Báo cáo khoa học: Molecular mechanisms of the phospho-dependent prolyl cis ⁄ trans isomerase Pin1 docx

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MINIREVIEWMolecular mechanisms of the phospho-dependent prolylcis⁄trans isomerase Pin1G. Lippens1, I. Landrieu1and C. Smet21 CNRS UMR 8576 Unite´de Glycobiologie Structurale et Fonctionnelle, Universite´des Sciences et Technologies de Lille 1-59655,Villeneuve d’Ascq, France2 Institut de Recherche Interdisciplinaire, CNRS, Lille, FranceIntroductionProlyl cis trans isomerases form a special class ofenzymes in many respects. Most other enzymes changethe covalent chemistry of their substrates (be it throughcleavage, or addition removal of a phosphate, acetylor methyl group), leading to a product that is distin-guishable by mass spectrometry or immunochemistryfrom the incoming molecular entity. The modificationimposed by prolyl cis trans isomerases is not covalent,but merely conformational. One therefore encountersmany technical difficulties when attempting to observetheir molecular action in vivo and even in vitro. Experi-ments with isolated proteins have indicated the involve-ment of two major classes of isomerases, thecyclophilins (Cyps) and the FK506-binding proteins(FKBPs), in the protein-folding process, where the con-formational state of a prolyl peptide bond can be arate-limiting step [1]. The importance of ‘binding ver-sus catalysis’ remains an open issue in many cases [2],partly because tinkering with the enzymatic activitythrough mutations also leads to changes in interactionparameters [3]. Assessing the role of the isomerizationfunction in vivo is even harder. In one of the mostintensively studied cases, the interaction between CypAand the Gag protein derived from the HIV virus, theKeywordscell cycle; CKS subunit; dynamics; enzyme;interaction module; phosphorylation; Pin1;prolyl cis trans isomerase; proteindegradation; protein structureCorrespondenceG. Lippens, CNRS UMR 8576 Unite´deGlycobiologie Structurale et Fonctionnelle,Universite´des Sciences et Technologies deLille 1-59655, Villeneuve d’Ascq Cedex,FranceFax: +33 3 20 43 65 55Tel: +33 3 20 33 42 71E-mail: Guy.Lippens@univ-lille1.fr(Received 5 June 2007, revised 3 August2007, accepted 17 August 2007)doi:10.1111/j.1742-4658.2007.06057.xSince its discovery 10 years ago, Pin1, a prolyl cis trans isomerase essentialfor cell cycle progression, has been implicated in a large number of molecu-lar processes related to human diseases, including cancer and Alzheimer’sdisease. Pin1 is made up of a WW interaction domain and a C-terminalcatalytic subunit, and several high-resolution structures are available thathave helped define its function. The enzymatic activity of Pin1 towardsshort 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 thatexamine its cis trans isomerase function using full-length protein substrates.The interpretation of this research has been further complicated by theobservation that many of its pSer Thr-Pro substrate motifs are located innatively unstructured regions of polypeptides, and are characterized byminor populations of the cis conformer. Finally, we review the data on thepossibility of alternative modes of substrate binding and the complex rolethat Pin1 plays in the degradation of its substrates. After considering theavailable work, it seems that further analysis is required to determinewhether binding or catalysis is the primary mechanism through which Pin1affects cell cycle progression.AbbreviationsAPP, 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 5211mere interaction of CypA with Gag might protect thevirion from an as yet poorly identified cellular integra-tion factor [4]. Whether the prolyl cis trans isomeraseactivity detected in vitro of CypA on Pro90 in the full-length Gag protein [5] plays a role in the viralrestriction process in vivo remains an open question. Inthe case of the Itk kinase, the cis trans isomerizationcatalyzed 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 regionbetween two SH3 domains of the Crp adaptor proteindetermines the autoinhibition of the domains [7]. Theselatter examples provide a structural basis for themanner in which prolyl cis trans isomerization couldact as a conformational switch in biological processes.Beyond the difficulties of characterizing this confor-mational switch in a cellular context and even more ina living organism, another problem with the molecularcharacterization of the prolyl-isomerase enzymes istheir redundancy in