MID1 catalyzes the ubiquitination of protein phosphatase 2A and mutations within its Bbox1 domain disrupt polyubiquitination of alpha4 but not of PP2Ac

Abstract

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.

Figure 1. Natural mutations of MID1 and birth anomalies.

A. Schematic representation of MID1 showing the locations of the various types of mutations. MID1 is a member of the TRIM family of proteins, consisting of seven distinct domains. The RING and Bbox domains coordinate two zinc ions each and adopt ββα-RING folds. The coiled-coil (CC) domain is important for MID1 dimerization. The CC contains a region that is required for microtubule association, denoted as the COS box . The C-terminal half contains the fibronectin type III (FNIII) and SPRY/B30.2 domains. Superimposed on the schematic structure are the four types natural mutations observed with patients with X-linked Opitz G Syndrome. B. Summary of all reported mutations, to date, of MID1 and the associated anomalies , , , , , , , . The four types of mutations observed are color-coded (red = point mutation, blue = frame shifts, magenta = termination/truncation, and black = deletion). Each column lists the different types of mutations (color coded) associated with each domain and grouped with the reported birth defects. X indicates a truncation mutation. The most common defects are hypertelorism, hyperspadias and cleft lip/palate based on the number of mutations associated with these defects. Other craniofacial anomalies that are not listed included low set ears, widows peak, heart defects and small jaw or chin to name a few.

Figure 2. MID1 catalyzed the ubiquitination of PP2Ac.

A. Western-blot (WB) showing in vitro ubiquitination of PP2Ac by MBP-tagged full-length MID1. (1) Lanes 1 and 2 show the results of ubiquitination assay for control reactions. In lane 1 ATP was omitted (no reaction) and in lane 2, only MBP was used. Lane 3 contains the results for the MPB-tagged MID1-catalyzed reaction. Two strong shifted bands corresponding to mono- and di-ubiquitinated PP2Ac and a faint smearing pattern indicative of poly-ubiquitinated PP2Ac were observed. (ii) Lane 1 shows the Biorad visible marker and lane 2 shows a control reaction in which MBP-MID1 was omitted. Lane 3 shows the ubiquitinated PP2Ac to confirm the molecular weights of the mono- and di-ubiquitinated products. B. (i) Ubiquitination of PP2Ac by MBP-tagged MID1 in the absence and presence of full-length alpha4. (ii) Ubiquitination of PP2Ac with increasing amounts of alpha4. The amounts of alpha4 were present at 1:1, 1:2 and 1:4 molar ratios with PP2Ac. C. Ubiquitination of PP2Ac by the MID1 RB1B2 protein (RING-Bbox1-Bbox2, MID1 residues 1–214). Lanes 1–6 show the results of control experiments in which a specific component of the assay was omitted, including the E1 activating enzyme (E1) (lane 1). Mono- and di-ubiquitinated PP2Ac were observed in lane 7, with all components of the reaction present. Background noise precludes detection of polyubiquitinated products. Antibody was specific for PP2Ac. D. Ubiquitination of PP2Ac by various constructs of the three N-terminal domains. These domains were expressed and purified similarly, and used at roughly the same concentration for the assays. Lane 1 uses the Bbox1-Bbox2 construct that includes the amino acids from the linker region between the RING and Bbox1 domains (LB1B2, residues 71–115). Lane 2 uses a RING domain (residues 1–92) that also includes a large position of the linker region. The RB1 and RB1B2 constructs consist of residues 1–164 and 1–214 respectively. E. Ubiquitination of PP2Ac in the presence of 10 different E2 conjugating enzymes.

Figure 3. RB1B2 with mutant Bbox domains possessed auto-ubiquitination activity.

(i) WB image showing the results of the E3 ligase auto-ubiquitination assay of RB1B2 harboring the C142S, C145T, C195F, and the mutation of Ala130 to Ser, Thr, and Val. Lane 1 does not contain the RB1B2 protein. (ii, iii) The WB images of the bands for the activated E2∼Ub and ubiquitin are shown separately; these bands were part of the same image as (i) but were excised due to their brightness and dynamic range problems for clarity. Antibody used with all four figures was specific for biotinylated ubiquitin. (iv) WB of the stock solutions of the various His-tagged RB1B2 constructs. No His-tagged RB1B2 protein was used in lane 1, which served as control in which the E3 was omitted. This image was taken from a separate gel because the antibody was specific for the His₆-tag.

Figure 4. RB1B2 with mutant Bbox domains catalyzed the ubiquitination of PP2Ac.

WB showing results of the ubiquitination reaction with PP2Ac using RB1B2 containing the various mutations within the Bbox domains. In lane 1, RB1B2 was omitted as control. At the bottom, the ability of the mutant RB1B2 constructs to catalyze the ubiquitination of PP2Ac is qualitatively noted. Antibody was specific against PP2Ac.

Figure 5. XLOS-observed mutations within Bbox1 disrupted polyubiquitination of alpha4.

A. Ubiquitination assay of alpha4 was performed with the RB1B2 construct containing (i) C142S, (ii) C145T, and (iii) C195F. B. Ubiquitination of alpha4 by RB1B2 with the (i) A130T, (ii) A130V and (iii) A130S mutations within the Bbox1 domain. The amount of alpha4 used for the assay with the A130T RB1B2 mutant was higher than that used for the A130V mutant assay. Nonetheless, the overall results for the two RB1B2 mutants are very similar. For comparison, wild type RB1B2 was shown to catalyze polyubiquitination of alpha4, as indicated by the smear/ladder pattern designated with the labeled alpha4∼Ub_n. Negative controls in which RB1B2 protein is omitted are shown for each reaction, as well. At the bottom of each figure, the ability of the mutant RB1B2 constructs to catalyze the ubiquitination of alpha4 is qualitatively noted. Antibody was specific for the C-terminal region of alpha4.

Figure 6. XLOS-observed mutations of highly conserved amino acids destabilized the Bbox structure.

A. Sequence alignment of the MID1 and MID2 Bbox1 domains from human, rat, mouse, chicken and zebrafish. Conserved A130, C142 and C145 residues are highlighted in red font and dark shading. At the bottom, the sequences of the various Bbox1 mutations are shown. B. Ribbon representation of the tertiary structure of the Bbox1-Bbox2 domains in tandem showing the coordinated zinc ions (red spheres) and their ligands. The structure shows the locations of the amino acids that were mutated, specifically residues Ala130, Cys142, Cys145 and Cys195. On the right, a zoomed view showing the relative proximity of Ala130 to the zinc binding Cys119 and Cys142 residues. Space filling representation of the side chain atoms of these three amino acids suggests that mutation of Ala130 to Thr and Val will induce steric clashes with the side chain atoms of Cys119 and Cys142, which can disrupt zinc-coordination.