Dataset Information

The interactome of the clathrin adaptor AP2 is modulated by the phosphorylation status of its µ2 subunit.

ABSTRACT: The clathrin adaptor AP2 is phosphorylated at a single site (Thr156) of its µ2 subunit. To gain insight how phosphorylation influences the interactome of AP2, we used human RPE cells which that were treated with LP-935509, an inhibitor of the adaptor-associated kinase AAK1. Kinase inhibition and loss of µ2 phosphorylation was verified with phospho-specific antibodies in westernblots of total cell extracts. We then used SILAC labelled cells and immune-isolated AP-2 and subjected bound proteins to mass spectrometry to identify all bound proteins and to determine phosphorylation-dependent changes in the interactome.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Retinal Pigment Epithelial Cell

SUBMITTER: Stefan Hoening

LAB HEAD: Stefan Höning

PROVIDER: PXD013468 | Pride | 2019-11-12

REPOSITORIES: Pride

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Publications

Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation.

Wrobel Antoni G AG Kadlecova Zuzana Z Kamenicky Jan J Yang Ji-Chun JC Herrmann Torsten T Kelly Bernard T BT McCoy Airlie J AJ Evans Philip R PR Martin Stephen S Müller Stefan S Salomon Susanne S Sroubek Filip F Neuhaus David D Höning Stefan S Owen David J DJ

Developmental cell 20190801 4

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding ...[more]

PMID: 31430451

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Project description:The emerging picture of transcriptional regulation is one of unexpected complexity. It is now clear that single transcription factors control hundreds, if not thousands, of direct targets by binding their genomic loci, but it is not understood how many of these are major players and how many are supporting cast. To address this, we leverage a well-characterized developmental network in Arabidopsis and map genome-wide binding of related proteins in multiple tissues. The transcription factor APETALA2 (AP2) has numerous functions, including roles in floral organ identity, seed development and stem cell maintenance. We focus on the role of AP2 in the floral transition and map direct targets on a genome-wide scale. We show that ap2 mutants flower early in long and short days, and that AP2 binds to many loci, most prominently floral pathway integrators, microRNAs and floral organ identity genes, many of which exhibit AP2-dependent transcription. Opposing, logical effects are evident in AP2 binding to two developmental microRNA genes that control AP2 expression, with AP2 positively regulating miR156 and negatively regulating miR172, forming a complex direct feedback loop, which also included all but one of the AP2-like miR172 target clade members. We also seek conserved targets by comparing the genome-wide direct target repertoire of AP2 with that of SCHLAFMÜTZE (SMZ), another member of the AP2-like miR172 target clade that shares partial redundancy, as evidenced by a hexuple mutant for the entire clade that flowered extremely early. Clear similarities and divergence are exposed in the AP2 and SMZ direct target repertoires. Finally, using an inducible expression system, we demonstrate that AP2 has dual molecular roles. It functions both as a transcriptional activator and repressor, directly inducing the expression of the floral repressor AGAMOUS-LIKE 15 (AGL15), and directly repressing the transcription of floral activators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). ChIP-Seq of two biological replicates for ATH-AP2 and respective control samples

Project description:The pathogenicity of the malaria parasites results from their ability to invade and remodel red blood cells (RBCs), expressing antigenic variant proteins for immune evasion and survival, and then to egress from the host cell. These sequential processes require concerted actions of a large number of proteins during the intraerythrocytic developmental cycle (IDC)(Boddey and Cowman, 2013; Cowman et al., 2017; Tan and Blackman, 2021), but the molecular basis of the required regulation is only partially understood. Here, we have characterized an essential Apicomplexan AP2 (ApiAP2) transcription factor (we refer to it as PfAP2-MRP; Master Regulator of Pathogenesis) that shows two peaks of expression during the IDC at 16- and 40-hour post-invasion (h.p.i.). When expression of PfAP2-MRP at 40 h.p.i. was disrupted using an inducible gene knockout approach, ∆PfAP2-MRP parasites unable to form mature merozoites and egress from the host RBCs owing to strong down-regulation of several known egress- and invasion-associated genes, in addition to several novel hypothetical genes thought to be involved in these key life cycle processes. Disruption of PfAP2-MRP expression at 16 h.p.i. results in transcriptional activation of the majority of silenced var genes observed at both bulk and single-cell level. This is also reflected by a significantly higher level of immuno-recognition of the exported proteins on the ∆PfAP2-MRP parasite-infected RBCs by pooled sera from malaria-exposed individuals from an endemic region. In addition, overexpression of many early gametocyte marker genes was also observed in ∆PfAP2-MRP parasites at both 40 h.p.i., and at 16 h.p.i. PfAP2-MRP directly regulates these genes by binding to their promoter region or indirectly through 14 other downstream AP2 transcription factors. Taken together, we conclude that PfAP2-MRP is an upstream transcriptional regulator that participates in mutually exclusive expression patterns shown by the var family of genes and is a critical determinant of parasite's growth during the IDC.

Dataset Information

The interactome of the clathrin adaptor AP2 is modulated by the phosphorylation status of its µ2 subunit.

Publications

Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation.

Similar Datasets

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