulldown assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.

The poly(A)-binding protein (PABP) is a unique translation initiation factor in that it binds to the mRNA 3 0 poly(A) tail and stimulates recruitment of the ribosome to the mRNA at the 5 0 end. PABP activity is tightly controlled by the PABP-interacting protein 2 (Paip2), which inhibits translation by displacing PABP from the mRNA. Here, we describe a close interplay between PABP and Paip2 protein levels in the cell. We demonstrate a mechanism for this co-regulation that involves an E3 ubiquitin ligase, EDD, which targets Paip2 for degradation. PABP depletion by RNA interference (RNAi) causes co-depletion of Paip2 protein without affecting Paip2 mRNA levels. Upon PABP knockdown, Paip2 interacts with EDD, which leads to Paip2 ubiquitination. Supporting a critical role for EDD in Paip2 degradation, knockdown of EDD expression by siRNA leads to an increase in Paip2 protein stability. Thus, we demonstrate that the turnover of Paip2 in the cell is mediated by EDD and is regulated by PABP. This mechanism serves as a homeostatic feedback to control the activity of PABP in cells.

The PABC domain is a peptide-binding domain that is specifically found in poly(A)-binding protein (PABP) and a HECT ubiquitin-protein isopeptide ligase (E3) known as HYD (hyperplastic discs), EDD (E3 isolated by differential display), or Rat100. The PABC domain of PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12-amino acid peptide motif, PAM2 (PABP-interacting motif 2). In contrast, little is known about the specificity or function of the domain from HYD. Here, we used isothermal calorimetry and surface plasmon resonance titrations to show that the PABC domain of HYD binds PAM2 peptides with micromolar affinity. NMR chemical shift perturbations were used to map the peptide-binding site in the PABC domain of HYD. The structural features of binding are very similar to those of the interactions with the domain of PABP, which explains the overlapping peptide specificity and binding affinity. We identified the anti-proliferative Tob proteins as potential binding partners of HYD. This was confirmed by glutathione S-transferase pulldown and immunoprecipitation experiments demonstrating the interaction with full-length Tob2. Altogether, our results point to a role of the PABC domain as a protein-protein interaction domain that brings together the processes of translation, ubiquitinmediated protein degradation, and cell cycle control.The tumor suppressor protein HYD (hyperplastic discs), also known as EDD (E3 3 isolated by differential display) or Rat100, is a member of the family of HECT (homologous to E6-associated protein carboxyl terminus) E3 ligases, which target specific proteins for ubiquitin-mediated proteolysis. The highly conserved ubiquitin/proteasome pathway controls the degradation of many critical regulatory proteins. Targeted proteins are post-translationally conjugated to a 76-residue ubiquitin moiety through a combined set of reactions involving activating (ubiquitin-activating enzyme), conjugating (ubiquitin carrier protein), and ligating (E3) enzymes. E3 enzymes physically interact with their substrates and are thus critical determinants of the specificity of ubiquitination. Two main groups of E3 ligases are the ring finger and HECT domain ligases (1, 2).In Drosophila, HYD is required for the regulation of cell proliferation during development (3). It has been inferred that HYD participates in signal transduction downstream of the signaling receptors to initiate and/or maintain proliferation as well as to terminate proliferation (3). Indeed, it has been shown that cells with mutations in HYD fail to properly terminate proliferation, leading to tumors. Furthermore, these mutations also result in developmental abnormalities such as adult sterility due to germ cell defects (4). HYD is frequently overexpressed in breast and ovarian cancers, supporting a role in cancer development (5, 6). HYD is also involved in DNA damage signaling, in which TopB1, a target for ubiquitinylation by HYD (7), co-localizes with BRAC1 at stalled replication for...