the cell. Indeed, a model organismsuch as yeast has as many as eight Cyps and fourFKBPs, and even knocking down all of them does notlead to a clear phenotype under normal conditions [8].The discovery of Ess1, a novel Saccharomyces cerevisiaeprolyl cis⁄ trans isomerase [9] that proved essential forcell division, was therefore highly relevant. Its humanhomolog was identified as a protein interacting withthe NIMA kinase during the G1⁄ S cell cycle stage, andwas called Pin1 for this reason [10]. Since its initialidentification, Pin1 has attracted a great deal ofattention, as the enzyme seems to be implicated invarious human diseases, ranging from cancer andneurodegenerative diseases to inflammation. Ratherthan adding to the list of excellent general reviews onPin1 [11–14] or to those reviews that emphasize its rolein cellular processes related to diseases [15–17], wefocus here on what is known about the molecularmechanisms of the enzyme action. Most importantly,we want to critically review the evidence that Pin1would or would not act as a prolyl cis trans isomerase.Molecular mechanisms of Pin1 actionThe initial X-ray structure of the catalytic domaincomplexed 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 66and 77 (human Pin1 numbering) is involved in bindingthe phosphate moiety of the substrate (Fig. 1). Thisloop is flexible, as suggested by its different conforma-tion in a second crystallographic structure, where thecatalytic domain was not complexed to a substratepeptide [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 aclosed 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 ofthis loop on the 100 ls to 1 ms time scale [22], but thecrystal structure of the Candida albicans Ess1 revealeda closed loop even in the absence of substrate [23]. Thedynamic character of this loop was recently shown byNMR relaxation dispersion measurements on thehuman enzyme during substrate binding [24] andFig. 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 residuesof 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 complementedby a sulfate ion (light blue). A poly(ethylene glycol) molecule (notshown) is sequestered between the catalytic domain and the WWdomain. Bottom: The complex between Pin1 and a Pol II CTDphospho-peptide (Protein Data Bank code: 1F8A [5]). The active siteloop adopts a more extended conformation, and the peptide inter-acts with the WW domain mainly through the phospho-Thr Promoiety.Molecular mechanisms of Pin1 G. Lippens et al.5212 FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBScatalysis [25]. Interestingly, in the case of CypA, thedynamic character of its active site was found to beessential for its catalytic function [3]. The fact thatPin1 can compensate for the lack of Ess1 in yeast wasexploited to further investigate the functional role ofthe different Pin1 residues. Behrsin et al. [26] appliedunigenic evolution, where the capacity of a randomlymutated pin1 gene to compensate for Ess1 in a yeaststrain devoid of the wild-type ess1 gene is screened.They showed that, in particular, Lys63 is essential forthe anchoring function of the phosphorylated sub-strate, whereby the K63A mutant affects the catalyticefficiency because of weaker binding. The two argi-nines in the loop, Arg68 and Arg69, would provide nomore than a single positive charge [26].A second important structural feature of the initialX-ray structure of Pin1 was the spatial proximity ofCys113 to the Ala-Pro bond (Fig. 1) [18]. Althoughthis initially suggested a catalytic mechanism through anucleophilic attack on the substrate carbonyl carbonby the Scof Cys113, the fact that the C113D mutantremains functional calls this mechanism into question[26]. The Cys113 residue, through its unusually lowpKavalue, might indeed maintain an overall electro-negative environment that is crucial for destabilizingthe 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 otherprolyl cis trans isomerases, with only its loop regionselecting for Pro residues preceded by a negativecharge. In the A. thaliana Pin1 homolog, we did notobserve significant chemical shift changes for theequivalent Cys70 upon saturation of the catalyticdomain with several phosphopeptides [22]. In yeast,the equivalent C120R mutant of Ess1 only preventedgrowth at the higher temperature of 37 °C [27].Finally, the chemically and structurally similar bindingpockets of Pin1 and FKBP and the structural resem-blance between their respective high-affinity inhibitors[28] further underscore