A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated Protein Kinase C regulation
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Pin1 as an essential prolyl cis/trans isomerase has attracted considerable attention because this enzyme family is implicated in cancer and neurodegenerative diseases. However, the requirement for its catalytic function remains a matter of dispute. The authors provide solid evidence that Pin1 modulates the activity of an important cell signaling kinase, Protein Kinase C, by a non-catalytic mechanism, acting as a chaperone to regulate the stability of this kinase.
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Abstract
Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P 3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and βII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of Pin1 complexed to the C-terminal tail of PKCβII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action.
Specifically, Pin1 adopts a compact conformation in which it engages two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, the latter being a non-canonical Pin1-interacting element. The structural information, combined with the results of extensive binding studies and in vivo experiments suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.
Impact statement
Integrated biophysical, structural, and in vivo approaches demonstrate a non-canonical and non-isomerizable binding motif-dependent mode of protein kinase C regulation by the peptidyl-prolyl isomerase Pin1 in mammalian cells.
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Author Response
Reviewer #1 (Public Review):
Reviewer 1: The structural part of this work is interesting, as it is the first structure of Pin1 with a ligand that bridges both domains. They might want to underline this - all other structures in the PDB have a single domain complex, but never both domains by a single longer peptide.
Done. We have highlighted the novelty of the structure in the abstract, introduction (page 5); and discussion (section “The Pin1-PKC interface is described by a novel bivalent interaction mode”, page 24).
Reviewer 1: I would however question the static representation of this structure - the 90{degree sign} kink in the peptide when complexed is probably one single snapshot, but I hardly believe the PPIase/WW domain orientation to be static. Unless the authors have additional information to stand by this …
Author Response
Reviewer #1 (Public Review):
Reviewer 1: The structural part of this work is interesting, as it is the first structure of Pin1 with a ligand that bridges both domains. They might want to underline this - all other structures in the PDB have a single domain complex, but never both domains by a single longer peptide.
Done. We have highlighted the novelty of the structure in the abstract, introduction (page 5); and discussion (section “The Pin1-PKC interface is described by a novel bivalent interaction mode”, page 24).
Reviewer 1: I would however question the static representation of this structure - the 90{degree sign} kink in the peptide when complexed is probably one single snapshot, but I hardly believe the PPIase/WW domain orientation to be static. Unless the authors have additional information to stand by this static structure, this point merits being commented on in the manuscript.
Done. Following the reviewer’s suggestion and to avoid the impression of “static” structure, we have added sentences that highlight the dynamic aspects of the complex evident from the entire ensemble representation of Figure 5-figure supplement 2:
Page 15 (Results):
“Of note, the linker region connecting the two domains retains its flexibility in the complex and confers some variability onto the relative positions of the WW and PPIase domains, as is evident from the ensemble representation of Figure 5-figure supplement 2. The complex exhibits novel structural features that distinguish it from all other structures of Pin1 complexes known to date. These features are highlighted in Fig. 6 using the lowest-energy structure of the ensemble.”
Page 24 (Discussion): “Moreover, the retention of linker flexibility in the Pin1::pV5bII complex suggests that Pin1 can potentially adopt minor “extended” states that would not be readily detectable by ensemble-averaged methods such as solution NMR.”
Also, in describing specific interactions in the section “Structural basis of the Pin1-PKCII C-term bivalent recognition mode”, we now note how many structures of the Pin1-pV5bII ensemble have those interactions.
Reviewer 1: I would like to point out to literature that described for example the non-canonical binding (Yeh ES, Lew BO & Means AR (2006) The loss of PIN1 deregulates cyclin E and sensitizes mouse embryo fibroblasts to genomic instability. J Biol Chem 281, 241-251. Pin1 recognizes cyclin E via a noncanonical pThr384- Gly385 motif [33] rather than the pThr380-Pro381 motif.). They mention briefly the absence of isomerase activity in similar TPP motifs, but this information might already come in the Results section.
