MINIREVIEW Molecular mechanisms of the phospho-dependent prolyl cis ⁄ trans isomerase Pin1 G. Lippens 1 , I. Landrieu 1 and C. Smet 2 1 CNRS UMR 8576 Unite ´ de Glycobiologie Structurale et Fonctionnelle, Universite ´ des Sciences et Technologies de Lille 1-59655, Villeneuve d’Ascq, France 2 Institut de Recherche Interdisciplinaire, CNRS, Lille, France Introduction Prolyl cis ⁄ trans isomerases form a special class of enzymes in many respects. Most other enzymes change the covalent chemistry of their substrates (be it through cleavage, or addition ⁄ removal of a phosphate, acetyl or methyl group), leading to a product that is distin- guishable by mass spectrometry or immunochemistry from the incoming molecular entity. The modification imposed by prolyl cis ⁄ trans isomerases is not covalent, but merely conformational. One therefore encounters many technical difficulties when attempting to observe their molecular action in vivo and even in vitro. Experi- ments with isolated proteins have indicated the involve- ment of two major classes of isomerases, the cyclophilins (Cyps) and the FK506-binding proteins (FKBPs), in the protein-folding process, where the con- formational state of a prolyl peptide bond can be a rate-limiting step [1]. The importance of ‘binding ver- sus catalysis’ remains an open issue in many cases [2], partly because tinkering with the enzymatic activity through mutations also leads to changes in interaction parameters [3]. Assessing the role of the isomerization function in vivo is even harder. In one of the most intensively studied cases, the interaction between CypA and the Gag protein derived from the HIV virus, the Keywords cell cycle; CKS subunit; dynamics; enzyme; interaction module; phosphorylation; Pin1; prolyl cis ⁄ trans isomerase; protein degradation; protein structure Correspondence G. Lippens, CNRS UMR 8576 Unite ´ de Glycobiologie Structurale et Fonctionnelle, Universite ´ des Sciences et Technologies de Lille 1-59655, Villeneuve d’Ascq Cedex, France Fax: +33 3 20 43 65 55 Tel: +33 3 20 33 42 71 E-mail: Guy.Lippens@univ-lille1.fr (Received 5 June 2007, revised 3 August 2007, accepted 17 August 2007) doi:10.1111/j.1742-4658.2007.06057.x Since its discovery 10 years ago, Pin1, a prolyl cis ⁄ trans isomerase essential for cell cycle progression, has been implicated in a large number of molecu- lar processes related to human diseases, including cancer and Alzheimer’s disease. Pin1 is made up of a WW interaction domain and a C-terminal catalytic subunit, and several high-resolution structures are available that have helped define its function. The enzymatic activity of Pin1 towards short peptides containing the pSer ⁄ Thr-Pro motif has been well docu- mented, and we discuss the available evidence for the molecular mecha- nisms of its isomerase activity. We further focus on those studies that examine its cis ⁄ trans isomerase function using full-length protein substrates. The interpretation of this research has been further complicated by the observation that many of its pSer ⁄ Thr-Pro substrate motifs are located in natively unstructured regions of polypeptides, and are characterized by minor populations of the cis conformer. Finally, we review the data on the possibility of alternative modes of substrate binding and the complex role that Pin1 plays in the degradation of its substrates. After considering the available work, it seems that further analysis is required to determine whether binding or catalysis is the primary mechanism through which Pin1 affects cell cycle progression. Abbreviations APP, amyloid precursor protein; CDK, cyclin-dependent kinase; CKS, cyclin-dependent kinase subunit; CTD, C-terminal domain; Cyp, cyclophilin; FKBP, FK506-binding protein; IRF, interferon regulatory factor; Pol II, polymerase II. FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS 5211 mere interaction of CypA with Gag might protect the virion from an as yet poorly identified cellular integra- tion factor [4]. Whether the prolyl cis ⁄ trans isomerase activity detected in vitro of CypA on Pro90 in the full- length Gag protein [5] plays a role in the viral restriction process in vivo remains an open question. In the case of the Itk kinase, the cis ⁄ trans isomerization catalyzed by CypA was shown to generate novel inter- action surfaces for two distinct molecular partners [6], and the conformation of a proline in the linker region between