the similarity between Pin1 andthe other peptide prolyl isomerases (PPIases).A second crystallographic structure (Fig. 1) with thePin1 WW domain complexed to a phospho-peptidederived from the C-terminal domain of polymerase II(Pol II CTD) indicated the trans conformation of theprolyl bond following the phosphorylated Ser [19].NMR spectroscopy confirmed that the cis conformerin a Cdc25-derived peptide could not interact with theWW 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 WWdomain and a phosphorylated peptide (V-pT668-P-E-E)derived from the APP cytoplasmic domain was probedby NMR spectroscopy [30]. For this latter phospho-peptide, the15N-labeled Glu670 was the primary probeof the interaction. Interestingly, the correlation peakscorresponding to both the trans and cis conformers ofthe pThr668-Pro prolyl bond were found to shift uponinteraction with the single WW domain, with the larg-est shift for the cis form. In an earlier study, the samegroup examined in great detail the structure of bothcis and trans conformers of the same peptide by NMRspectroscopy [31]. The local structure of the cis con-former of this peptide is characterized by a hydrogenbond between the amide proton of Val667 and theGlu671 side chain carboxyl group. This particularmotif might be recognized by the Pin1 WW domain,or, alternatively, the presence of two glutamate resi-dues downstream of the proline could lead the WWdomain to read the cis form in a reversed manner. Theinitial screen for Pin1 substrates, which identified it asa phospho-dependent prolyl cis trans isomerase,indicated that, at least for some peptides, using aglutamate (but not aspartate) rather than a phospho-Ser Thr group before the critical proline did notdecrease Pin1 enzymatic efficiency [32]. However, wefound that a Tau mutant carrying multiple Glu-Promotifs did not significantly interact with the Pin1WW domain (G. Lippens, I. Landrieu, C. Smet andR. Brandt, unpublished results). Further structuralcharacterization of the complex between the Pin1 WWdomain and the amyloid peptide will be necessary, andmight form a novel starting point for the developmentof WW domain inhibitors.Even more surprising is the recent finding that Pin1could recognize cyclin E via a noncanonical pThr384-Gly385 motif [33] rather than the pThr380-Pro381motif. The main argument was that the latterpThr380-Pro381 motif is buried in the yeast Cdc4molecular surface that was determined by X-ray crys-tallography [34]. In this structure of the peptide–CDC4complex, however, Pin1 is missing, whereas in thestudy on cyclin E degradation, Pin1 was brought in bythe phospho-cyclin E and not by the CDC4a compo-nent of the final complex (Fig. 2) [35]. Therefore, theoutcome of the molecular competition between Pin1and CDC4 for the same phosphorylated motif is notclear, and still leaves open the possibility that Pin1could interfere with the cyclin E–CDC4 interface. Pin1was proposed to isomerize the peptide bond betweenPro381 and Pro382. The concomittant structural rear-rangement would cause cyclin E to approach thedistant E2 ligase of a different SKP Cullin F-boxprotein (SCF)Cdc4ccomplex. During our work on theG. Lippens et al. Molecular mechanisms of Pin1FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS 5213neuronal Tau protein, we studied by NMR spectroscopya peptide containing the sequence (LP) pT217(PPT),which matches the optimal L I-L I P-pT-P CDC4phospho-degron sequence [36], and moreover stronglyresembles the cyclin E peptide (LL)pT380(PPQ) thatwas crystallized at the CDC4 interface [34]. In the Taupeptide, 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 notshow detectable levels of cis conformer (Fig. 3), and thisdipeptide did not interact with Pin1. Moreover, whereasa catalytic amount of Pin1 greatly enhanced the isomeri-zation rate for the pThr212-Pro213 bond, we did notdetect any Pin1-catalyzed exchange peak for the neigh-boring pThr217-Pro218 bond in the same peptide(Fig. 3). The mechanistic details of how CDC4a couldoverrule 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 substratesConcerning the function of Pin1, a first intriguingobservation is that many of its substrates meet the cri-teria for the recently identified class of intrinsicallyunfolded proteins. Although unstructured regions inproteins or fully unstructured proteins have beenknown since the beginning of structural biology, onlyrecently have they been identified as a true class ofproteins that challenge the