Done. We have incorporated this information in the Discussion section, page 25 (last paragraph).
Reviewer 1: The expression levels of Pin1 and PKCa are amazingly linear (Fig 7A), but when they overexpress WT Pin1 in a KO line, with 3-4 times higher overexpression, the PKCa levels are hardly higher than in the original WT cell line.
We thank Reviewer 1 for raising this interesting point. Our simple interpretation of the data is that physiological expression of Pin1 in the cell model we use is a limiting factor in the stimulated PKCa degradation pathway, but that Pin1 is no longer a limiting factor at higher expression levels. We now include this point in the Discussion, page 26.
Reviewer 1: Also, the levels in the W34A/R68A/R69A (abolishing both WW and PPIase binding functions) are surprising, why would PKCa levels rise above the level found in the Pin1 KO cells?
This result remains a puzzle but, as we are including all independent biological replicates in the analysis, the data are the data. Moreover, by assessing the functional complementation data to the KO by two-tailed t-test (see last point below), this effect does not reach statistical significance. Nonetheless, as the result is reproducible, we now comment on this effect in the Results, page 21. One speculation is this triple mutant has dominant negative properties imposed on some limiting factor in PKCa degradation that are revealed in the absence of WT Pin1. Considerably more work needs to be done to settle this issue. However, in light of the fact that this result does not conflict with the structural/biochemical data (rather, it is consistent with it), we hope this positive response satisfies the Reviewer.
Reviewer 1: Finally, if even slight overexpression of the C113S catalytically inactive mutant leads to more efficient PKCa degradation than overexpression of the WT Pin1 (Figure 7C), it is hard to interpret. The conclusion that Pin1-mediated regulation of PKCa requires a bivalent interaction mode of Pin1 with PKCa independent of its catalytic activity do depend on these data, so they merit further analysis.
We certainly had no intention of concluding that the C113S catalytically inactive mutant is more efficient with regard to promoting PKCa degradation than overexpression of the WT Pin1. That overstates the data. We concede that our organization of the Pin1 rescue data in the original Fig 7C confused the issue, and that the original text also invited conclusions that overstate the result. To correct this problem, we reorganized Fig. 7C to simplify the presentation by comparing the complementation data to the KO. All statistical comparisons are now to the KO cell line (not to WT as before) and we employ the two-tailed t-test to compare the data. Statistical significance is attained only for reconstituted WT and C113S Pin1 expression. The text is also appropriately revised to describe the results clearly. We trust the Reviewer agrees that the C113S data are compelling and are consistent with a noncanonical (noncatalytic) mode of PKCa regulation by Pin1. This is a major point of Fig 7C as it links the structural/biochemical data to a cellular context.
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eLife assessment
Pin1 as an essential prolyl cis/trans isomerase has attracted considerable attention because this enzyme family is implicated in cancer and neurodegenerative diseases. However, the requirement for its catalytic function remains a matter of dispute. The authors provide solid evidence that Pin1 modulates the activity of an important cell signaling kinase, Protein Kinase C, by a non-catalytic mechanism, acting as a chaperone to regulate the stability of this kinase.
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Reviewer #1 (Public Review):
When writing a short review on the function of Pin1 some 15 years ago (Lippens et al., Febs J 2007), we concluded the introduction by the following sentence: "..., it seems that further analysis is required to determine whether binding or catalysis is the primary mechanism through which Pin1 affects cell cycle progression." In the present manuscript, the authors provide experimental evidence for the Pin1/PKC interaction that tips the balance towards interaction and not catalysis.
