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Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase.

ABSTRACT: Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.

SUBMITTER: Karman Z

PROVIDER: S-EPMC7776573 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase.

Karman Zoltan Z Rethi-Nagy Zsuzsanna Z Abraham Edit E Fabri-Ordogh Lilla L Csonka Akos A Vilmos Peter P Debski Janusz J Dadlez Michal M Glover David M DM Lipinszki Zoltan Z

Open biology 20201223 12

Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Her ...[more]

PMID: 33352067

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Project description:Significance statementMutations in hepatocyte nuclear factor-1 β ( HNF1B ) are the most common monogenic causes of congenital renal malformations. HNF1B is necessary to directly reprogram fibroblasts to induced renal tubule epithelial cells (iRECs) and, as we demonstrate, can induce ectopic pronephric tissue in Xenopus ectodermal organoids. Using these two systems, we analyzed the effect of HNF1B mutations found in patients with cystic dysplastic kidney disease. We found cross-species conserved targets of HNF1B, identified transcripts that are differentially regulated by the patient-specific mutant protein, and functionally validated novel HNF1B targets in vivo . These results highlight evolutionarily conserved transcriptional mechanisms and provide insights into the genetic circuitry of nephrogenesis.BackgroundHepatocyte nuclear factor-1 β (HNF1B) is an essential transcription factor during embryogenesis. Mutations in HNF1B are the most common monogenic causes of congenital cystic dysplastic renal malformations. The direct functional consequences of mutations in HNF1B on its transcriptional activity are unknown.MethodsDirect reprogramming of mouse fibroblasts to induced renal tubular epithelial cells was conducted both with wild-type HNF1B and with patient mutations. HNF1B was expressed in Xenopus ectodermal explants. Transcriptomic analysis by bulk RNA-Seq identified conserved targets with differentially regulated expression by the wild-type or R295C mutant. CRISPR/Cas9 genome editing in Xenopus embryos evaluated transcriptional targets in vivo .ResultsHNF1B is essential for reprogramming mouse fibroblasts to induced renal tubular epithelial cells and induces development of ectopic renal organoids from pluripotent Xenopus cells. The mutation R295C retains reprogramming and inductive capacity but alters the expression of specific sets of downstream target genes instead of diminishing overall transcriptional activity of HNF1B. Surprisingly, targets associated with polycystic kidney disease were less affected than genes affected in congenital renal anomalies. Cross-species-conserved transcriptional targets were dysregulated in hnf1b CRISPR-depleted Xenopus embryos, confirming their dependence on hnf1b .ConclusionsHNF1B activates an evolutionarily conserved program of target genes that disease-causing mutations selectively disrupt. These findings provide insights into the renal transcriptional network that controls nephrogenesis.

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Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase.

Publications

Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase.

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