sequence–structure–functionparadigm [38]. Phosphorylation in such regions is arecurring theme, and transforms them into effectiveanchoring points for novel components in the multi-protein complexes that govern the fate of the cell [39].Unfortunately, for many of these complexes, we donot yet know how these intrinsically unstructureddomains exert their molecular function. For Cdc25, forexample, one of Pin1’s most extensively studied sub-strates, it is not clear how phosphorylation at theThr48 Thr67 sites regulates the phosphatase activity;the same is true for Tau, a neuronal protein involvedin tubulin polymerization. Tau loses its ability to poly-merize tubulin after phosphorylation at the Thr231position, and Pin1 can restore this function [40].Understanding the binding of Tau to tubulin and itsmodulation by phosphorylation will be necessarybefore we can evaluate the role of Pin1 in this complexprocess. Structural studies on peptides derived fromthe two proteins, Cdc25 and Tau, have shown thatonly a low percentage of the prolines downstream ofthe phospho-Thr Ser residues adopt the cis confor-mation, typically 3–10% [41,42], and at least for theFig. 2. Schematic view of the parallelbetween Pin1 and CKS in protein degrada-tion. Top: Model of the SCFCDC4E3 ligaseand the role of Pin1. Pin1 is brought in withthe cyclin E substrate, through interactionwith the pSer384-Pro motif. When the com-plex contains the CDC4a isoform, the iso-merase activity of Pin1 leads to cyclin Edissociation, and allows association with anovel CDC4c complex, where ubiquitin addi-tion would occur (adapted from Brazin et al.[6]). Bottom: Model of the SCFSkp2E3 ligase, where CKS1 associates with theSkp2 protein and hence forms an integralpart of the E3 ligase that recognizes itsphosphorylated p27kip1substrate (adaptedfrom 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 FEBSfull-length Tau protein, our preliminary NMR dataindicate 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 bestabilized by the surrounding (folded) structure. It wassuggested that multiple phosphorylations might createa locally strained conformation [43], favoring thecis conformation of one or more prolines, but NMRstudies have as yet not detected a prevalent cis con-former in peptides carrying two or more of thesemotifs [37,44]. Because many of the Pin1-recognizedphosphorylation motifs are in unstructured regions, wethus can reasonably expect conformational heterogene-ity at the level of its substrate pSer Thr-Pro prolylbonds, with the cis form being the less common one.The predominant trans conformation at the pThr ⁄Ser-Pro bonds combined with the Pin1-mediatedincrease in immunoreactivity of the MPM-2 antibody[45] towards its substrates suggests that the antibodycould recognize the cis conformer of the pThr-Proprolyl bond in substrates such as phospho-Cdc25 orphospho-Tau. Indeed, Pin1 as an isolated enzymewould merely lower the energetic barrier separatingboth conformations without changing their relativepopulations. However, when coupled to anothermolecular process that is conformer dependent (suchas protease sensitivity or antibody recognition),isomerization could catalyze changes in the relativetPro213Fig. 3. NOESY spectrum of the triply phosphorylated SRSRpT212PpS214LPpT217PPTR peptide of Tau. The cis conformation for the Pro219 isbelow the limit of detection. Upon addition of a catalytic amount of Pin1, enhanced cis trans isomerization of the pThr212-Pro213 peptidebond 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 at4.42 p.p.m.), whereas in the same spectrum, the equivalent exchange peak for the pThr217-Pro218 bond (which should be in the green boxat 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 Pin1FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS 5215populations, as one of the forms would continuouslydisappear from the pool of free peptides. Structuralcharacterization of the MPM-2 antibody with aphospho-peptide substrate might be very informativein 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 crystalstructure of a cyclin-dependent kinase (CDK)2 ⁄cyclin A kinase [47] revealed the existence of the transconformation of the Ser-Pro bond in the peptide sub-strate. Similarly, the major proline-directed phospha-tase PP2A recognizes and dephosphorylates only thetrans conformer of its phosphorylated peptide sub-strate [46]. Pin1 could thus enhance the molecularfunction of this phosphatase by speeding up thecis–trans interconversion rate, as was proposed