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 (α4) is a key regulator of protein phosphatase 2A (PP2A) and mTOR in steps essential for cell-cycle progression. α4 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 α4 is associated with hepatocellular carcinomas, breast cancer, and invasive adenocarcinomas. Here, we provide data suggesting that α4 is regulated by ubiquitin-dependent degradation mediated by MID1. In cells stably expressing a dominant-negative form of MID1, significantly elevated levels of α4 were observed. Treatment of cells with the specific proteasome inhibitor, lactacystin, resulted in a 3-fold increase in α4 in control cells and a similar level in mutant cells. Using in vitro assays, individual MID1 E3 domains facilitated monoubiquitination of α4, 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-α4 interaction and the subsequent polyubiquitination of α4, indicating that direct binding to Bbox1 was necessary for the polyubiquitination of α4. The mutant had little impact on the RING E3 ligase functionality of MID1. Mass spectrometry data confirmed Western blot analysis that ubiquitination of α4 occurs only within the last 105 amino acids. These novel findings identify a new role for MID1 and a mechanism of regulation of α4 that is likely to impact the stability and activity level of PP2Ac.     

MID1 Catalyzes the Ubiquitination of Protein Phosphatase 2A and Mutations within Its Bbox1 Domain Disrupt Polyubiquitination of Alpha4 but Not of PP2Ac

MID1 is a microtubule-associated protein that belongs to the TRIM family. MID1 functions as an ubiquitin E3 ligase, and recently was shown to catalyze the polyubiquitination of, alpha4, a protein regulator of protein phosphatase 2A (PP2A). It has been hypothesized that MID1 regulates PP2A, requiring the intermediary interaction with alpha4. Here we report that MID1 catalyzes the in vitro ubiquitination of the catalytic subunit of PP2A (PP2Ac) in the absence of alpha4. In the presence of alpha4, the level of PP2Ac ubiquitination is reduced. Using the MID1 RING-Bbox1-Bbox2 (RB1B2) construct containing the E3 ligase domains, we investigate the functional effects of mutations within the Bbox domains that are identified in patients with X-linked Opitz G syndrome (XLOS). The RB1B2 proteins harboring the C142S, C145T, A130V/T mutations within the Bbox1 domain and C195F mutation within the Bbox2 domain maintain auto-polyubiquitination activity. Qualitatively, the RB1B2 proteins containing these mutations are able to catalyze the ubiquitination of PP2Ac. In contrast, the RB1B2 proteins with mutations within the Bbox1 domain are unable to catalyze the polyubiquitination of alpha4. These results suggest that unregulated alpha4 may be the direct consequence of these natural mutations in the Bbox1 domain of MID1, and hence alpha4 could play a greater role to account for the increased amount of PP2A observed in XLOS-derived fibroblasts.     

XLOS-Observed Mutations of MID1 Bbox1 Domain Cause Domain Unfolding

MID1 catalyzes the ubiquitination of the protein alpha4 and the catalytic subunit of protein phosphatase 2A. Mutations within the MID1 Bbox1 domain are associated with X-linked Opitz G syndrome (XLOS). Our functional assays have shown that mutations of Ala130 to Val or Thr, Cys142 to Ser and Cys145 to Thr completely disrupt the polyubiquitination of alpha4. Using NMR spectroscopy, we characterize the effect of these mutations on the tertiary structure of the Bbox1 domain by itself and in tandem with the Bbox2 domain. The mutation of either Cys142 or Cys145, each of which is involved in coordinating one of the two zinc ions, results in the collapse of signal dispersion in the HSQC spectrum of the Bbox1 domain indicating that the mutant protein structure is unfolded. Each mutation caused the coordination of both zinc ions, which are ∼13 Å apart, to be lost. Although Ala130 is not involved in the coordination of a zinc ion, the Ala130Thr mutant Bbox1 domain yields a poorly dispersed HSQC spectrum similar to those of the Cys142Ser and Cys145Thr mutants. Interestingly, neither cysteine mutation affects the structure of the adjacent Bbox2 domain when the two Bbox domains are engineered in their native tandem Bbox1-Bbox2 protein construct. Dynamic light scattering measurements suggest that the mutant Bbox1 domain has an increased propensity to form aggregates compared to the wild type Bbox1 domain. These studies provide insight into the mechanism by which mutations observed in XLOS affect the structure and function of the MID1 Bbox1 domain.     

