Determinants of protein phosphatase 1β substrate specificity for MyPhoNE motif-containing proteins

Phosphoprotein phosphatase 1 (PP1) forms holoenzymes composed of a catalytic subunit (PP1c) and one or two of over 200 regulatory subunits (PP1Rs). Humans express four conserved PP1c isoforms: PP1cα, PP1cβ/δ, and splice variants PP1cγ1 and PP1cγ2. To systematically characterize PP1c isoform-specific interactions, we employed mass spectrometry to identify PP1cα, PP1cβ, and PP1cγ interacting proteins, determine their isoform specificity, and assess and quantify their abundance within the PP1 holoenzyme pool. Our data show that PP1c forms hundreds of dimeric and trimeric holoenzymes, but the 10 most abundant PP1Rs make up 74% of PP1 holoenzymes, and they are highly uniform among PP1c isoforms. A key exception is myosin phosphatase N-terminal element (MyPhoNE)-containing PP1Rs, which form abundant holoenzyme complexes exclusively with PP1cβ. To define the determinants of MYPT1-PP1cβ specificity, we systematically assessed the contributions of MYPT1-PP1cβ interactions. First, we generated PP1cβ-PP1cγ chimeras and PP1cβ Tyr 305/Tyr307 point mutations to test the contribution of the PP1 C-terminal residues, and secondly, we used PP1cβ Thr197Gln (T197Q) mutation to test the effect of the MYPT1:MyPhoNE-specific interaction. Using genome editing, we demonstrate that PP1cβ T197Q-expressing cells exhibit altered PP1 holoenzyme composition and phosphorylation signaling, including increased phosphorylation of the Polo-like kinase 1 (Plk1) activation loop. Our studies further the understanding of the PP1c isoform-specific preference and demonstrate how a single amino acid change can alter PP1 holoenzyme composition and phosphorylation signaling, potentially explaining how recently discovered PP1cβ clinical variants impact PP1 biology.