forboth Tau and Cdc25 [46]. Indeed, for the small phos-pho-peptides that are used in most studies, a pool ofmainly 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 mainlycis conformers takes less time in the presence of Pin1[46], and this unambiguously involves Pin1’s prolylcis trans isomerase activity. For the in vitro phosphor-ylated full-length Tau and CDC25, however, no effortwas made to prepare a similar large pool of cis con-formers. We found at the peptide level that thepThr231-Pro bond of Tau is mainly in the trans form[37]. Extending our combined in vitro phosphorylationand NMR spectroscopy of full-length Tau fromprotein kinase A [49] to a CDK kinase, we havepreliminary data that the pThr231-Pro bond in thisCDK-phosphorylated full-length Tau also adopts thetrans conformation to a major extent (I. Landrieu,L. Amniai and G. Lippens, unpublished results).Isomerization from cis to trans hence cannot beinvoked any more as the sole mechanism promotingthe accelerated dephosphorylation by PP2A. Pin1might in an as yet unidentified manner favor the inter-action between PP2A and its phosphorylated sub-strates, and hence stimulate their dephosphorylationwithout necessarily requiring its catalytic prolylcis trans isomerase activity.Role of Pin1 in protein stabilityRegulation of protein degradation seems to be an all-important role for Pin1, and as such, a remarkableparallel with the CKS (CDK subunit) family can beestablished. CKS targets the activated CDK complextowards phosphorylated substrates such as CDC25,and is as such essential for the entry of Xenopus laevisegg extracts into mitosis [50]. Well characterized struc-turally, CKS proteins can be found in different confor-mations with regard to their last b-strand. Foldedback on itself in a monomeric compact form [51], theC-terminal b-strand in the swapped dimer is locked ina second monomer [52]. The structural differencesbetween compact monomer and swapped dimer aremainly limited to the conformation of the Glu-Prodipeptide in the hinge region between the last b-strandand the core of CKS. The crystal structure of the com-plex of CKS with CDK2 cyclin A clearly showed thatonly the monomeric, compact CKS could bind to theCDK subunit [53], the swapped dimer giving rise toimportant steric clashes preventing the interaction.Pin1 antagonizes the stimulatory role of CKS in mito-sis entry [50], and we initially assumed that this wasthrough a direct interaction with the Glu-Pro hingemotif and subsequent conformational transitionbetween both structural forms. Experiments proved thehypothesis wrong, as we did not obtain any evidenceof 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 phosphorylatedpeptide between the Pin1 WW domain and the CKSbinding module [54], and showed that the interactionsurfaces were quite similar in terms of amino acidcomposition and structure (Fig. 4).As well as a comparable role in regulating the phos-phorylation state of CDC25 or other substrates, theparallel between the WW and CKS interactiondomains can be drawn further when considering theirrespective implications for the ubiquitination processdirecting proteins towards degradation. Indeed, humanCks1 was identified as an important factor in theSCFSkp2ubiquitin E3 ligase. SCFSkp2plays a role inthe degradation of the CDK inhibitor p27kip1in lateG1phase after phosphorylation on its Thr187 residue[55,56]. CKS1 interacts both with Skp2 and with ap27kip1-derived pThr187 peptide [57], and hence playsthe role of an adaptor protein that is an integral partof the E3 ligase (Fig. 2). Similarly, Schizosaccharo-myces pombe p13suc1binds 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 interactionof cyclin E with the SCFCdc4complex. It stimulatesubiquitin addition to cyclin E by the SCFCdc4cE3 ligase, after releasing the same cyclin E from thecomplex with SCFCdc4a[33]. Pin1 would, however, notbe part of the initial SCFCdc4a ccomplex, but wouldbe brought in by the substrate itself (Fig. 2). Theenhanced cyclin E degradation by Pin1 contrasts withMolecular mechanisms of Pin1 G. Lippens et al.5216 FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBSits role in cyclin D stability. Pin1 overexpression wasfound to stabilize this cyclin at both the protein andmRNA levels [59]. A similar stabilizing effect wasdescribed for Emi1, an inhibitor of the APC complexrequired to induce S-phase and M-phase entry by driv-ing cyclin A and cyclin B accumulation. Pin1 preventsits association with SCFbTrcp, and hence stabilizesEmi1 