ASPP proteins discriminate between PP1 catalytic subunits through their SH3 domain and the PP1 C-tail

Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP (apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. Here we show that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. We solve the crystal structure of the human ASPP2/PP1 complex and show that ASPP2 recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1.

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The crystallographic data sets were indexed, scaled and merged with xia2. Molecular replacement was achieved by using the atomic coordinates of human PP1 and human ASPP2 structures in PHASER. Refinement was carried out using PHENIX. Model building was carried out in Coot, model validation used PROCHECK and figures were prepared using the PyMOL Molecular Graphics System, Version 2.0 (Schrödinger, LLC). See the Methods section for further details and references. NMR spectra were processed with NMRPipe or Topspin 4.0.1 (Bruker) and analyzed using either CARA (http://www.cara.nmr.ch) or Sparky (http://www.cgl.ucsf.edu/home/sparky). Chemical shift referencing, CSI/SSP analysis and secondary structure propensity calculations were performed as described previously. See the Methods section for further details and references. Image analysis was performed using ImageJ Statistical analysis was performed with Prism7 Graphpad. Mass Spectrometry: Acquired spectra were searched using the MaxQuant software package version 1.5.2.8 embedded with the Andromeda search engine 69against human proteome reference dataset (http:/www.uniprot.org/) extended with reverse decoy sequences.
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Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The NMR chemical shifts have been deposited in the BioMagResBank, www.bmrb.wisc.edu (accession no. 27464). The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 6GHM). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012378.

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