Structure of the Protein Phosphatase 2A Holoenzyme

SummaryProtein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B′/B56/PR61. Surprisingly, the B′/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B′/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B′/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A

Methylation-Dependent Binding of the Epstein-Barr Virus BZLF1 Protein to Viral Promoters

The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection

Centralspindlin assembly and 2 phosphorylations on MgcRacGAP by Polo-like kinase 1 initiate Ect2 binding in early cytokinesis

<div><p>Cytokinesis is the final step of cell division which partitions genetic and cytosolic content into daughter cells. Failed cytokinesis causes polyploidy, genetic instability, and cancer. Kinases use phosphorylation to regulate the timing and location of the cytokinetic furrow. Polo-like kinase 1 (Plk1) is an essential mitotic kinase that triggers cytokinesis by phosphorylating MgcRacGAP to create a docking site for Ect2 at the central spindle. Ect2 binds to MgcRacGAP via its N-terminal BRCT domain (BRCA1 C-terminal), which docks at specific phosphorylated residues. Here we investigate the minimal Plk1-dependent phosphorylation sites required for cytokinesis onset. We demonstrate that phosphorylation of the major MgcRacGAP site, S157, is necessary but not sufficient to bind the Ect2 BRCT domain. Phosphorylation of an additional residue on MgcRacGAP at S164 is also required to elicit efficient binding. Surprisingly, BRCT binding additionally requires MKLP1 and its cognate interacting N-terminal domain of MgcRacGAP. Our findings indicate that central spindle assembly and 2 Plk1-dependent phosphorylations are required to establish efficient binding of the Ect2 BRCT in early cytokinesis. We propose that these requirements establish a high threshold to restrain premature or ectopic cytokinesis.</p></div

Mechanisms of the Scaffold Subunit in Facilitating Protein Phosphatase 2A Methylation

<div><p>The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A–C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.</p></div

Electrostatic interactions between the A-subunit and LCMT-1.

<p>(<b>A</b>) Structural model of the PP2A core enzyme-LCMT-1 complex built from the structure of the PP2Ac-LCMT-1 complex and the A-subunit with morphed conformation. The electrostatic potential for the A-subunit (left panel) and LCMT-1 (right panel) is shown. The corresponding regions on the A-subunit and LCMT-1 for potential electrostatic interactions are circled (i and ii). The A-subunit residues that directly contact LCMT-1 in the model, Arg183 and Arg258, are shown. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086955#pone.0086955.s003" target="_blank">Movie S1</a>. (<b>B</b>) The effect of the A-subunit mutations to Arg183 and Arg258 on methylation of the PP2A core enzyme. Results from 5–7 independent experiments were summarized and the average K_m ± std. dev. and the P value were calculated and shown. <b>(C)</b> FRET assay measured changes in the distance between the N- and C-termini of the A-subunit in the core enzyme prior to and after addition of an excess molar amount of LCMT-1 or a stoichiometric amount of PR70 (108–575). Representative results were shown with mean ± SEM calculated from triplicate.</p

The effect of the N-terminal HEAT repeats of the A-subunit on methylation.

<p>(<b>A</b>) Schematic representation of internal truncations of the N-terminal HEAT repeats to reduce the length of the A-subunit N-terminal structure. (<b>B</b>) Normalized concentrations of PP2Ac assembled with full-length or truncated A-subunits used for kinetic analysis of PP2A methylation in (<b>C</b>). (<b>C</b>) Kinetics of methylation of PP2A core enzymes with varied length of the A-subunit. Results from 6–7 independent experiments were summarized and the average K_m ± std. dev. was calculated and shown in the table on the right. (<b>D</b>) Correlation between the number of deleted N-terminal HEAT repeats and the K_m of methylation of PP2A core enzyme by LCMT-1. Statistical test of this correlation was performed and the P value was calculated and shown. (<b>E</b>) Structure of the core enzyme portion of PP2A holoenzyme (PDB ID: 2NPP). The A-subunit, PP2Ac, and PP2Ac-tail are colored green, blue, and magenta, respectively. The positions of the HEAT-repeats where truncations were made are indicated by numbers.</p