when this latter is phosphorylated on its Ser10-Pro motif [60]. In yet another case, the Drosophilahomolog Dodo was shown to facilitate the degradationof the transcription factor CF2 [61]. Finally, the neuro-nal Tau protein also contains the aforementionedphospho-degron sequence (LPT217PPLSP). AlthoughPin1 has as yet not been implicated directly in its deg-radation, phosphorylated Tau is targeted for proteaso-mal degradation through the E3 ubiquitin ligaseCHIP, complexed to an Hsc70 moiety [62], whereCHIP would selectively ubiquitinate (natively)unfolded proteins by collaborating with the molecularchaperone [63]. If this CDC4 phospho-degron sequenceon Tau is physiologically phosphorylated and recog-nized by the ubiquitin ligase, this would close the circleof Pin1’s preference for unfolded substrates. In anycase, the WW domain of Pin1 is an excellent exampleof the complex roles played by protein–protein inter-action modules [64], and whether the catalytic domainis a second interaction domain or rather a genuineenzyme awaits further elucidation.Functional overlap of Pin1 with otherprolyl cis⁄trans isomerases?Regulation of the transcription machinery was earlydescribed as an essential function of Ess1 [65,66]. Itsinteraction with the numerous YSPTSPS heptapeptiderepeats of the Pol II CTD [67,68], although of weakR12W29S13R99R30Q78W82Fig. 4. Molecular surface (left) and ribbondiagrams (right) of the Pin1 WW domain(top) or CKS (p13Suc1) interaction domain.Color coding is according to the chemicalshift changes observed with a CDC25-derived phosphopeptide. The residueswhose chemical shift is most affected uponpeptide binding (red) are Arg12 and Trp29for the WW domain, and Arg30, Gln78 andTrp82 for p13Suc1. Blue color indicates theabsence of chemical shift changes.G. Lippens et al. Molecular mechanisms of Pin1FEBS Journal 274 (2007) 5211–5222 ª 2007 The Authors Journal compilation ª 2007 FEBS 5217affinity when these are tested as isolated peptidesin vitro [69], could lead to the assembly of differentmolecular complexes when mRNA is transcribed. Ess1would regulate the phosphorylation state of the CTD,and the yeast Fcp1 phosphatase plays a similar role asPP2A in this process. However, conflicting results havebeen reported, with Ess1 either stimulating CTDdephosphorylation by Fcp1 [70] or inhibiting this sameprocess [71,72]. Differences in the exact nature of thesubstrate (full-length Pol II or the isolated CTDdomain) might explain this discrepancy [72]. Fcp1 wasfound to more effectively dephosphorylated the pSer5position [70], whereas the WW domain of Pin1 bindswith slightly better affinity the pSer2 position [69]. Thissuggests a sequential mechanism, with preliminarybinding of the WW domain to the pSer2-Pro motifand subsequent isomerization by the catalytic domainat the Ser5 position. The yeast Ess1 protein, however,prefers both to bind and isomerize pSer5-Pro overpSer2-Pro [73]. A role of Pin1 in transcriptional regu-lation through the (de)stabilization of complexesformed at the CTD of Pol II was suggested by theobservation that Ess1 and the yeast Nedd4 ubiquitinligase Rsp5 compete for the largest subunit of theRNA Pol II, possibly through their respective WWdomains [74].Using a number of ess1 temperature-sensitivemutants, two groups unexpectedly discovered thatCypA can functionally replace Ess1 [66,75]. Bothprolyl cis trans isomerases could catalyze protein con-formational changes essential for the assembly and oractivity of the Sin3–Rpd3 histone deacetylase complex,but not through binding and or catalytic actiontowards the same peptide motifs [76]. Ess1 interactsdirectly with the Sin3 component, and downregulatesin this manner the deacetylase activity of Rpd3,whereas CypA would drive the equilibrium towardsthe formation of a Sin3–Rpd3–Sap30 complex.Whereas this provides the first evidence of crosstalkamong different PPIase families, the observation of abasal 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 inflammatoryresponse towards antiviral double-stranded RNA. Pin1interacts with the pSer339-Pro340 motif on interferonregulatory 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-Promotif of IRF-3 is interrupted by a Leucine, despitebeing in one of the best conserved regions betweenboth transcription factors. Pin1 no longer recognizesthis motif, and no IRF-4 regulation by Pin1 has beenreported. However, IRF-4 is regulated by FKBP52, amember of the FK5060-binding prolyl cis trans isome-rases [79]. The tetratricopeptide