two SH3 domains of the Crp adaptor protein determines the autoinhibition of the domains [7]. These latter examples provide a structural basis for the manner in which prolyl cis ⁄ trans isomerization could act as a conformational switch in biological processes. Beyond the difficulties of characterizing this confor- mational switch in a cellular context and even more in a living organism, another problem with the molecular characterization of the prolyl-isomerase enzymes is their redundancy in the cell. Indeed, a model organism such as yeast has as many as eight Cyps and four FKBPs, and even knocking down all of them does not lead to a clear phenotype under normal conditions [8]. The discovery of Ess1, a novel Saccharomyces cerevisiae prolyl cis⁄ trans isomerase [9] that proved essential for cell division, was therefore highly relevant. Its human homolog was identified as a protein interacting with the NIMA kinase during the G 1 ⁄ S cell cycle stage, and was called Pin1 for this reason [10]. Since its initial identification, Pin1 has attracted a great deal of attention, as the enzyme seems to be implicated in various human diseases, ranging from cancer and neurodegenerative diseases to inflammation. Rather than adding to the list of excellent general reviews on Pin1 [11–14] or to those reviews that emphasize its role in cellular processes related to diseases [15–17], we focus here on what is known about the molecular mechanisms of the enzyme action. Most importantly, we want to critically review the evidence that Pin1 would or would not act as a prolyl cis ⁄ trans isomerase. Molecular mechanisms of Pin1 action The initial X-ray structure of the catalytic domain complexed with an Ala-Pro dipeptide and a sulfate ion [18] suggested an important role for two structural ele- ments in catalysis. First, the loop between residues 66 and 77 (human Pin1 numbering) is involved in binding the phosphate moiety of the substrate (Fig. 1). This loop is flexible, as suggested by its different conforma- tion in a second crystallographic structure, where the catalytic domain was not complexed to a substrate peptide [19]. Heteronuclear NOE data on human Pin1, however, indicated only a limited decrease in the flexi- bility of this loop on peptide binding [20] or even a closed conformation in the absence of substrate [21]. Our NMR data on the Arabidopsis thaliana analog, Pin1At, showed severe line broadening in the equiva- lent stretch, giving weight to the dynamic character of this loop on the 100 ls to 1 ms time scale [22], but the crystal structure of the Candida albicans Ess1 revealed a closed loop even in the absence of substrate [23]. The dynamic character of this loop was recently shown by NMR relaxation dispersion measurements on the human enzyme during substrate binding [24] and Fig. 1. Top: Molecular structure of Pin1 (Protein Data Bank code: 1Pin [4]), showing the catalytic domain (green), the WW domain (red), and the linker region (yellow). The positively charged residues of the active site loop (Lys63, Arg68, and Arg69) are in blue, whereas the presumed catalytic Cys113 is in pink. The Ala-Pro pep- tide in the active site is indicated by sticks, and is complemented by a sulfate ion (light blue). A poly(ethylene glycol) molecule (not shown) is sequestered between the catalytic domain and the WW domain. Bottom: The complex between Pin1 and a Pol II CTD phospho-peptide (Protein Data Bank code: 1F8A [5]). The active site loop adopts a more extended conformation, and the peptide inter- acts with the WW domain mainly through the phospho-Thr ⁄ Pro moiety. Molecular mechanisms of Pin1 G. Lippens et al. 5212 FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS catalysis [25]. Interestingly, in the case of CypA, the dynamic character of its active site was found to be essential for its catalytic function [3]. The fact that Pin1 can compensate for the lack of Ess1 in yeast was exploited to further investigate the functional role of the different Pin1 residues. Behrsin et al. [26] applied unigenic evolution, where the capacity of a randomly mutated pin1 gene to compensate for Ess1 in a yeast strain devoid of the wild-type ess1 gene is screened. They showed that, in particular, Lys63 is essential for the anchoring function of the phosphorylated sub- strate, whereby the K63A mutant affects the catalytic efficiency because of weaker binding. The two argi- nines in the