PP2A-B\u2032 holoenzyme substrate recognition, regulation and role in cytokinesis

Cheng-Guo Wu,Jenny Day,Hui Chen,Feng Guo,Benjamin M Johnson ,Alka Choudhary,Ziqing Lin,Yitong Li,Eduard Resch,Ylva Ivarsson,Ying Ge,Irene M Ong ,Aiping Zheng,Sean J Mcilwain ,Woo‐Jong Lee ,Ting‐Jia Gu ,Vikash Kumar Yadav ,Mark E Burkard ,Michael Rowse,Qingge Xu,Yongna Xing

doi:10.1038/celldisc.2017.27

Abstract

Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase; it forms diverse heterotrimeric holoenzymes that counteract kinase actions. Using a peptidome that tiles the disordered regions of the human proteome, we identified proteins containing [LMFI]xx[ILV]xEx motifs that serve as interaction sites for B′-family PP2A regulatory subunits and holoenzymes. The B′-binding motifs have important roles in substrate recognition and in competitive inhibition of substrate binding. With more than 100 novel ligands identified, we confirmed that the recently identified LxxIxEx B′α-binding motifs serve as common binding sites for B′ subunits with minor variations, and that S/T phosphorylation or D/E residues at positions 2, 7, 8 and 9 of the motifs reinforce interactions. Hundreds of proteins in the human proteome harbor intrinsic or phosphorylation-responsive B′-interaction motifs, and localize at distinct cellular organelles, such as midbody, predicting kinase-facilitated recruitment of PP2A-B′ holoenzymes for tight spatiotemporal control of phosphorylation at mitosis and cytokinesis. Moroever, Polo-like kinase 1-mediated phosphorylation of Cyk4/RACGAP1, a centralspindlin component at the midbody, facilitates binding of both RhoA guanine nucleotide exchange factor (epithelial cell transforming sequence 2 (Ect2)) and PP2A-B′ that in turn dephosphorylates Cyk4 and disrupts Ect2 binding. This feedback signaling loop precisely controls RhoA activation and specifies a restricted region for cleavage furrow ingression. Our results provide a framework for further investigation of diverse signaling circuits formed by PP2A-B′ holoenzymes in various cellular processes.

Highlights

Cellular processes and normal physiological functions require coordination of numerous covalent modifications, such as protein phosphorylation
We confirm that the LxxIxEx is the preferred short linear motifs (SLiMs) for interactions with different B′ regulatory subunits, and we find that the specificity is highly similar between the isolated regulatory subunits and the holoenzymes
In addition to the LxxIxEx-containing ligands recently identified as B′α by affinity purification coupled to mass spectrometry (AP-MS) [12], we identified more than 100 new putative substrates or binding proteins for B′ family, suggesting that proteomic peptide phage display (ProP-PD) is a powerful approach for rapid identification of SLiMs for interaction with diverse phosphatase 2A (PP2A) holoenzymes

Summary

Introduction

Cellular processes and normal physiological functions require coordination of numerous covalent modifications, such as protein phosphorylation. While many kinases have well-characterized preferences for short linear motifs (SLiMs; 3–10 amino-acid stretches) for substrate targeting [3, 4], there is a scarcity of knowledge on substrate recognition of phosphatase complexes, which has been a critical barrier for understanding spatiotemporal control of protein phosphorylation in cellular signaling. Protein phosphatase 2A (PP2A) is an essential and highly abundant serine/threonine phosphatase in eukaryotic cells, counteracting diverse kinases in cell growth, cell proliferation, apoptosis, cytoskeleton dynamics and stress response [5,6,7]. The dearth of knowledge on substrate recognition by diverse PP2A holoenzymes has greatly hindered the dissection of PP2A function in diverse cellular signalings

Methods

Results

Conclusion