Gerburg Wulf

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.

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.

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.

The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response

p53 is activated in response to various genotoxic stresses resulting in cell cycle arrest or apoptosis. It is well documented that DNA damage leads to phosphorylation and activation of p53 (refs 1–3), yet how p53 is activated is still not fully understood. Here we report that DNA damage specifically induces p53 phosphorylation on Ser/Thr-Pro motifs, which facilitates its interaction with Pin1, a member of peptidyl-prolyl isomerase. Furthermore, the interaction of Pin1 with p53 is dependent on the phosphorylation that is induced by DNA damage. Consequently, Pin1 stimulates the DNA-binding activity and transactivation function of p53. The Pin1-mediated p53 activation requires the WW domain, a phosphorylated Ser/Thr-Pro motif interaction module, and the isomerase activity of Pin1. Moreover, Pin1-deficient cells are defective in p53 activation and timely accumulation of p53 protein, and exhibit an impaired checkpoint control in response to DNA damage. Together, these data suggest a mechanism for p53 regulation in cellular response to genotoxic stress.

Phosphorylation-specific prolyl isomerization: is there an underlying theme?

The prolyl isomerase Pin1 is a conserved enzyme that is intimately involved in diverse biological processes and pathological conditions such as cancer and Alzheimers disease. By catalysing cis–trans interconversion of certain motifs containing phosphorylated serine or threonine residues followed by a proline residue (pSer/Thr-Pro), Pin1 can have profound effects on phosphorylation signalling. The structural and functional differences that result from cis–trans isomerization of specific pSer/Thr-Pro motifs probably underlie most, if not all, Pin1-dependent actions. Phosphorylation-dependent prolyl isomerization by Pin1 remains a unique mode for the modulation of signal transduction. Here, we provide an overview of the plethora of regulatory events that involve this unique enzyme, with a particular focus on oncogenic signalling and neurodegeneration.

PIN1 Is an E2F Target Gene Essential for Neu/Ras-Induced Transformation of Mammary Epithelial Cells

ABSTRACT Oncogenes Neu/HER2/ErbB2 and Ras can induce mammary tumorigenesis via upregulation of cyclin D1. One major regulatory mechanism in these oncogenic signaling pathways is phosphorylation of serines or threonines preceding proline (pSer/Thr-Pro). Interestingly, the pSer/Thr-Pro motifs in proteins exist in two completely distinct cis and trans conformations, whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. By isomerizing pSer/Thr-Pro bonds, Pin1 can regulate the conformation and function of certain phosphorylated proteins. We have previously shown that Pin1 is overexpressed in breast tumors and positively regulates cyclin D1 by transcriptional activation and posttranslational stabilization. Moreover, in Pin1 knockout mice, mammary epithelial cells fail to undergo massive proliferation during pregnancy, as is the case in cyclin D1 null mice. These results indicate that Pin1 is upregulated in breast cancer and may be involved in mammary tumors. However, the mechanism of Pin1 overexpression in cancer and its significance in cell transformation remain largely unknown. Here we demonstrate that PIN1 expression is mediated by the transcription factor E2F and enhanced by c-Neu and Ha-Ras via E2F. Furthermore, overexpression of Pin1 not only confers transforming properties on mammary epithelial cells but also enhances the transformed phenotypes of Neu/Ras-transformed mammary epithelial cells. In contrast, inhibition of Pin1 suppresses Neu- and Ras-induced transformed phenotypes, which can be fully rescued by overexpression of a constitutively active cyclin D1 mutant that is refractory to the Pin1 inhibition. Thus, Pin1 is an E2F target gene that is essential for the Neu/Ras-induced transformation of mammary epithelial cells through activation of cyclin D1.

PKM2 isoform-specific deletion reveals a differential requirement for pyruvate kinase in tumor cells.

The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.

Role of Pin1 in the Regulation of p53 Stability and p21 Transactivation, and Cell Cycle Checkpoints in Response to DNA Damage

DNA damage leads to stabilization and accumulation of p53, which plays a pivotal role in transcriptional activation of p21 and cell cycle arrest. The increase in p53 stability depends critically on its phosphorylation on serine/threonine residues, including those preceding a proline (Ser(P)/Thr-Pro). The Ser(P)/Thr-Pro moiety exists in the two distinct cis andtrans conformations and their conversion is catalyzed specifically by the prolyl isomerase Pin1. Pin1 regulates the conformation and function of certain phosphorylated proteins and plays an important role in cell cycle regulation, oncogenesis, and Alzheimers disease. However, nothing is known about the role of Pin1 in DNA damage. Here we found that DNA damage enhanced the interaction between Pin1 and p53, which depended on the WW domain in Pin1 and Ser33/46-Pro motifs in p53. Furthermore, Pin1 regulates the stability of p53 and its transcriptional activity toward the p21 promoter. As a result, p53 and p21 barely increased after DNA damage in Pin1 knock-out embryonic fibroblasts or in neoplastic cells depleted of Pin1. Moreover, Pin1 null cells displayed significant defects in cell cycle checkpoints induced by DNA damage. These results demonstrate a new role of Pin1 in regulating p53 function during DNA damage.