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.

Structural and functional observations of the P151L MID1 mutation reveal alpha4 plays a significant role in X‐linked Opitz Syndrome

“…As shown previously, the B-box1 domain is required for the binding and poly-ubiquitination of alpha4 but not PP2A [1,11]. As shown previously, the B-box1 domain is required for the binding and poly-ubiquitination of alpha4 but not PP2A [1,11].…”

“…The B-box1 domain is critical for poly-ubiquitination of alpha4 and PP2Ac [1,11]. The structure of the B-box1 domain is similar to RING E3 ligase domains [22][23][24][25][26], consisting of two b-strands and one a-helix and held together by the coordination of two zinc ions (Fig.…”

“…1). We recently showed that the A130T, C142S and C145T XLOSobserved mutations resulted in complete unfolding of the tertiary structure and disrupted MID1 E3 ligase function and targeting of alpha4 [1,27]. We recently showed that the A130T, C142S and C145T XLOSobserved mutations resulted in complete unfolding of the tertiary structure and disrupted MID1 E3 ligase function and targeting of alpha4 [1,27].…”

Two Novel Pathogenic MID1 Variants and Genotype-Phenotype Correlation Reanalysis in X-Linked Opitz G/BBB Syndrome

“…MID1 consists of the RING finger (RING), zinc-binding B-box-1 (B1) and B-box-2 (B2) domains, followed by a coiled-coil region (CC) and C-terminal subgroup one signature (COS), fibronectin type III repeat (FN3) and PRY-SPRY domains [Quaderi et al, 1997;Du et al, 2014]. Functionally impaired MID1 variants have a lower affinity for microtubules, causing degradation of protein phosphatase 2A (PP2A), a microtubule-associated protein.…”

Hedgehog-dependent E3-ligase Midline1 regulates ubiquitin-mediated proteasomal degradation of Pax6 during visual system development

“…Patients suffer from multiple malformations of the ventral midline as a consequence of mutations in the MID1 gene (25,26). In previous studies, Mid1 has been described to regulate protein phosphatase 2A (PP2A) stability (27)(28)(29)(30). PP2A/α4 is not the only known substrate, because recently it was shown that Mid1 interacts with the GLI regulator Fu, leading to a cytoplasmic retention of GLI3 in cancer cells (31).…”