Understanding the antagonism of retinoblastoma protein dephosphorylation by PNUTS provides insights into the PP1 regulatory code

Abstract

The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets by associating with nearly 200 regulatory proteins to form highly specific holoenzymes. However, how these proteins direct PP1 specificity and the ability to predict how these PP1 interacting proteins bind PP1 from sequence alone is still missing. PP1 nuclear targeting subunit (PNUTS) is a PP1 targeting protein that, with PP1, plays a central role in the nucleus, where it regulates chromatin decondensation, RNA processing, and the phosphorylation state of fundamental cell cycle proteins, including the retinoblastoma protein (Rb), p53, and MDM2. The molecular function of PNUTS in these processes is completely unknown. Here, we show that PNUTS, which is intrinsically disordered in its free form, interacts strongly with PP1 in a highly extended manner. Unexpectedly, PNUTS blocks one of PP1's substrate binding grooves while leaving the active site accessible. This interaction site, which we have named the arginine site, allowed us to define unique PP1 binding motifs, which advances our ability to predict how more than a quarter of the known PP1 regulators bind PP1. Additionally, the structure shows how PNUTS inhibits the PP1-mediated dephosphorylation of critical substrates, especially Rb, by blocking their binding sites on PP1, insights that are providing strategies for selectively enhancing Rb activity.nuclear phosphatases | enzyme regulation | enzyme specificity | X-ray crystal structure | nuclear magnetic resonance P rotein phosphatase 1 (PP1; ∼38.5 kDa), a single-domain protein, is the most widely expressed and abundant serine/threonine phosphatase (1). By dephosphorylating a variety of protein substrates, PP1 regulates diverse biological processes, including protein synthesis, muscle contraction, carbohydrate metabolism, neuronal signaling and, of specific interest for this work, cell-cycle progression. Although the intrinsic substrate specificity of PP1 is very low, by interacting with regulatory proteins (∼200 biochemically confirmed PP1 interactors), PP1 achieves high specificity (2-5). The majority of PP1 regulators and some substrates bind PP1 via a primary PP1-binding motif, the RVxF motif, which binds to a hydrophobic groove on PP1 ∼20 Å distal from its catalytic center (6). Outside of the RVxF motif, PP1 regulatory proteins mostly lack any apparent sequence similarity. Thus, additional interaction sites, such as the SILK (7), the MyPhoNE (8), and the recently identified ΦΦ motif (9) can only be identified by structural analysis, a major challenge for a comprehensive understanding of PP1 regulation. Only when the primary sequences of PP1 regulators are correlated with specific PP1 binding modes and activities will the PP1 interactome, and the biological processes it regulates, become a viable drug target for the multitude of PP1-associated diseases, such as multiple cancers.One of the key PP1 regulatory targeting proteins in the nucleus, in addition to nuclear inhibitor of PP1 (NIPP1) and Repoman, is the...

Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase

“…2D). The K d of GADD34 552-567 for PP1 was ~6.5-fold weaker than that determined for some PP1 regulators, namely PNUTS and spinophilin (Choy et al, 2014; Ragusa et al, 2010) (8.7 nM and 9.3 nM, respectively), but close to that for NIPP1 (K d of 73 nM) (O’Connell et al, 2012). Competitive fluorescence anisotropy experiments with a longer GADD34 construct confirmed that GADD34 552-567 constitutes the complete PP1 binding domain (Fig.…”

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

“…[FW]. NCX1 sequences were also screened for the presence of the ⌽ 1 ⌽ 2 and/or arginine motifs, as defined by Choy et al (36). Lasergene (DNA Star, Madison, WI) was used for protein alignments.…”

“…Therefore, in addition to the RVXF PP1-binding motif, the partial ⌽ 1 ⌽ 2 motif and the conserved Arg-428 on NCX1 may form part of a high affinity PP1-binding site. The Arg motif in the PP1 nuclear targeting subunit (PNUTS)-PP1 holoenzyme has been shown to work as a substrate selectivity filter, by blocking access to the C-terminal binding groove (36). Therefore, dephosphorylation of pSer-68-PLM may not require binding to the C-terminal groove, and PP1 interaction sites such as SILK (55) and MyPhoNE (56) were not found in NCX1.…”

Biophysical Analysis of the N-Terminal Domain from the Human Protein Phosphatase 1 Nuclear Targeting Subunit PNUTS Suggests an Extended Transcription Factor TFIIS-Like Fold

“…We investigated this question through a preliminary NMR study. The 1 H, 15 N Heteronuclear Single Quantum Coherence (HSQC) spectrum of 15 N-labelled PNUTS showed high dispersion of well-defined backbone NH resonances with uniform intensity of the signals (Fig 4).…”

“…The PNUTS C-terminal region contains a zinc finger domain implying a possible interaction with nucleic acids [14], although this domain in PNUTS is not known to bind either RNA or DNA. The polypeptide region between the TFIIS LW and zinc finger domains in PNUTS is highly unstructured and plays a conserved role in binding to PP1 [15][16][17].…”