Download PDF (restricted access) Download PDF (external access)

Biochemical Journal

Publication date: 2013-02
Volume: 449 Pages: 649 - 59
ISSN: 0264-6021, 1470-8728 PMID: 23088536
DOI: 10.1042/BJ20120506
Publisher: Published by Portland Press on behalf of the Biochemical Society

Author:

Skene-Arnold, Tamara D
Luu, Hue Anh ; Uhrig, R Glen ; De Wever, Veerle ; Nimick, Mhairi ; Maynes, Jason ; Fong, Andrea ; James, Michael NG ; Trinkle-Mulcahy, Laura ; Moorhead, Greg B ; Holmes, Charles FB

Keywords:

Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Apoptosis Regulatory Proteins, Humans, Models, Molecular, Molecular Sequence Data, Multiprotein Complexes, Mutagenesis, Site-Directed, Neoplasms, Protein Interaction Domains and Motifs, Protein Phosphatase 1, Rabbits, Recombinant Proteins, Sequence Homology, Amino Acid, Surface Plasmon Resonance, Tumor Suppressor Protein p53, src Homology Domains, 06 Biological Sciences, 11 Medical and Health Sciences, 03 Chemical Sciences, Biochemistry & Molecular Biology

Abstract:

The serine/threonine PP-1c (protein phosphatase-1 catalytic subunit) is regulated by association with multiple regulatory subunits. Human ASPPs (apoptosis-stimulating proteins of p53) comprise three family members: ASPP1, ASPP2 and iASPP (inhibitory ASPP), which is uniquely overexpressed in many cancers. While ASPP2 and iASPP are known to bind PP-1c, we now identify novel and distinct molecular interactions that allow all three ASPPs to bind differentially to PP-1c isoforms and p53. iASPP lacks a PP-1c-binding RVXF motif; however, we show it interacts with PP-1c via a RARL sequence with a Kd value of 26 nM. Molecular modelling and mutagenesis of PP-1c-ASPP protein complexes identified two additional modes of interaction. First, two positively charged residues, Lys260 and Arg261 on PP-1c, interact with all ASPP family members. Secondly, the C-terminus of the PP-1c α, β and γ isoforms contain a type-2 SH3 (Src homology 3) poly-proline motif (PxxPxR), which binds directly to the SH3 domains of ASPP1, ASPP2 and iASPP. In PP-1cγ this comprises residues 309-314 (PVTPPR). When the Px(T)PxR motif is deleted or mutated via insertion of a phosphorylation site mimic (T311D), PP-1c fails to bind to all three ASPP proteins. Overall, we provide the first direct evidence for PP-1c binding via its C-terminus to an SH3 protein domain.