PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

Reviewer #1 (Public review):

Summary:

In this manuscript the Treisman and colleagues address the question of how protein phosphatase 1 (PP1) regulatory subunits (or PP1-interacting protein (PIPs)) confer specificity on the PP1 catalytic subunit which by itself possesses little substrate specificity. In prior work the authors showed that the PIP Phactrs confers specificity by remodelling a hydrophobic groove immediately adjacent to the PP1 catalytic site through residues within the RVxF- ø ø -R-W string of Phactrs. Specifically, the residues proximal and including the 'W' of the RVxF- ø ø -R-W string remodel the hydrophobic groove. Other residues of the RVxF- ø ø -R-W string (i.e. the RVxF- ø ø -R) are not involved in this remodelling.

The authors suggest that the RVxF- ø ø -R-W string is a conserved feature of many PIPs including PNUTS, Neurabin/spinophilin and R15A. However, from a sequence and structural perspective, only the RVxF- ø ø -R- is conserved. The W is not conserved in most and in the R15A structure (PDB:7NZM) the Trp side chain points away from the hydrophobic channel - this could be a questionable interpretation due to model-building into the low-resolution cryo-EM map (4 A).

In this paper, the authors convincingly show that Neurabin confers substrate specificity through interactions of its PDZ domain with the PDZ domain-binding motif (PBM) of 4E-BP. They show the PBM motif is required for Neurabin to increase PP1 activity towards 4E-BP and a synthetic peptide modelled on 4E-BP and also a synthetic peptide based on IRSp53 with a PBM added. The PBM of 4E-BP1 confers high affinity binding to the Neurabin PDZ domain. A crystal structure of a PP1-4E-BP1 fusion with Neurabin shows that the PBM of 4E-BP interacts with the PDZ domain of Neurabin. No interactions of 4E-BP and the catalytic site of PP1 are observed. Cell biology work showed that Neurabin-PP1 regulates the TOR signalling pathway by dephosphorylating 4E-BPs.

Strengths:

This work demonstrates convincingly using a variety of cell biology, proteomics, biophysics and structural biology that the PP1 interacting protein Neurabin confers specificity on PP1 through an interaction of its PDZ domain with a PDZ-binding motif of 4E-BP1 proteins. Remodelling of the hydrophobic groove of the PP1 catalytic subunit is not involved in Neurabin-dependent substrate specificity, in contrast to how Phactrs confers specificity on PP1. The active site of the Neurabin/PP1 complex does not recognise residues in the vicinity of the phospho-residue, thus allowing for multiple phospho-sites on 4E-BP to be dephosphorylated by Neurabin/PP1. This contrasts with substrate specificity conferred by the Phactrs PIP that confers specificity of Phactrs/PP1 towards its substrates in a sequence-specific context by remodelling the hydrophobic groove immediately adjacent to the catalytic. The structural and biochemical insights are used to explore the role of Neurabin/PP1 in dephosphorylation 4E-BPs in vivo, showing that Neurabin/PP1 regulates the TOR signalling pathway, specifically mTORC1-dependent translational control.

Weaknesses:

The only weakness is the suggestion that a conserved RVxF- ø ø -R-W string exists in PIPs. The 'W' is not conserved in sequence and 3 dimensions in most of the PIPs discussed in this manuscript. The lack of conservation of the W would be consistent with the finding based on multiple PP1-PIP structures that apart from Phactrs, no other PIP appears to remodel the PP1 hydrophobic channel.

Reviewer #2 (Public review):

This manuscript explores the molecular mechanisms that are involved in substrate recognition by the PP1 phosphatase. The authors previously showed that the PP1 interacting protein (PPI), PhactrI, conferred substrate specificity by remodelling the PP1 hydrophobic substrate groove. In this work, the authors aimed to understand the key determinant of how other PIPs, Neurabin and Spinophilin, mediate substrate recognition.

