title: Detection and characterization of the in vitro e3 ligase activity of the human MID1 protein.
abstract: Human MID1 (midline-1) is a microtubule-associated protein that is postulated to target the catalytic subunit of protein phosphatase 2A for degradation. It binds alpha4 that then recruits the catalytic subunit of protein phosphatase 2A. As a member of the TRIM (tripartite motif) family, MID1 has three consecutive zinc-binding domains-RING (really interesting new gene), Bbox1, and Bbox2-that have similar ββα-folds. Here, we describe the in vitro characterization of these domains individually and in tandem. We observed that the RING domain exhibited greater ubiquitin (Ub) E3 ligase activity compared to the Bbox domains. The amount of autopolyubiquitinated products with RING-Bbox1 and RING-Bbox1-Bbox2 domains in tandem was significantly greater than those of the individual domains. However, no polyubiquitinated products were observed for the Bbox1-Bbox domains in tandem. Using mutants of Ub, we observed that these MID1 domain constructs facilitate Ub chain elongation via Lys63 of Ub. In addition, we observed that the high-molecular-weight protein products were primarily due to polyubiquitination at one site (Lys154) on the Bbox1 domain of the RING-Bbox1 and RING-Bbox1-Bbox2 constructs. We observed that MID1 E3 domains could interact with multiple E2-conjugating enzymes. Lastly, a 45-amino-acid peptide derived from the C-terminus of alpha4 that binds tightly to Bbox1 was observed to be monoubiquitinated in the assay and appears to down-regulate the amount of polyubiquitinated products formed. These studies shed light on MID1 E3 ligase activity and show how its three zinc-binding domains can contribute to MID1's overall function.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Han
authors.forename: Haijuan
authors.initials: H
authors.lastname: Du
authors.forename: Michael A
authors.initials: MA
authors.lastname: Massiah
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008868:Microtubule Proteins / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D008868:Microtubule Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D025801:Ubiquitin; C109052:MID1 protein, human; D044767:Ubiquitin-Protein Ligases