The MID1 E3 Ligase Catalyzes the Polyubiquitination of Alpha4 (α4), a Regulatory Subunit of Protein Phosphatase 2A (PP2A) NOVEL INSIGHTS INTO MID1-MEDIATED REGULATION of PP2A

Alpha4 (alpha 4) is a key regulator of protein phosphatase 2A (PP2A) and mTOR in steps essential for cell-cycle progression. alpha 4 forms a complex with PP2A and MID1, a microtubule-associated ubiquitin E3 ligase that facilitates MID1-dependent regulation of PP2A and the dephosphorylation of MID1 by PP2A. Ectopic overexpression of alpha 4 is associated with hepatocellular carcinomas, breast cancer, and invasive adenocarcinomas. Here, we provide data suggesting that alpha 4 is regulated by ubiquitin-dependent degradation mediated by MID1. In cells stably expressing a dominant-negative form of MID1, significantly elevated levels of alpha 4 were observed. Treatment of cells with the specific proteasome inhibitor, lactacystin, resulted in a 3-fold increase in alpha 4 in control cells and a similar level in mutant cells. Using in vitro assays, individual MID1 E3 domains facilitated monoubiquitination of alpha 4, whereas full-length MID1 as well as RING-Bbox1 and RING-Bbox1-Bbox2 constructs catalyzed its polyubiquitination. In a novel non-biased functional screen, we identified a leucine to glutamine substitution at position 146 within Bbox1 that abolished MID1-alpha 4interaction and the subsequent polyubiquitination of alpha 4, indicating that direct binding to Bbox1 was necessary for the polyubiquitination of alpha 4. The mutant had little impact on the RING E3 ligase functionality of MID1. Mass spectrometry data confirmed Western blot analysis that ubiquitination of alpha 4 occurs only within the last 105 amino acids. These novel findings identify a new role for MID1 and a mechanism of regulation of alpha 4 that is likely to impact the stability and activity level of PP2Ac.