The authors generated a few PP1-PIP fusion constructs, undertook TMT phosphoproteomics and validated their method using PP1-Phactr1/2/3/4 fusion constructs. Using this method, the authors identified phsophorylation sites controlled by PP1-Neurabin and focussed their work on 4E-BP1, thereby linking PP1-Neurabin to mTORC1 signalling. Upon validating that PP1-Neurabin dephosphorylates 4E-BP1, they determined that 4E-BP1 PBM binds to the PDZ domain of Neurabin with an affinity that was greater than 30-fold as compared to other substrates. PP1-Neurabin dephosphorylated 4E-BP1WT and IRSp53WT with a catalytic efficiency much greater than PP1 alone. However, PP1-Neurabin bound to 4E-BP1 and IRSp53 mutants lacking the Neurabin PDZ domain with a catalytic efficiency lesser than that observed with 4E-BP1WT. These results indicate the involvement of the PDZ domain in facilitating substrate recruitment by PP1-Neurabin. Interestingly, PP1-Phactr1 dephosphorylation of 4E-BP1 phenocopies PP1 alone, while PP1-Phactr1 dephosphorylates IRSp53 to a much higher extent than PP1 alone. These results highlight the importance of the PDZ domain and also shed light on how different PP1-PIP holoenzymes mediate substrate recognition using distinct mechanisms. The authors also show that the remodelling of the hydrophobic PP1 substrate groove which is essential for substrate recognition by PP1-Phactr1, was not required by PP1-Neurabin. Additionally, the authors also resolved the structure of a PP1-4E-BP1 fusion with the PDZ-containing C-terminal of Neurabin and observed that the Neurabin/PP1-4E-BP1 complex structure was oriented at 21{degree sign} to that in the unliganded Spinophilin/PP1 complex (resolved by Ragusa et al., 2010) owing to a slight bend in the C-terminal section that connects it to the RVxF-ΦΦ-R-W string. Since no interaction was observed with the remodelled PP1-Neurabin hydrophobic groove, the authors utilised AlphaFold3 to further answer this. They observed a high confidence of interaction between the groove and phosphorylated substrate and a low confidence of interaction between the groove and unphosphorylated substrate, thereby suggesting that the hydrophobic groove remodelling is not involved in PP1-Neurabin recognition and dephosphorylation of 4E-BP1.

In this work, the authors provide novel insights into how Neurabin depends on the interaction between its PDZ domain and PBM domains of potential substrates to mediate its recruitment by PP1. Additionally, they uncover a novel PP1-Neurabin substrate, 4E-BP1. They systematically employ phosphoproteomics, biochemical, and structural methods to investigate substrate specificity in a robust fashion. Furthermore, the authors also compare the interactions between PP1-Neurabin to 4E-BP1 and IRSp53 (PP1-Phactr1 substrate) with PP1-Phactr1, to showcase the specificity of the mode of action employed by these complexes in mediating substrate specificity. The authors employ an innovative PP1-PIP fusion strategy previously explored by Oberoi et al., 2016 and the authors themselves in Fedoryshchak et al., 2020. Although this method, allows for a more controlled investigation of the interactions between PP1-PIPs and its substrates, this methodology may not fully recapitulate the interactions that may occur in a physiological setting. This could potentially be overcome by studying the interactions of the full proteins using classical biochemical approaches in cell lines. Furthermore, the authors have substantially characterised the importance of the PDZ domain using their fusion constructs, however, I believe that further exploration into either structural or AlphaFold3 modelling of PBM domain substrate mutants, or a Neurabin PDZ-domain mutant might further strengthen this claim. Overall, the paper makes a substantial contribution to understanding substrate recognition and specificity in PP1-PIP complexes. The study's innovative methods, biological relevance, and mechanistic insights are strengths, but whether this mechanism occurs in a physiological context is unclear.

Reviewer #3 (Public review):

Protein Phosphatase 1 (PP1), a vital member of the PPP superfamily, drives most cellular serine/threonine dephosphorylation. Despite PP1's low intrinsic sequence preference, its substrate specificity is finely tuned by over 200 PP1-interacting proteins (PIPs), which employ short linear motifs (SLIMs) to bind specific PP1 surface regions. By targeting PP1 to cellular sites, modifying substrate grooves, or altering surface electrostatics, PIPs influence substrate specificity. Although many PIP-PP1-substrate interactions remain uncharacterized, the Phactr family of PIPs uniquely imposes sequence specificity at dephosphorylation sites through a conserved "RVxF-ΦΦ-R-W" motif. In Phactr1-PP1, this motif forms a hydrophobic pocket that favors substrates with hydrophobic residues at +4/+5 in acidic contexts (the "LLD motif"), a specificity that endures even in PP1-Phactr1 fusions. Neurabin/Spinophilin remodel PP1's hydrophobic groove in distinct ways, creating unique holoenzyme surfaces, though the impact on substrate specificity remains underexplored. This study investigates Neurabin/Spinophilin specificity via PDZ domain-driven interactions, showing that Neurabin/PP1 specificity is governed more by PDZ domain interactions than by substrate sequence, unlike Phactr1/PP1.