loop, Arg68 and Arg69, would provide no more than a single positive charge [26]. A second important structural feature of the initial X-ray structure of Pin1 was the spatial proximity of Cys113 to the Ala-Pro bond (Fig. 1) [18]. Although this initially suggested a catalytic mechanism through a nucleophilic attack on the substrate carbonyl carbon by the S c of Cys113, the fact that the C113D mutant remains functional calls this mechanism into question [26]. The Cys113 residue, through its unusually low pK a value, might indeed maintain an overall electro- negative environment that is crucial for destabilizing the double bond character of the pThr-Pro bond [26]. Further evidence is available for the catalytic mecha- nism of Pin1 resembling more closely that of the other prolyl cis ⁄ trans isomerases, with only its loop region selecting for Pro residues preceded by a negative charge. In the A. thaliana Pin1 homolog, we did not observe significant chemical shift changes for the equivalent Cys70 upon saturation of the catalytic domain with several phosphopeptides [22]. In yeast, the equivalent C120R mutant of Ess1 only prevented growth at the higher temperature of 37 °C [27]. Finally, the chemically and structurally similar binding pockets of Pin1 and FKBP and the structural resem- blance between their respective high-affinity inhibitors [28] further underscore the similarity between Pin1 and the other peptide prolyl isomerases (PPIases). A second crystallographic structure (Fig. 1) with the Pin1 WW domain complexed to a phospho-peptide derived from the C-terminal domain of polymerase II (Pol II CTD) indicated the trans conformation of the prolyl bond following the phosphorylated Ser [19]. NMR spectroscopy confirmed that the cis conformer in a Cdc25-derived peptide could not interact with the WW domain [29]. Despite these findings, recently pre- sented data hint at other potential binding modes. First, when the role of Pin1 in amyloid precursor pro- tein (APP) processing and ensuing b-amyloid produc- tion was studied, the interaction between the WW domain and a phosphorylated peptide (V-pT 668 -P-E-E) derived from the APP cytoplasmic domain was probed by NMR spectroscopy [30]. For this latter phospho- peptide, the 15 N-labeled Glu670 was the primary probe of the interaction. Interestingly, the correlation peaks corresponding to both the trans and cis conformers of the pThr668-Pro prolyl bond were found to shift upon interaction with the single WW domain, with the larg- est shift for the cis form. In an earlier study, the same group examined in great detail the structure of both cis and trans conformers of the same peptide by NMR spectroscopy [31]. The local structure of the cis con- former of this peptide is characterized by a hydrogen bond between the amide proton of Val667 and the Glu671 side chain carboxyl group. This particular motif might be recognized by the Pin1 WW domain, or, alternatively, the presence of two glutamate resi- dues downstream of the proline could lead the WW domain to read the cis form in a reversed manner. The initial screen for Pin1 substrates, which identified it as a phospho-dependent prolyl cis ⁄ trans isomerase, indicated that, at least for some peptides, using a glutamate (but not aspartate) rather than a phospho- Ser ⁄ Thr group before the critical proline did not decrease Pin1 enzymatic efficiency [32]. However, we found that a Tau mutant carrying multiple Glu-Pro motifs did not significantly interact with the Pin1 WW domain (G. Lippens, I. Landrieu, C. Smet and R. Brandt, unpublished results). Further structural characterization of the complex between the Pin1 WW domain and the amyloid peptide will be necessary, and might form a novel starting point for the development of WW domain inhibitors. Even more surprising is the recent finding that Pin1 could recognize cyclin E via a noncanonical pThr384- Gly385 motif [33] rather than the pThr380-Pro381 motif. The main argument was that the latter pThr380-Pro381 motif is buried in the yeast Cdc4 molecular surface that was determined by X-ray crys- tallography [34]. In this structure of the peptide–CDC4 complex, however, Pin1 is missing, whereas in the study on cyclin E degradation, Pin1 was brought in by the phospho-cyclin E and not by the CDC4a compo- nent of the final complex (Fig. 2) [35]. Therefore, the outcome of the molecular competition between Pin1 and CDC4 for the same phosphorylated