title: The E3 ubiquitin ligase- and protein phosphatase 2A (PP2A)-binding domains of the Alpha4 protein are both required for Alpha4 to inhibit PP2A degradation.
abstract: Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electron-electron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. Structurally, Alpha4 differs from its yeast homolog, Tap42, in two important ways: 1) the position of the helix containing the PP2Ac-binding residues is in a more open conformation, showing flexibility in this region; and 2) Alpha4 contains a ubiquitin-interacting motif. The effects of wild-type and mutant Alpha4 on PP2Ac ubiquitination and stability were examined in mammalian cells by performing tandem ubiquitin-binding entity precipitations and cycloheximide chase experiments. Our results reveal that both the C-terminal Mid1-binding domain and the PP2Ac-binding determinants are required for Alpha4-mediated protection of PP2Ac from polyubiquitination and degradation.
authors: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors: Michele
authors: M
authors: LeNoue-Newton
authors: Guy R
authors: GR
authors: Watkins
authors: Ping
authors: P
authors: Zou
authors: Katherine L
authors: KL
authors: Germane
authors: Lisa R
authors: LR
authors: McCorvey
authors: Brian E
authors: BE
authors: Wadzinski
authors: Benjamin W
authors: BW
authors: Spiller
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C490467:IGBP1 protein, human; C092377:Igbp1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D010750:Phosphoproteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; C517189:PPP2CA protein, human; D054648:Protein Phosphatase 2