Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits.

Thomas Kruse,Marie Kveiborg,Dimitriya Hristoforova Garvanska,Arminja N Kettenbach,Jacob Samsøe‐Petersen,Isha Nasa,Jeanette Schwarz,Blanca Lopez‐Mendez,Denise Nikodemus,Sebastian Peter Gnosa,Emil Peter Thrane Hertz,Hanna Sofia Pena,Jakob Nilsson,Hieu Nguyen,Jamin B Hein

doi:10.15252/embj.2019103695

Abstract

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B‐subunits. Only a limited number of PP2A‐regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site‐specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A‐B56 and PP2A‐B55 holoenzymes. Strikingly, we find that B‐subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A‐B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A‐B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.

Highlights

phosphatase 2A (PP2A) is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits
To understand principles of PP2A specificity, we focused on PP2AB56 which is a major tumor suppressor (Janssens et al, 2005; Eichhorn et al, 2009)
Provided sufficient specificity and potency, we reasoned that such an inhibitor could be used to displace PP2A-B56 from all cellular LxxIxE containing interactors and, to interrogate the phosphoproteome regulated by this phosphatase

Summary

Introduction

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. A limited number of PP2Aregulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site-specific dephosphorylation and of its tumor suppressor functions. We develop phosphoproteomic strategies for global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. We find that B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A-B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function

Methods

Results

Conclusion