motif is not clear, and still leaves open the possibility that Pin1 could interfere with the cyclin E–CDC4 interface. Pin1 was proposed to isomerize the peptide bond between Pro381 and Pro382. The concomittant structural rear- rangement would cause cyclin E to approach the distant E2 ligase of a different SKP ⁄ Cullin ⁄ F-box protein (SCF) Cdc4c complex. During our work on the G. Lippens et al. Molecular mechanisms of Pin1 FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS 5213 neuronal Tau protein, we studied by NMR spectroscopy a peptide containing the sequence (LP) pT 217 (PPT), which matches the optimal L ⁄ I-L ⁄ I ⁄ P-pT-P CDC4 phospho-degron sequence [36], and moreover strongly resembles the cyclin E peptide (LL)pT380(PPQ) that was crystallized at the CDC4 interface [34]. In the Tau peptide, despite significant proportions of cis conform- ers for the three proline residues preceded by a phos- pho-Thr [37], the Pro218-Pro219 peptide bond did not show detectable levels of cis conformer (Fig. 3), and this dipeptide did not interact with Pin1. Moreover, whereas a catalytic amount of Pin1 greatly enhanced the isomeri- zation rate for the pThr212-Pro213 bond, we did not detect any Pin1-catalyzed exchange peak for the neigh- boring pThr217-Pro218 bond in the same peptide (Fig. 3). The mechanistic details of how CDC4a could overrule the strict phospho-Ser ⁄ Thr dependence of Pin1, be it for binding or for prolyl cis⁄ trans isomerization, therefore await further structural elucidation. Structural features of Pin1 substrates Concerning the function of Pin1, a first intriguing observation is that many of its substrates meet the cri- teria for the recently identified class of intrinsically unfolded proteins. Although unstructured regions in proteins or fully unstructured proteins have been known since the beginning of structural biology, only recently have they been identified as a true class of proteins that challenge the sequence–structure–function paradigm [38]. Phosphorylation in such regions is a recurring theme, and transforms them into effective anchoring points for novel components in the multi- protein complexes that govern the fate of the cell [39]. Unfortunately, for many of these complexes, we do not yet know how these intrinsically unstructured domains exert their molecular function. For Cdc25, for example, one of Pin1’s most extensively studied sub- strates, it is not clear how phosphorylation at the Thr48 ⁄ Thr67 sites regulates the phosphatase activity; the same is true for Tau, a neuronal protein involved in tubulin polymerization. Tau loses its ability to poly- merize tubulin after phosphorylation at the Thr231 position, and Pin1 can restore this function [40]. Understanding the binding of Tau to tubulin and its modulation by phosphorylation will be necessary before we can evaluate the role of Pin1 in this complex process. Structural studies on peptides derived from the two proteins, Cdc25 and Tau, have shown that only a low percentage of the prolines downstream of the phospho-Thr ⁄ Ser residues adopt the cis confor- mation, typically 3–10% [41,42], and at least for the Fig. 2. Schematic view of the parallel between Pin1 and CKS in protein degrada- tion. Top: Model of the SCF CDC4 E3 ligase and the role of Pin1. Pin1 is brought in with the cyclin E substrate, through interaction with the pSer384-Pro motif. When the com- plex contains the CDC4a isoform, the iso- merase activity of Pin1 leads to cyclin E dissociation, and allows association with a novel CDC4c complex, where ubiquitin addi- tion would occur (adapted from Brazin et al. [6]). Bottom: Model of the SCF Skp2 E3 ligase, where CKS1 associates with the Skp2 protein and hence forms an integral part of the E3 ligase that recognizes its phosphorylated p27 kip1 substrate (adapted from Sarkar et al. [7] and Dolinski [8]). Molecular mechanisms of Pin1 G. Lippens et al. 5214 FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS full-length Tau protein, our preliminary NMR data indicate that the same low population of cis conform- ers is found in the full-length protein. For the cis con- former to be the predominant one, it needs to be stabilized by the surrounding (folded) structure. It was suggested that multiple phosphorylations might create a locally strained conformation [43], favoring the cis conformation of one or more prolines, but NMR studies have as yet not detected a