Structural and Biochemical Characterization of Human PR70 in Isolation and in Complex with the Scaffolding Subunit of Protein Phosphatase 2A

Abstract

Protein Phosphatase 2A (PP2A) is a major Ser/Thr phosphatase involved in the regulation of various cellular processes. PP2A assembles into diverse trimeric holoenzymes, which consist of a scaffolding (A) subunit, a catalytic (C) subunit and various regulatory (B) subunits. Here we report a 2.0 Å crystal structure of the free B’’/PR70 subunit and a SAXS model of an A/PR70 complex. The crystal structure of B’’/PR70 reveals a two domain elongated structure with two Ca2+ binding EF-hands. Furthermore, we have characterized the interaction of both binding partner and their calcium dependency using biophysical techniques. Ca2+ biophysical studies with Circular Dichroism showed that the two EF-hands display different affinities to Ca2+. In the absence of the catalytic C-subunit, the scaffolding A-subunit remains highly mobile and flexible even in the presence of the B’’/PR70 subunit as judged by SAXS. Isothermal Titration Calorimetry studies and SAXS data support that PR70 and the A-subunit have high affinity to each other. This study provides additional knowledge about the structural basis for the function of B’’ containing holoenzymes.

B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability

“…There were 7 Dutch patients (from RUMC: cases 1, 8-9, and 11; from UMC Utrecht: cases 7, 15, and 16). Six of these cases were identified through routine diagnostic exome sequencing the absence of the C subunit (32). It may be of interest to study if fingolimod (FTY720), a PP2A activator and immunosuppressant that is licensed for treatment of multiple sclerosis (33)(34)(35), or FDA-approved compounds of the phenothiazine family that were recently discovered as PP2A activators (36) may improve brain function in these patients.…”

PP2A as a master regulator of the cell cycle

“…The Bα subunit is a 7-bladed β-propeller with a hairpin that extends to interact with the side face of the N-terminal HEAT repeats of the A-subunit (Xu et al ., 2008). Recently, the high-resolution structure of a B″ holoenzyme associated with PR70 and two structures of B″ family subunits in isolation were finally solved (Dovega et al ., 2014; Wlodarchak et al ., 2013). These structures show that the B″ subunits are distinct from other families and consist of a multi-domain arrangement with two prominent calcium binding EF hands and a hydrophobic interacting motif.…”

“…These structures show that the B″ subunits are distinct from other families and consist of a multi-domain arrangement with two prominent calcium binding EF hands and a hydrophobic interacting motif. One of the EF hands directly contacts the top ridge of the scaffold subunit and is important for A–B″ binding (Dovega et al ., 2014; Wlodarchak et al ., 2013). The N-terminal hydrophobic motif binds to the N-terminal side surface of the A-subunit while the C-terminal domain interacts with PP2Ac.…”

Recurrent PPP2R1A Mutations in Uterine Cancer Act through a Dominant-Negative Mechanism to Promote Malignant Cell Growth

“…For instance, N-terminal deletions of Aa inhibit C binding (14), and B55/Ba and B56/B 0 g3 do not bind to a C-terminally truncated Aa (15,20), while PR72/B" does (20). This suggests cooperativity between specific B-type subunits and C in binding the A subunit, while other B-type subunits may bind A independently from C (21). Particularly the conserved C-terminal tail of the C subunit provides additional, stabilizing contacts with B55/B and most B56/B 0 subunits, but not B56/B 0 d, PR72/B" or striatin/ B"', to promote holoenzyme assembly (22).…”