Protein phosphatase 2A modulates podocyte maturation and glomerular functional integrity in mice

Xiujuan Zhu,Cunji Gao,Jing Zhou,Chengxian Xu,Weiqiang Lin,Yuhong Ye,Jianhua Mao,Yingying Zhang

doi:10.1186/s12964-019-0402-y

Abstract

BackgroundProtein phosphorylation & dephosphorylation are ubiquitous cellular processes that allow for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a multifunction phosphatase that is well expressed in all cell types of kidney during early renal development, though its functions in kidney remains to be elucidated.MethodsPP2A conditional knock-out mice was generated with PP2A fl/fl mice that were crossed with Podocin-Cre mice. The phenotype of Pod-PP2A–KO mice (homozygous for the floxed PP2A allele with Podocin-Cre) and littermate PP2A fl/fl controls (homozygous for the PP2A allele but lacking Podocin-Cre) were further studied. Primary podocytes isolated from the Pod-PP2A-KO mice were cultured and they were then employed with sing label-free nano-LC − MS/MS technology on a Q-exactive followed by SIEVE processing to identify possible target molecular entities for the dephosphorylation effect of PP2A, in which Western blot and immunofluorescent staining were used to analyze further.ResultsPod-PP2A–KO mice were developed with weight loss, growth retardation, proteinuria, glomerulopathy and foot process effacement, together with reduced expression of some slit diaphragm molecules and cytoskeleton rearrangement of podocytes. Y box protein 1 (YB-1) was identified to be the target molecule for dephosphorylation effect of PP2A. Furthermore, YB-1 phosphorylation was up-regulated in the Pod-PP2A–KO mice in contrast to the wild type controls, while total and un-phosphorylated YB-1 both was moderately down-regulated in podocytes from the Pod-PP2A-KO mice.ConclusionOur study revealed the important role of PP2A in regulating the development of foot processes and fully differentiated podocytes whereas fine-tuning of YB-1 via a post-translational modification by PP2A regulating its activity might be crucial for the functional integrity of podocytes and glomerular filtration barrier.Graphic abstract

Highlights

Protein phosphorylation & dephosphorylation are ubiquitous cellular processes that allow for the nuanced and reversible regulation of protein activity
The mutant mice manifest a sequelae of podocyte and glomerular abnormalities, which included weight loss, growth retardation, proteinuria, renal dysplasia, glomerulopathy and foot process (FP) effacement, together with reduced expression of slit diaphragm molecule and cytoskeleton rearrangement of podocytes
phosphatase 2A (PP2A) loss in podocyte isolated from Pod-PP2A–Knock out (KO) mice was confirmed by Western blot (Fig. 1c), by tail genotyping (Fig. 1d) and by immunofluorescences (Fig. 1e)

Summary

Introduction

Protein phosphorylation & dephosphorylation are ubiquitous cellular processes that allow for the nuanced and reversible regulation of protein activity. Podocytes exhibit a unique cytoskeletal architecture that is fundamentally linked to their function in maintaining kidney filtration barrier [2]. Many molecules such as Nephrin [3], CD2AP [4], Podocin [5, 6] and TRPC6 [7] have been identified to play a key role in keeping the functional integrity of glomerular filtration and preventing plasma protein leakage to urine. Protein phosphatase 2A (PP2A) is a multifunction phosphatase that is ubiquitously expressed in eukaryotic cells which consists of a complex with three subunits including a scaffold subunit A, a catalytic subunit C, and a regulatory subunit B [8,9,10]. PP2A was suggested to regulate various cellular processes such as signal transduction, cytoskeleton dynamics, promoting cell transformation [11], and angiogenesis [12, 13]

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