prevalent cis con- former in peptides carrying two or more of these motifs [37,44]. Because many of the Pin1-recognized phosphorylation motifs are in unstructured regions, we thus can reasonably expect conformational heterogene- ity at the level of its substrate pSer ⁄ Thr-Pro prolyl bonds, with the cis form being the less common one. The predominant trans conformation at the pThr ⁄ Ser-Pro bonds combined with the Pin1-mediated increase in immunoreactivity of the MPM-2 antibody [45] towards its substrates suggests that the antibody could recognize the cis conformer of the pThr-Pro prolyl bond in substrates such as phospho-Cdc25 or phospho-Tau. Indeed, Pin1 as an isolated enzyme would merely lower the energetic barrier separating both conformations without changing their relative populations. However, when coupled to another molecular process that is conformer dependent (such as protease sensitivity or antibody recognition), isomerization could catalyze changes in the relative tPro213 Fig. 3. NOESY spectrum of the triply phosphorylated SRSRpT 212 PpS 214 LPpT 217 PPTR peptide of Tau. The cis conformation for the Pro219 is below the limit of detection. Upon addition of a catalytic amount of Pin1, enhanced cis ⁄ trans isomerization of the pThr212-Pro213 peptide bond leads to an additional red peak (red box, peak connecting the trans Pro213 Ha resonance at 4.94 p.p.m. and the cis Pro213 Ha at 4.42 p.p.m.), whereas in the same spectrum, the equivalent exchange peak for the pThr217-Pro218 bond (which should be in the green box at 4.72 p.p.m., trans Pro218 Ha, and 5.20 p.p.m., cis Pro218 Ha) was not detected. G. Lippens et al. Molecular mechanisms of Pin1 FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS 5215 populations, as one of the forms would continuously disappear from the pool of free peptides. Structural characterization of the MPM-2 antibody with a phospho-peptide substrate might be very informative in this aspect, and shed further light on this issue. Another well-studied process for which Pin1’s cata- lytic activity has been put forward is dephosphoryla- tion by the PP2A phosphatase [46]. The crystal structure of a cyclin-dependent kinase (CDK)2 ⁄ cyclin A kinase [47] revealed the existence of the trans conformation of the Ser-Pro bond in the peptide sub- strate. Similarly, the major proline-directed phospha- tase PP2A recognizes and dephosphorylates only the trans conformer of its phosphorylated peptide sub- strate [46]. Pin1 could thus enhance the molecular function of this phosphatase by speeding up the cis–trans interconversion rate, as was proposed for both Tau and Cdc25 [46]. Indeed, for the small phos- pho-peptides that are used in most studies, a pool of mainly cis conformers can be obtained by lithium ⁄ trifluoroethanol stabilization and⁄ or selective proteo- lytic cleavage of the major pool of trans conformers [46,48]. The dephosphorylation of this pool of mainly cis conformers takes less time in the presence of Pin1 [46], and this unambiguously involves Pin1’s prolyl cis ⁄ trans isomerase activity. For the in vitro phosphor- ylated full-length Tau and CDC25, however, no effort was made to prepare a similar large pool of cis con- formers. We found at the peptide level that the pThr231-Pro bond of Tau is mainly in the trans form [37]. Extending our combined in vitro phosphorylation and NMR spectroscopy of full-length Tau from protein kinase A [49] to a CDK kinase, we have preliminary data that the pThr231-Pro bond in this CDK-phosphorylated full-length Tau also adopts the trans conformation to a major extent (I. Landrieu, L. Amniai and G. Lippens, unpublished results). Isomerization from cis to trans hence cannot be invoked any more as the sole mechanism promoting the accelerated dephosphorylation by PP2A. Pin1 might in an as yet unidentified manner favor the inter- action between PP2A and its phosphorylated sub- strates, and hence stimulate their dephosphorylation without necessarily requiring its catalytic prolyl cis ⁄ trans isomerase activity. Role of Pin1 in protein stability Regulation of protein degradation seems to be an all- important role for Pin1, and as such, a remarkable parallel with the CKS (CDK subunit) family can be established. CKS targets the activated CDK complex towards phosphorylated substrates such as CDC25, and is as such essential for the entry of Xenopus laevis egg extracts into mitosis [50]. Well characterized