Kun Ping Lu

The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein

One of the neuropathological hallmarks of Alzheimers disease is the neurofibrillary tangle, which contains paired helical filaments (PHFs) composed of the microtubule-associated protein tau,. Tau is hyperphosphorylated in PHFs,, and phosphorylation of tau abolishes its ability to bind microtubules and promote microtubule assembly,. Restoring the function of phosphorylated tau might prevent or reverse PHF formation in Alzheimers disease. Phosphorylation on a serine or threonine that precedes proline (pS/T–P) alters the rate of prolyl isomerization and creates a binding site for the WW domain of the prolyl isomerase Pin1 (refs 8,9,10,11, 12,13,14). Pin1 specifically isomerizes pS/T–P bonds and regulates the function of mitotic phosphoproteins,. Here we show that Pin1 binds to only one pT–P motif in tau and co-purifies with PHFs, resulting in depletion of soluble Pin1 in the brains of Alzheimers disease patients. Pin1 can restore the ability of phosphorylated tau to bind microtubules and promote microtubule assembly in vitro. As depletion of Pin1 induces mitotic arrest and apoptotic cell death, sequestration of Pin1 into PHFs may contribute to neuronal death. These findings provide a new insight into the pathogenesis of Alzheimers disease.

Structural and Functional Analysis of the Mitotic Rotamase Pin1 Suggests Substrate Recognition Is Phosphorylation Dependent

The human rotamase or peptidyl-prolyl cis-trans isomerase Pin1 is a conserved mitotic regulator essential for the G2/M transition of the eukaryotic cell cycle. We report the 1.35 A crystal structure of Pin1 complexed with an AlaPro dipeptide and the initial characterization of Pin1s functional properties. The crystallographic structure as well as pH titration studies and mutagenesis of an active site cysteine suggest a catalytic mechanism that includes general acid-base and covalent catalysis during peptide bond isomerization. Pin1 displays a preference for an acidic residue N-terminal to the isomerized proline bond due to interaction of this acidic side chain with a basic cluster. This raises the possibility of phosphorylation-mediated control of Pin1-substrate interactions in cell cycle regulation.

Structural basis for phosphoserine-proline recognition by group IV WW domains.

Pin1 contains an N-terminal WW domain and a C-terminal peptidyl-prolyl cis-trans isomerase (PPIase) domain connected by a flexible linker. To address the energetic and structural basis for WW domain recognition of phosphoserine (P.Ser)/phosphothreonine (P.Thr)- proline containing proteins, we report the energetic and structural analysis of a Pin1–phosphopeptide complex. The X-ray crystal structure of Pin1 bound to a doubly phosphorylated peptide (Tyr-P.Ser-Pro-Thr-P.Ser-Pro-Ser) representing a heptad repeat of the RNA polymerase II large subunits C-terminal domain (CTD), reveals the residues involved in the recognition of a single P.Ser side chain, the rings of two prolines, and the backbone of the CTD peptide. The side chains of neighboring Arg and Ser residues along with a backbone amide contribute to recognition of P.Ser. The lack of widespread conservation of the Arg and Ser residues responsible for P.Ser recognition in the WW domain family suggests that only a subset of WW domains can bind P.Ser-Pro in a similar fashion to that of Pin1.

Regulation of NF-κB Signaling by Pin1-Dependent Prolyl Isomerization and Ubiquitin-Mediated Proteolysis of p65/RelA

The transcription factor NF-kappaB is activated by the degradation of its inhibitor IkappaBalpha, resulting in its nuclear translocation. However, the mechanism by which nuclear NF-kappaB is subsequently regulated is not clear. Here we demonstrate that NF-kappaB function is regulated by Pin1-mediated prolyl isomerization and ubiquitin-mediated proteolysis of its p65/RelA subunit. Upon cytokine treatment, Pin1 binds to the pThr254-Pro motif in p65 and inhibits p65 binding to IkappaBalpha, resulting in increased nuclear accumulation and protein stability of p65 and enhanced NF-kappaB activity. Significantly, Pin1-deficient mice and cells are refractory to NF-kappaB activation by cytokine signals. Moreover, the stability of p65 is controlled by ubiquitin-mediated proteolysis, facilitated by a cytokine signal inhibitor, SOCS-1, acting as a ubiquitin ligase. These findings uncover two important mechanisms of regulating NF-kappaB signaling and offer new insight into the pathogenesis and treatment of some human diseases such as cancers.

A Structural Basis for Substrate Specificities of Protein Ser/Thr Kinases: Primary Sequence Preference of Casein Kinases I and II, NIMA, Phosphorylase Kinase, Calmodulin- Dependent Kinase II, CDK5, and Erk1

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease

Protein phosphorylation regulates many cellular processes by causing changes in protein conformation. The prolyl isomerase PIN1 has been identified as a regulator of phosphorylation signalling that catalyses the conversion of specific phosphorylated motifs between the two completely distinct conformations in a subset of proteins. PIN1 regulates diverse cellular processes, including growth-signal responses, cell-cycle progression, cellular stress responses, neuronal function and immune responses. In line with the diverse physiological roles of PIN1, it has also been linked to several diseases that include cancer, Alzheimers disease and asthma, and thus it might represent a novel therapeutic target.

