The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac–α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. In this report, we show that alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. We also identify a novel ubiquitin-interacting motif (UIM) within alpha4 (amino acid residues [46][47][48][49][50][51][52][53][54][55][56][57][58][59][60] and analyze the interaction between alpha4 and ubiquitin using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, we demonstrate that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4.Protein serine/threonine phosphatase 2A (PP2A) is an abundant cellular enzyme with numerous substrates that modulate a wide variety of cellular functions. Considering the multitude of cellular processes under the control of PP2A, it is not surprising that several different mechanisms exist to regulate phosphatase activity. These regulatory mechanisms include association with specific regulatory subunits and post-translational modifications of PP2Ac (i.e. phosphorylation, carboxymethylation, and ubiquitination) (1-3). Both biochemical and structural studies of PP2A have provided key mechanistic insights to explicate regulation of phosphatase holoenzyme composition and activity via phosphorylation and carboxymethylation (1,2,4,5); however, little is known about PP2Ac ubiquitination beyond the initial report demonstrating the polyubiquitination and degradation of microtubule-associated PP2Ac (3). The E3 ubiquitin ligase responsible for targeting PP2Ac for proteasome degradation

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert ␣-D-ribose 5-phosphate (ribose 5-phosphate) and ␣-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Enzyme-catalyzed phosphoryl transfer forms the basis for many biological, bioenergetic, and regulatory processes and is one of the most common cellular reactions (1). Numerous enzyme families have evolved mechanistically distinct solutions for phosphoryl transfer (2). Phosphomutases are phosphotransfer enzymes that rearrange the position of phosphate within a substrate molecule through either intramolecular (i.e. the phosphate is transferred to a different position on the same molecule) or intermolecular phosphoryl transfer (i.e. the phosphate is transferred from one substrate molecule to another).Bacterial phosphopentomutases (PPMs) 3 (EC 5.4.2.7) interconvert ribose 1-phosphate and ribose 5-phosphate, which bridges glucose metabolism and RNA biosynthesis (3). The importance of this reaction has recently been underscored by the observation that targeted deletion of the gene encoding PPM in the pathogen Francisella tularensis (deoB) results in markedly decreased virulence (4). PPMs appear to be biochemically and structurally distinct from their human congeners (5, 6), making them potential targets for antibiotic development.Sequence clustering classifies prokaryotic PPMs within the alkaline phosphatase superfamily of metalloenzymes, which includes a range of functionally diverse enzymes such as cofactor-independent phosphoglycerate mutase, phosphodiesterase, and estrone and aryl sulfatases (7). The majority of alkaline phosphatase superfamily enzymes catalyze a hydrolase reaction; however, both PPM (5) and the cofactor-independent phosphoglycerate mutase catalyze phosphomutase reactions (8, 9).All previously characterized alkaline phosphatase superfamily members follow a unified general reaction mechanism ( Fig. 1) (10). In alkaline phosphatase itself (11, 12), the catalytically competent enzyme has an unphosphorylated catalytic nucleophile, Ser-102 (Fig. 1, state 1). Turnover is initiated when the metallocenter activates a phosphoester donor substrate (R D -OPO 3 H Ϫ ) (Fig. 1, state 2) to transfer the phosphoryl group to the hydroxyl of Ser-102 (Fig. 1, state 3). This results in a covalent phosphoenzyme intermediate (E-OPO 3 H Ϫ ) (Fig. 1, state 4). A second phosphoryl transfer from the enzyme to the acceptor water molecule (Fig. 1, states 5 and 6) completes the reaction cycle. This general reaction mechanism has also been verified * This work was supported, in whole or in part, by National Institutes of Health Grants GM079419 (to T. M. I.), GM077189 (to B. O. B.), GM051366 (to B. E. W.), DK070787 (to B. E. W.), T32 NS07491 (to T. D. P.), T32 GM008320 (to T. D. P.), T90 DA022873 (to D. P. N.), and T32 GM07628 (to G. R. W.). This work was also supported by a pilot award funded by the Vanderbilt Instit...

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 electronelectron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. PP2A3 is a ubiquitous serine/threonine phosphatase involved in the regulation of numerous cell signaling pathways and cellular functions, including proliferation, cytoskeletal rearrangement, apoptosis, and cell migration (1-3). Several pathologies have been linked to dysregulation of PP2Ac, including Alzheimer disease, cancer, and diabetes (4 -8). The activity of PP2Ac is tightly controlled in vivo via association with regulatory subunits, interactions with other cellular proteins, and various post-translational modifications (9 -12). PP2A regulatory subunits play a critical role in determining phosphatase activity and substrate selectivity, as well as directing the subcellular localization of the PP2A holoenzyme (3). PP2A exists primarily as a heterotrimeric holoenzyme consisting of a structural A-subunit, a variable regulatory B-subunit, and PP2Ac. However, an atypical pool of PP2Ac exists in complex with the regulatory subunit Alpha4 that binds directly to PP2Ac in the absence of the A-and B-subunits (13-16). Recent studies have shown that Alpha4 plays a crucial role in the control of PP2A ubiquitination and stability (12,17,18).Alpha4, a multidomain protein with similarity to Tap42 from yeast, was initially discovered as a 52-kDa phosphoprotein in B-cell receptor complexes (16,19). Both Alpha4 and Tap42 consist of an N-terminal domain that contains the residues important for PP2Ac binding (20) and a C-terminal domain that is protease-sensitive and intrinsically disordered (21). The C-terminal domain of Alpha4 binds Mid1, a putative E3 ligase (12,22). Alpha4 regulates all three type 2A protein phosphatases (PP2Ac, PP4, and PP6), modulating both catalytic activity and expression levels (13,14,17,23). In addition to its association with PP2A family members, Alpha4 associates and co-localizes with Mid1, a putative E3 ubiquitin ligase thought to facilitate PP2Ac polyubiquitination (12,22). The C terminus of Alpha4 and the B-box1 domain of the Mid1 protein mediate the association between Mid1 and Alpha4 (12,22). Mutations in Mid1 have been linked to Opitz syndrome, a developmental disorder (24,25). At the cellular level, mutations in Mid1 lead to decreases in ubiquitination and degradation of PP2Ac, especially microtubule-associated PP2Ac (12, 26).Alpha4 serves as a scaffold for PP2Ac and Mid1 and protects PP2Ac fr...

Background: ␣4 binds to the PP2A catalytic subunit and the microtubule-associated E3 ligase MID1. Results: MID1-dependent monoubiquitination promotes calpain-mediated cleavage of ␣4, altering its phosphatase regulatory function. Conclusion: Defects in this regulatory process may underlie the MAP hypophosphorylation and hyperphosphorylation seen in Opitz syndrome and Alzheimer disease. Significance: Pharmacological agents that interfere with ␣4 monoubiquitination or cleavage are potential therapeutics to treat Alzheimer disease.