struc- turally, CKS proteins can be found in different confor- mations with regard to their last b-strand. Folded back on itself in a monomeric compact form [51], the C-terminal b-strand in the swapped dimer is locked in a second monomer [52]. The structural differences between compact monomer and swapped dimer are mainly limited to the conformation of the Glu-Pro dipeptide in the hinge region between the last b-strand and the core of CKS. The crystal structure of the com- plex of CKS with CDK2 ⁄ cyclin A clearly showed that only the monomeric, compact CKS could bind to the CDK subunit [53], the swapped dimer giving rise to important steric clashes preventing the interaction. Pin1 antagonizes the stimulatory role of CKS in mito- sis entry [50], and we initially assumed that this was through a direct interaction with the Glu-Pro hinge motif and subsequent conformational transition between both structural forms. Experiments proved the hypothesis wrong, as we did not obtain any evidence of an interaction between Pin1 and this Glu-Pro dipep- tide of CKS. We did, however, show in vitro competi- tion for the same Cdc25-derived phosphorylated peptide between the Pin1 WW domain and the CKS binding module [54], and showed that the interaction surfaces were quite similar in terms of amino acid composition and structure (Fig. 4). As well as a comparable role in regulating the phos- phorylation state of CDC25 or other substrates, the parallel between the WW and CKS interaction domains can be drawn further when considering their respective implications for the ubiquitination process directing proteins towards degradation. Indeed, human Cks1 was identified as an important factor in the SCF Skp2 ubiquitin E3 ligase. SCF Skp2 plays a role in the degradation of the CDK inhibitor p27 kip1 in late G 1 phase after phosphorylation on its Thr187 residue [55,56]. CKS1 interacts both with Skp2 and with a p27 kip1 -derived pThr187 peptide [57], and hence plays the role of an adaptor protein that is an integral part of the E3 ligase (Fig. 2). Similarly, Schizosaccharo- myces pombe p13 suc1 binds to the activated anaphase- promoting complex (APC) ⁄ cyclosome [58]. Pin1 intervenes in a complex manner with the degra- dation of cyclin E through regulation of the interaction of cyclin E with the SCF Cdc4 complex. It stimulates ubiquitin addition to cyclin E by the SCF Cdc4c E3 ligase, after releasing the same cyclin E from the complex with SCF Cdc4a [33]. Pin1 would, however, not be part of the initial SCF Cdc4a ⁄ c complex, but would be brought in by the substrate itself (Fig. 2). The enhanced cyclin E degradation by Pin1 contrasts with Molecular mechanisms of Pin1 G. Lippens et al. 5216 FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS its role in cyclin D stability. Pin1 overexpression was found to stabilize this cyclin at both the protein and mRNA levels [59]. A similar stabilizing effect was described for Emi1, an inhibitor of the APC complex required to induce S-phase and M-phase entry by driv- ing cyclin A and cyclin B accumulation. Pin1 prevents its association with SCF bTrcp , and hence stabilizes Emi1 when this latter is phosphorylated on its Ser10- Pro motif [60]. In yet another case, the Drosophila homolog Dodo was shown to facilitate the degradation of the transcription factor CF2 [61]. Finally, the neuro- nal Tau protein also contains the aforementioned phospho-degron sequence (LPT 217 PPLSP). Although Pin1 has as yet not been implicated directly in its deg- radation, phosphorylated Tau is targeted for proteaso- mal degradation through the E3 ubiquitin ligase CHIP, complexed to an Hsc70 moiety [62], where CHIP would selectively ubiquitinate (natively) unfolded proteins by collaborating with the molecular chaperone [63]. If this CDC4 phospho-degron sequence on Tau is physiologically phosphorylated and recog- nized by the ubiquitin ligase, this would close the circle of Pin1’s preference for unfolded substrates. In any case, the WW domain of Pin1 is an excellent example of the complex roles played by protein–protein inter- action modules [64], and whether the catalytic domain is a second interaction domain or rather a genuine enzyme awaits further elucidation. Functional overlap of Pin1 with other prolyl cis ⁄ trans isomerases? Regulation of the transcription machinery was early described as an essential function of Ess1 [65,66]. Its interaction with the numerous YSPTSPS