Structure of the MID1 tandem B-boxes reveals an interaction reminiscent of intermolecular ring heterodimers

The tripartite motif (TRIM) protein family, defined by N-terminal RING, B-box, and coiled-coil (RBCC) domains, consists of either a single type 2 B-box domain or tandem B-box domains of type 1 and type 2 (B1B2). Here, we report the first structure of the B-box domains in their native tandem orientation. The B-boxes are from Midline-1, a putative ubiquitin E3 ligase that is required for the proteosomal degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). This function of MID1 is facilitated by the direct binding of Alpha4, a regulatory subunit of PP2Ac, to B-box1, while B-box2 appears to influence this interaction. Both B-box1 and B-box2 bind two zinc atoms in a cross-brace motif and adopt a similar betabetaalpha structure reminiscent of the RING, PHD, ZZ, and U-box domains, although they differ from each other and with RING domains in the spacing of their zinc-binding residues. The two B-box domains pack against each other with the interface formed by residues located on the structured loop consisting of the two antiparallel beta-strands. The surface area of the interface is 188 A2 (17% of the total surface). Consistent with the globular structure, the Tm of the tandem B-box domain (59 degrees C) is higher than the individual domains, supporting a stable interaction between the B-box 1 and 2 domains. Notably, the interaction is reminiscent of the interaction of recently determined RING dimers, suggesting the possibility of an evolutionarily conserved role for B-box2 domains in regulating functional RING-type folds.     

In vitro and in vivo interaction of AtRma2 E3 ubiquitin ligase and auxin binding protein 1

E3 ubiquitin (Ub) ligases play diverse roles in cellular regulation in eukaryotes. Three homologous AtRmas (AtRma1, AtRma2, and AtRma3) were recently identified as ER-localized Arabidopsis homologs of human RING membrane-anchor E3 Ub ligase. Here, auxin binding protein 1 (ABP1), one of the auxin receptors in Arabidopsis, was identified as a potential substrate of AtRma2 through a yeast two-hybrid assay. An in vitro pull-down assay confirmed the interaction of full-length AtRma2 with ABP1. AtRma2 was transiently expressed in tobacco (Nicotiana benthamiana) plants through an Agrobacterium-mediated infiltration method and bound ABP1 in vivo. In vitro ubiquitination assays revealed that bacterially-expressed AtRma2 ubiquitinated ABP1. ABP1 was poly-ubiquitinated in tobacco cells and its stability was significantly increased in the presence of MG132, a 26S proteasome inhibitor. This suggests that ABP1 is controlled by the Ub/26S proteasome system. Therefore, AtRma2 is likely involved in the cellular regulation of ABP1 expression levels.     

TRAF-interacting protein (TRIP) is a RING-dependent ubiquitin ligase

TRAF-interacting protein (TRIP) was initially identified as a TRAF1- and TRAF2-binding partner that inhibited NF-kappaB activation without a known mechanism. Inspection of the TRIP sequence revealed an N-terminal RING domain, which is found in many E3 ubiquitin (Ub) ligases. We show that TRIP is a RING-dependent Ub ligase that undergoes auto-ubiquitination and requires an intact RING domain. Both TRIP and its RING mutant interact with TRAF1, 2, 3, 5, and 6, but failed to interact with CYLD and NIK. Stable expression of TRIP or a RING mutant did not affect IKK activation induced by TNF or IL-1 and had no affect on TNF-induced apoptosis. Similarly, RANKL-induced signaling and osteoclastogenesis were not affected by TRIP or its RING mutant. Interestingly, TRIP expression was down regulated during the late stages of osteoclastogenesis. Taken together, our results demonstrate that TRIP is a novel RING-dependent Ub ligase and a binding partner for TRAFs.     

Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.     

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn²⁺ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.