repeats of FKBP52mediate the interaction with IRF-4 and hence might bethe equivalent of Pin1’s WW domain, whereas itscatalytic domain could induce structural changes inthe N-terminal proline-rich domain of IRF-4. In thesame field of immunology, Pin1 also regulates theproduction of such proinflammatory cytokines asgranulocyte–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. Pin1interferes with this interaction, and hence stimulatescytokine 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 beenextensively explored. Phosphorylation at the Ser16 resi-due in the WW domain prevents its interaction withphosphorylated substrates [82], and thereby partiallyinactivates the function of Pin1. Polo-like kinase-1-mediated phosphorylation, on the other hand, stabi-lizes Pin1 by inhibiting its ubiquitination [83]. Pin1stability and regulated activity itself hence intervene inits complex relationship with phosphorylation.Conclusions and perspectivesThe list of potential substrates of Pin1 seems never-ending, and one wonders how one single protein couldbe involved in such a variety of cellular processes. Wecan only propose some possibilities. First, the WWdomain is clearly not very selective with regard to itsmolecular targets. Its binding pocket mostly sequestersthe phosphate moiety and the proline side chain(Figs 1 and 4), whereas other amino acids around thismotif only marginally contribute to the binding affin-ity. When studying phosphorylated peptides derivedfrom Tau, we found that the best binder was actuallythe dipeptide pThr-Pro, with a KDof 100 lm [84]. Par-allel studies with Pol II CTD-derived peptides haveshown 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, aslong as it is in a rather unstructured region. Second,the weak affinity precludes the formation of stablecomplexes, and leaves room for the Pin1 molecule tosample a large number of potential substrates duringits half-life. Finally, the group of S. Hanes, who wasthe first to describe the Sacch. cerevisiae parvulin Ess1Molecular 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 copynumbers in the cell, at least under normal growth con-ditions. Indeed, they found that although wild-typeyeast cells contain on the order of 200 000 moleculesof Ess1 per cell, a level lower than 400 molecules percell is sufficient for growth, leaving plenty of Ess1molecules for many substrates [73]. Only under certainconditions of stress does the large pool of Ess1 seemessential 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 certaincases might be more complex [85]. Certainly, Pin1overexpression correlates strongly with poor prognosisin a variety of cancers, and these clinical data cannotbe overlooked [86]. Nonetheless, Pin1 also stabilizesp53 and increases its transcriptional activity, which isessential to counteract oncogenesis [87,88]. At the cel-lular level, its role in cyclin E and c-Myc degradationor 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 themouse lines might lead to different outcomes for thesame mutation, making the construction of a singlecoherent framework more problematic [89]. As is thecase for p53, where the relative levels of protein andits inhibitors activators can lead to subtle but signifi-cant differences between results in cell and animalmodels [90], careful analysis of in vivo models will beneeded to validate all data acquired in vitro or in cellmodels before drawing conclusions on Pin1’s role incancer. Finally, in the context of Alzheimer’s disease,Pin1 was shown to have a beneficial role, as it restoresthe capacity of Cdc2-phosphorylated Tau to polymer-ize tubulin into microtubules [40]. However, the tan-gles of Tau and other amyloid species, althoughcharacteristic in Alzheimer’s disease and correlatingwell with cognitive decline, are now seen in a newlight by the scientific community. Over a period of10 years, they have shifted from being an importantcause of the disease towards consituting a cellulardefense against the toxic oligomeric but soluble spe-cies, although these latter still await clear identifica-tion [91]. Could Pin1 be intended primarily as aprotective mechanism, recognizing aberrant phosphor-ylated Ser Thr-Pro motifs and targeting them throughinteraction or conformational change towards dephos-porylation, degradation, or aggregation? Is prolylcis trans isomerization required for this function? andcould this mechanism go awry in certain diseases suchas cancer? 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