Their main data concern the interaction between the V5 domains of two PKC isoenzymes (alpha and betaII) and Pin1. This V5 domain can be further separated into a Turn Motif (TM) and a Hydrophobic Motif (HM), that both can be phosphorylated on specific positions. Phosphorylation in the TM occurs on a TPP motif, and in agreement with …
Reviewer #1 (Public Review):
When writing a short review on the function of Pin1 some 15 years ago (Lippens et al., Febs J 2007), we concluded the introduction by the following sentence: "..., it seems that further analysis is required to determine whether binding or catalysis is the primary mechanism through which Pin1 affects cell cycle progression." In the present manuscript, the authors provide experimental evidence for the Pin1/PKC interaction that tips the balance towards interaction and not catalysis.
Their main data concern the interaction between the V5 domains of two PKC isoenzymes (alpha and betaII) and Pin1. This V5 domain can be further separated into a Turn Motif (TM) and a Hydrophobic Motif (HM), that both can be phosphorylated on specific positions. Phosphorylation in the TM occurs on a TPP motif, and in agreement with previous results on the same motif in Tau, Pin1 cannot isomerize efficiently the TP amide bond when the residue following the proline is another proline. Phosphorylation of the HM is not proline directed but occurs on a serine flanked by 2 aromatic residues (FSF or FSY, according to the isoenzyme). They dissect in detail the interaction of both motifs with the WW and PPIase domains and conclude that the fully phosphorylated V5 peptide binds Pin1 in a directional mode, with the TM binding to the WW domain and the HM to the PPIase domain.
In the absence of crystals of the complex, they solve a structure by NMR, and use selectively labeled peptides (and probably a lot of NMR time) to obtain a structural model. Finally, they provide functional data by silencing/overepxressing Pin1 and inactive mutants (both at the level of its WW domain and the PPIase domain) in HEK293T cells and evaluating the PKCalpha homeostasis.
The structural part of this work is interesting, as it is the first structure of Pin1 with a ligand that bridges both domains. They might want to underline this - all other structures in the PDB have a single domain complex, but never both domains by a single longer peptide. I would however question the static representation of this structure - the 90{degree sign} kink in the peptide when complexed is probably one single snapshot, but I hardly believe the PPIase/WW domain orientation to be static. Unless the authors have additional information to stand by this static structure, this point merits being commented on in the manuscript.
I would like to point out to literature that described for example the non-canonical binding (Yeh ES, Lew BO & Means AR (2006) The loss of PIN1 deregulates cyclin E and sensitizes mouse embryo fibroblasts to genomic instability. J Biol Chem 281, 241-251. Pin1 recognizes cyclin E via a noncanonical pThr384- Gly385 motif [33] rather than the pThr380-Pro381 motif.). They mention briefly the absence of isomerase activity in similar TPP motifs, but this information might already come in the Results section.
The weakest part seems the in vivo data. Although this is not the main focus of this lab, there is some issues that could be addressed. The expression levels of Pin1 and PKCa are amazingly linear (Fig 7A), but when they overexpress WT Pin1 in a KO line, with 3-4 times higher overexpression, the PKCa levels are hardly higher than in the original WT cell line. Also, the levels in the W34A/R68A/R69A (abolishing both WW and PPIase binding functions) are surprising, why would PKCa levels rise above the level found in the Pin1 KO cells? Finally, if even slight overexpression of the C113S catalytically inactive mutant leads to more efficient PKCa degradation than overexpression of the WT Pin1 (Figure 7C), it is hard to interpret. The conclusion that Pin1-mediated regulation of PKCa requires a bivalent interaction mode of Pin1 with PKCa independent of its catalytic activity do depend on these data, so they merit further analysis.
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Reviewer #2 (Public Review):
Chen, Dixit et al. report on the first structure of a bivalent interaction between a natural interaction partner of Pin1: the C-terminal tail of PKC phosphorylated at two sites. The biggest strength of the paper is the impressive amount of NMR-based structural data that is sound and clearly reported. The authors strive to propose a novel non-catalytic mechanistic role for Pin1 that is supported by cell culture models and somewhat by the interaction assays, however, in my eyes, they fell short in proving their mechanistic hypothesis. Nevertheless, the potential ways Pin1 may modulate PKC's activity is nicely discussed.
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