A significant strength of this work is the use of PP1-PIP fusion proteins to effectively model intact PP1•PIP holoenzymes by replicating the interactions that remodel the PP1 interface and confer site-specific substrate specificity. When combined with proteomic analyses to assess phospho-site depletion in mammalian cells, these fusions offer critical insights into holoenzyme specificity, revealing new candidate substrates for Neurabin and Spinophilin. The studies present compelling evidence that the PDZ domain of PP1-Neurabin directs its specificity, with the remodelled PP1 hydrophobic groove interactions having minimal impact. This mechanism is supported by structural analysis of the PP1-4E-BP1 substrate fusion bound to a Neurabin construct, highlighting the 4E-BP1/PDZ interaction. This work delivers crucial insights into PP1-PIP holoenzyme function, combining biochemical, proteomic, and structural approaches. It validates the PP1-PIP fusion protein model as a powerful tool, suggesting it may extend to studying additional holoenzymes. While an extremely useful model, it must be considered unlikely the PP1-PIP fusions fully recapitulate the specificity and regulation of the holoenzyme.

Author response:

We are very pleased to see these positive reviews of our preprint.

Reviewers 1 and 3 raise issues around PIP-PP1 interactions.

(1) Role of the “RVxF-ΦΦ-R-W string”

Most PIPs interact with the globular PP1 catalytic core through short linear interaction motifs (SLiMs) and Choy et al (PNAS 2014) previously showed that many PIPs interact with PP1 through conserved trio of SLiMs, RVxF-ΦΦ-R, which is also present in the Phactrs.

Previous structural analysis showed the trajectory of the PPP1R15A/B, Neurabin/Spinphilin (PPP1R9A/B), and PNUTS (PPP1R10) PIPs across the PP1 surface encompasses not only the RVxF-ΦΦ-R trio, but also additional sequences C-terminal to it (Chen et al, eLife, 2015). This extended trajectory is maintained in the Phactr1-PP1 complex (Fedoryshchak et al, eLife (2020). Based on structural alignment we proposed the existence of an additional hydrophobic “W” SLiM that interacts with the PP1 residues I133 and Y134.

The extended “RVxF-ΦΦ-R-W” interaction brings sequences C-terminal to the “W” SLiM into the vicinity of the hydrophobic groove that adjoins the PP1 catalytic centre. In the Phactr1/PP1 complex, these sequences remodel the groove, generating a novel pocket that facilitates sequence-specific substrate recognition.

This raises the possibility that sequences C-terminal to the extended “RVxF-ΦΦ-R-W string” in the other complexes also confer sequence-specific substrate recognition, and our study aims to test this hypothesis. Indeed, the hydrophobic groove structures of the Neurabin/Spinophilin/PP1 and Phactr1/PP1 complexes differ significantly (Ragusa et al, 2010; see Fedoryshchak et al 2020, Fig2 FigSupp1).

(2) Orientation of the W side chain

Reviewer 1 points out that in the substrate-bound PP1/PPP1R15A/Actin/eIF2 pre-dephosphorylation complex the W sidechain is inverted with respect to its orientation in PP1-PPP1R15B complex (Yan et al, NSMB 2021). The authors proposed that this may reflect the role of actin in assembly of the quaternary complex. This does not necessarily invalidate the notion that sequences C-terminal to the “W” motif might play a role in actin-independent substrate recognition, and we therefore consider our inclusion of the R15A/B fusions in our analysis to be reasonable.

(3) Conservation of W

The motif ‘W’ does not mandate tryptophan - Phactrs and PPP1R15A/B indeed have W at this position but Neurabin/spinophilin contain VDP, which makes similar interactions. Similarly the _“_RVxF” motifs in Phactr1, Neurabin/Spinophilin, PPP1R15A/B and PNUTS are LIRF, KIKF, KV(R/T)F and TVTW respectively.

In our revision, we will present comparisons of the differentially remodelled/modified PP1 hydrophobic groove in the various complexes, discuss the different orientations of the tryptophan in the previously published PPP1R15A/PP1 and PPP1R15B/PP1 structures. We will also address the other issues raised by the referees.