?4 phosphoprotein (also known as IGBP1) is a component of the mammalian target-of-rapamycin (mTOR) pathway that controls the initiation of translation and cell-cycle progression in response to nutrients and growth factors. Aberrant signaling of the mTOR pathway has been reported in many cancers. ?4 interacts with the catalytic subunit of protein phosphtase 2A (PP2Ac) to mediate the dephosphorylation of eukaryotic initiation factor 4E-binding protein1 (4E-BP1) and p70S6 kinase (p70S6K). Our laboratory has reported that EDD E3 ubiquitin ligase (EDD/UBR5) and poly(A)-binding protein (PABP) are novel binding partners of ?4 phosphoprotein. In the present study, the interaction of EDD and PABP with ?4 was confirmed in human MCF-7 breast cancer and African green monkey COS-1 kidney cell lines, using immunoprecipitation and immunoblotting (IP/IB) analysis. However, co-IP of total MCF-7 cell lysates with anti-EDD antibodies revealed that EDD does not physically interact with PP2Ac. Several ?4 deletion constructs, that contained either the N-terminal or C-terminal regions of ?4, were transfected into MCF-7 and COS-1 cells. Co-IP studies with anti-EDD and PABP antibodies revealed that EDD interacts with the C-terminal region of ?4 whereas PABP, like PP2Ac, binds to the N-terminal region. EDD and PABP were found to interact with ?4 in both quiescent and actively growing cells. EDD is known to ubiquitinate poly(A)-binding protein-interacting protein 2 (Paip2), targeting it for proteosomal degradation. Paip2 is an antagonist of PABP activity. When ?4 levels in MCF-7 cells were knocked down using small interfering RNA (siRNA), there was no effect on EDD protein levels. There was also no effect on Paip2 levels, indicating that ?4 is not involved in the EDD- mediated ubiquitination of Paip2. Knockdown of EDD gene expression by siRNA did not alter mono-ubiquitination of ?4, indicating that ?4 is not a substrate of EDD. However, knockdown of EDD gene expression decreased poly-ubiquitination of PP2Ac and increased the overall cellular levels of PP2Ac, suggesting PP2Ac as a novel substrate of EDD. The present study suggests a potential role for ?4 in PABP-mediated initiation of mRNA translation. Furthermore, this study suggests a role for EDD in regulating PP2Ac levels through its interaction with ?4. In summary, the ?4 partners EDD, PABP and PP2Ac interact at specific regions of ?4. PP2Ac, but not ?4, is a substrate of EDD. The interaction of PABP with ?4 suggests a potential role for ?4 in PABP-mediated initiation of mRNA translation.