Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins

Abstract

The serine/threonine phosphatase protein phosphatase 2A (PP2A) plays an essential role in many aspects of cellular functions and has been shown to be an important tumor suppressor. The core enzyme of PP2A comprises a 65 kDa scaffolding subunit and a 36 kDa catalytic subunit. Here we report the crystal structures of the PP2A core enzyme bound to two of its inhibitors, the tumor-inducing agents okadaic acid and microcystin-LR, at 2.6 and 2.8 A resolution, respectively. The catalytic subunit recognizes one end of the elongated scaffolding subunit by interacting with the conserved ridges of HEAT repeats 11-15. Formation of the core enzyme forces the scaffolding subunit to undergo pronounced structural rearrangement. The scaffolding subunit exhibits considerable conformational flexibility, which is proposed to play an essential role in PP2A function. These structures, together with biochemical analyses, reveal significant insights into PP2A function and serve as a framework for deciphering the diverse roles of PP2A in cellular physiology.

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…The two Glu64 substitutions result in the inability of Aa to bind to the PR61/ B 0 a, g and d subunits, and the Arg418 and the frame-shift mutants display no binding to Ca and little or no binding to all B-type subunits [12,29,38]. These biochemical data can be explained in light of recent structural findings: Glu64 forms a hydrogen bond with the Ca C terminus, thereby stabilizing the A-PR61/B 0 g1 interface [36,37] (see Box 1, Figure Ib), Arg418 maps to the A-C interface and forms a hydrogen bond with Glu67 of Ca [34,36]. In addition, partial knockdown of the Aa subunit results in selective loss of PP2A holoenzymes containing the PR61/B 0 g subunit [29,30], rendering cells tumorigenic via increased Ser62 phosphorylation and stabilization of c-Myc [5,29,30].…”

“…33 No.3 previous biochemical and mutagenesis studies [1], the structure of Aa revealed that it binds to the Ca subunit via the intra-repeat loops of HEAT-repeats 11-15 (Box 1, Figure Ia), which causes a major conformational switch in Aa. This switch occurs mainly at the inter-repeat loops between repeats 11 and 12, and 12 and 13, resulting in a horseshoe-or C-shaped conformation of the Aa subunit [34]. The Ca subunit itself adopts an a/b fold typical of the serine/threonine-specific phosphoprotein phosphatase (PPP) family of phosphatases, which includes protein phosphatase 1 and calcineurin [35].…”

“…The Ca subunit itself adopts an a/b fold typical of the serine/threonine-specific phosphoprotein phosphatase (PPP) family of phosphatases, which includes protein phosphatase 1 and calcineurin [35]. The active site contains two catalytic metal ions and points towards the end of the Aa subunit to which the regulatory B-type subunit probably binds [34]. The configuration of PR61/B 0 g within the holoenzyme is also solved: it binds the intra-repeat loops of HEAT-repeats 2-7 on the same apical side of the horseshoe as the C subunit [36,37] (Box 1, Figure Ia).…”

“…Of note, methylation requirements for holoenzyme assembly in vitro do not necessarily reflect the in vivo requirements. Indeed, whereas the methylation requirement for PP2A T55 assembly is well established in vivo, the in vitro requirements for holoenzyme assembly are less stringent [34,37,50]. This difference might have arisen because most in vitro assembly studies use an inactive conformation of PP2A C, which was essential for obtaining sufficient amounts for crystallization but which is known to have a different affinity for interaction partners to that of the active form [45,46].…”

The 3D Structure of Protein Phosphatase 2A: New Insights into a Ubiquitous Regulator of Cell Signaling

“…Consequently, a series of recent reports on the 3D structures of multimeric forms of PP2A have had a major impact on our understanding of this important signaling molecule. A report by Shi and colleagues solved the structure of the AC core dimer bound to inhibitory toxins (4). Two additional papers, one by Cho and Xu (5) and a second from Shi and colleagues (6), coincidently report the structure of the same PP2A holoenzyme composed of the AC core dimer associated with the B56␥ regulatory subunit.…”