heptapeptide repeats of the Pol II CTD [67,68], although of weak R12 W29 S13 R99 R30 Q78 W82 Fig. 4. Molecular surface (left) and ribbon diagrams (right) of the Pin1 WW domain (top) or CKS (p13 Suc1 ) interaction domain. Color coding is according to the chemical shift changes observed with a CDC25- derived phosphopeptide. The residues whose chemical shift is most affected upon peptide binding (red) are Arg12 and Trp29 for the WW domain, and Arg30, Gln78 and Trp82 for p13 Suc1 . Blue color indicates the absence of chemical shift changes. G. Lippens et al. Molecular mechanisms of Pin1 FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS 5217 affinity when these are tested as isolated peptides in vitro [69], could lead to the assembly of different molecular complexes when mRNA is transcribed. Ess1 would regulate the phosphorylation state of the CTD, and the yeast Fcp1 phosphatase plays a similar role as PP2A in this process. However, conflicting results have been reported, with Ess1 either stimulating CTD dephosphorylation by Fcp1 [70] or inhibiting this same process [71,72]. Differences in the exact nature of the substrate (full-length Pol II or the isolated CTD domain) might explain this discrepancy [72]. Fcp1 was found to more effectively dephosphorylated the pSer5 position [70], whereas the WW domain of Pin1 binds with slightly better affinity the pSer2 position [69]. This suggests a sequential mechanism, with preliminary binding of the WW domain to the pSer2-Pro motif and subsequent isomerization by the catalytic domain at the Ser5 position. The yeast Ess1 protein, however, prefers both to bind and isomerize pSer5-Pro over pSer2-Pro [73]. A role of Pin1 in transcriptional regu- lation through the (de)stabilization of complexes formed at the CTD of Pol II was suggested by the observation that Ess1 and the yeast Nedd4 ubiquitin ligase Rsp5 compete for the largest subunit of the RNA Pol II, possibly through their respective WW domains [74]. Using a number of ess1 temperature-sensitive mutants, two groups unexpectedly discovered that CypA can functionally replace Ess1 [66,75]. Both prolyl cis ⁄ trans isomerases could catalyze protein con- formational changes essential for the assembly and ⁄ or activity of the Sin3–Rpd3 histone deacetylase complex, but not through binding and ⁄ or catalytic action towards the same peptide motifs [76]. Ess1 interacts directly with the Sin3 component, and downregulates in this manner the deacetylase activity of Rpd3, whereas CypA would drive the equilibrium towards the formation of a Sin3–Rpd3–Sap30 complex. Whereas this provides the first evidence of crosstalk among different PPIase families, the observation of a basal enzymatic activity towards phosphorylated sub- strates in cell lysates from Pin1 – ⁄ – knockout mice [77] hints at a direct functional overlap. An intriguing com- plementarity is further found in the inflammatory response towards antiviral double-stranded RNA. Pin1 interacts with the pSer339-Pro340 motif on interferon regulatory factor (IRF)-3, leading ultimately to its deg- radation and ensuing impaired production of inter- feron-b [78]. In the homologous IRF-4, the Ser-Pro motif of IRF-3 is interrupted by a Leucine, despite being in one of the best conserved regions between both transcription factors. Pin1 no longer recognizes this motif, and no IRF-4 regulation by Pin1 has been reported. However, IRF-4 is regulated by FKBP52, a member of the FK5060-binding prolyl cis ⁄ trans isome- rases [79]. The tetratricopeptide repeats of FKBP52 mediate the interaction with IRF-4 and hence might be the equivalent of Pin1’s WW domain, whereas its catalytic domain could induce structural changes in the N-terminal proline-rich domain of IRF-4. In the same field of immunology, Pin1 also regulates the production of such proinflammatory cytokines as granulocyte–macrophage colony-stimulating factor [80], interleukin-2 and interferon-c [81]. The ARE-con- taining cytokine mRNAs interact with AUF1 factors, and this interaction targets them for degradation. Pin1 interferes with this interaction, and hence stimulates cytokine production. In the resting eosinophils, how- ever, Pin1’s activity is suppressed through phosphory- lation on one or more of its own Ser ⁄ Thr residues. Regulation of Pin1 function through phosphoryla- tion is indeed an important topic that has not been extensively explored. Phosphorylation at the Ser16 