Pin1-Dependent Prolyl Isomerization Regulates Dephosphorylation of Cdc25C and Tau Proteins

The reversible protein phosphorylation on serine or threonine residues that precede proline (pSer/Thr-Pro) is a key signaling mechanism for the control of various cellular processes, including cell division. The pSer/Thr-Pro moiety in peptides exists in the two completely distinct cis and trans conformations whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. Previous results suggest that Pin1 might regulate the conformation and dephosphorylation of its substrates. However, it is not known whether phosphorylation-dependent prolyl isomerization occurs in a native protein and/or affects dephosphorylation of pSer/Thr-Pro motifs. Here we show that the major Pro-directed phosphatase PP2A is conformation-specific and effectively dephosphorylates only the trans pSer/Thr-Pro isomer. Furthermore, Pin1 catalyzes prolyl isomerization of specific pSer/Thr-Pro motifs both in Cdc25C and tau to facilitate their dephosphorylation by PP2A. Moreover, Pin1 and PP2A show reciprocal genetic interactions, and prolyl isomerase activity of Pin1 is essential for cell division in vivo. Thus, phosphorylation-specific prolyl isomerization catalyzed by Pin1 is a novel mechanism essential for regulating dephosphorylation of certain pSer/Thr-Pro motifs.

Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1.

Phosphorylation on serines or threonines preceding proline (Ser/Thr‐Pro) is a major signaling mechanism. The conformation of a subset of phosphorylated Ser/Thr‐Pro motifs is regulated by the prolyl isomerase Pin1. Inhibition of Pin1 induces apoptosis and may also contribute to neuronal death in Alzheimers disease. However, little is known about the role of Pin1 in cancer or in modulating transcription factor activity. Here we report that Pin1 is strikingly overexpressed in human breast cancers, and that its levels correlate with cyclin D1 levels in tumors. Overexpression of Pin1 increases cellular cyclin D1 protein and activates its promoter. Furthermore, Pin1 binds c‐Jun that is phosphorylated on Ser63/73‐Pro motifs by activated JNK or oncogenic Ras. Moreover, Pin1 cooperates with either activated Ras or JNK to increase transcriptional activity of c‐Jun towards the cyclin D1 promoter. Thus, Pin1 is up‐regulated in human tumors and cooperates with Ras signaling in increasing c‐Jun transcriptional activity towards cyclin D1. Given the crucial roles of Ras signaling and cyclin D1 overexpression in oncogenesis, our results suggest that overexpression of Pin1 may promote tumor growth.

The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-β production

Neuropathological hallmarks of Alzheimers disease are neurofibrillary tangles composed of tau and neuritic plaques comprising amyloid-β peptides (Aβ) derived from amyloid precursor protein (APP), but their exact relationship remains elusive. Phosphorylation of tau and APP on certain serine or threonine residues preceding proline affects tangle formation and Aβ production in vitro. Phosphorylated Ser/Thr-Pro motifs in peptides can exist in cis or trans conformations, the conversion of which is catalysed by the Pin1 prolyl isomerase. Pin1 has been proposed to regulate protein function by accelerating conformational changes, but such activity has never been visualized and the biological and pathological significance of Pin1 substrate conformations is unknown. Notably, Pin1 is downregulated and/or inhibited by oxidation in Alzheimers disease neurons, Pin1 knockout causes tauopathy and neurodegeneration, and Pin1 promoter polymorphisms appear to associate with reduced Pin1 levels and increased risk for late-onset Alzheimers disease. However, the role of Pin1 in APP processing and Aβ production is unknown. Here we show that Pin1 has profound effects on APP processing and Aβ production. We find that Pin1 binds to the phosphorylated Thr 668-Pro motif in APP and accelerates its isomerization by over 1,000-fold, regulating the APP intracellular domain between two conformations, as visualized by NMR. Whereas Pin1 overexpression reduces Aβ secretion from cell cultures, knockout of Pin1 increases its secretion. Pin1 knockout alone or in combination with overexpression of mutant APP in mice increases amyloidogenic APP processing and selectively elevates insoluble Aβ42 (a major toxic species) in brains in an age-dependent manner, with Aβ42 being prominently localized to multivesicular bodies of neurons, as shown in Alzheimers disease before plaque pathology. Thus, Pin1-catalysed prolyl isomerization is a novel mechanism to regulate APP processing and Aβ production, and its deregulation may link both tangle and plaque pathologies. These findings provide new insight into the pathogenesis and treatment of Alzheimers disease.

Pin1 regulates turnover and subcellular localization of β-catenin by inhibiting its interaction with APC

Phosphorylation on a serine or threonine residue preceding proline (Ser/Thr-Pro) is a key regulatory mechanism, and the conformation of certain phosphorylated Ser/Thr-Pro bonds is regulated specifically by the prolyl isomerase Pin1. Whereas the inhibition of Pin1 induces apoptosis, Pin1 is strikingly overexpressed in a subset of human tumours. Here we show that Pin1 regulates β-catenin turnover and subcellular localization by interfering with its interaction with adenomatous polyposis coli protein (APC). A differential-display screen reveals that Pin1 increases the transcription of several β-catenin target genes, including those encoding cyclin D1 and c-Myc. Manipulation of Pin1 levels affects the stability of β-catenin in vitro. Furthermore, β-catenin levels are decreased in Pin1-deficient mice but are increased and correlated with Pin1 overexpression in human breast cancer. Pin1 directly binds a phosphorylated Ser-Pro motif next to the APC-binding site in β-catenin, inhibits its interaction with APC and increases its translocation into the nucleus. Thus, Pin1 is a novel regulator of β-catenin signalling and its overexpression might contribute to the upregulation of β-catenin in tumours such as breast cancer, in which APC or β-catenin mutations are not common.