resi- due in the WW domain prevents its interaction with phosphorylated substrates [82], and thereby partially inactivates the function of Pin1. Polo-like kinase-1- mediated phosphorylation, on the other hand, stabi- lizes Pin1 by inhibiting its ubiquitination [83]. Pin1 stability and regulated activity itself hence intervene in its complex relationship with phosphorylation. Conclusions and perspectives The list of potential substrates of Pin1 seems never- ending, and one wonders how one single protein could be involved in such a variety of cellular processes. We can only propose some possibilities. First, the WW domain is clearly not very selective with regard to its molecular targets. Its binding pocket mostly sequesters the phosphate moiety and the proline side chain (Figs 1 and 4), whereas other amino acids around this motif only marginally contribute to the binding affin- ity. When studying phosphorylated peptides derived from Tau, we found that the best binder was actually the dipeptide pThr-Pro, with a K D of 100 lm [84]. Par- allel studies with Pol II CTD-derived peptides have shown similar results, with only a two-fold better affin- ity for the pSer5-Pro motive over the pSer2-Pro motif [66]. We thus believe that the WW domain will recog- nize in vitro basically any pThr ⁄ pSer-Pro pattern, as long as it is in a rather unstructured region. Second, the weak affinity precludes the formation of stable complexes, and leaves room for the Pin1 molecule to sample a large number of potential substrates during its half-life. Finally, the group of S. Hanes, who was the first to describe the Sacch. cerevisiae parvulin Ess1 Molecular mechanisms of Pin1 G. Lippens et al. 5218 FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS [9], has described a large redundancy of protein copy numbers in the cell, at least under normal growth con- ditions. Indeed, they found that although wild-type yeast cells contain on the order of 200 000 molecules of Ess1 per cell, a level lower than 400 molecules per cell is sufficient for growth, leaving plenty of Ess1 molecules for many substrates [73]. Only under certain conditions of stress does the large pool of Ess1 seem essential for growth, which probably brings us proba- bly to the situation existing in human diseases. Whereas the detrimental role of Pin1 in human dis- eases, and especially cancer, seemed at first to be evi- dent, recent findings suggest that the picture in certain cases might be more complex [85]. Certainly, Pin1 overexpression correlates strongly with poor prognosis in a variety of cancers, and these clinical data cannot be overlooked [86]. Nonetheless, Pin1 also stabilizes p53 and increases its transcriptional activity, which is essential to counteract oncogenesis [87,88]. At the cel- lular level, its role in cyclin E and c-Myc degradation or Emi1 stabilization would equally point to a protec- tive role as a conditional tumor suppressor. Yeh et al. have pointed out that the genetic background of the mouse lines might lead to different outcomes for the same mutation, making the construction of a single coherent framework more problematic [89]. As is the case for p53, where the relative levels of protein and its inhibitors ⁄ activators can lead to subtle but signifi- cant differences between results in cell and animal models [90], careful analysis of in vivo models will be needed to validate all data acquired in vitro or in cell models before drawing conclusions on Pin1’s role in cancer. Finally, in the context of Alzheimer’s disease, Pin1 was shown to have a beneficial role, as it restores the capacity of Cdc2-phosphorylated Tau to polymer- ize tubulin into microtubules [40]. However, the tan- gles of Tau and other amyloid species, although characteristic in Alzheimer’s disease and correlating well with cognitive decline, are now seen in a new light by the scientific community. Over a period of 10 years, they have shifted from being an important cause of the disease towards consituting a cellular defense against the toxic oligomeric but soluble spe- cies, although these latter still await clear identifica- tion [91]. Could Pin1 be intended primarily as a protective mechanism, recognizing aberrant phosphor- ylated Ser ⁄ Thr-Pro motifs and targeting them through interaction or conformational change towards dephos- porylation, degradation, or aggregation? 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