Landscape of Pin1 in the cell cycle

Pin1 is a peptidyl-prolyl isomerase which plays a critical role in many diseases including cancer and Alzheimer''s disease. The essential role of Pin1 is to affect stability, localization or function of phosphoproteins by catalyzing structural changes. Among the collection of Pin1 substrates, many have been shown to be involved in regulating cell cycle progression. The cell cycle disorder caused by dysregulation of these substrates is believed to be a common phenomenon in cancer. A number of recent studies have revealed possible functions of several important Pin1-binding cell cycle regulators. Investigating the involvement of Pin1 in the cell cycle may assist in the development of future cancer therapeutics. In this review, we summarize current knowledge regarding the network of Pin1 substrates and Pin1 regulators in cell cycle progression. In G1/S progression, cyclin D1, RB, p53, p27, and cyclin E are all well-known cell cycle regulators that are modulated by Pin1. During G2/M transition, our lab has shown that Aurora A suppresses Pin1 activity through phosphorylation at Ser16 and cooperates with hBora to modulate G2/M transition. We conclude that Pin1 may be thought of as a molecular timer which modulates cell cycle progression networks.     

Pin1 regulates centrosome duplication, and its overexpression induces centrosome amplification, chromosome instability, and oncogenesis

Phosphorylation on Ser/Thr-Pro motifs is a major mechanism regulating many events involved in cell proliferation and transformation, including centrosome duplication, whose defects have been implicated in oncogenesis. Certain phosphorylated Ser/Thr-Pro motifs can exist in two distinct conformations whose conversion in certain proteins is catalyzed specifically by the prolyl isomerase Pin1. Pin1 is prevalently overexpressed in human cancers and is important for the activation of multiple oncogenic pathways, and its deletion suppresses the ability of certain oncogenes to induce cancer in mice. However, little is known about the role of Pin1 in centrosome duplication and the significance of Pin1 overexpression in cancer development in vivo. Here we show that Pin1 overexpression correlates with centrosome amplification in human breast cancer tissues. Furthermore, Pin1 localizes to and copurifies with centrosomes in interphase but not mitotic cells. Moreover, Pin1 ablation in mouse embryonic fibroblasts drastically delays centrosome duplication without affecting DNA synthesis and Pin1 inhibition also suppresses centrosome amplification in S-arrested CHO cells. In contrast, overexpression of Pin1 drives centrosome duplication and accumulation, resulting in chromosome missegregation, aneuploidy, and transformation in nontransformed NIH 3T3 cells. More importantly, transgenic overexpression of Pin1 in mouse mammary glands also potently induces centrosome amplification, eventually leading to mammary hyperplasia and malignant mammary tumors with overamplified centrosomes. These results demonstrate for the first time that the phosphorylation-specific isomerase Pin1 regulates centrosome duplication and its deregulation can induce centrosome amplification, chromosome instability, and oncogenesis.     

Conformational selection in the recognition of phosphorylated substrates by the catalytic domain of human Pin1

Post-translational phosphorylation and the related conformational changes in signaling proteins are responsible for regulating a wide range of subcellular processes. Human Pin1 is central to many of these cell signaling pathways in normal and aberrant subcellular processes, catalyzing cis-trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. Pin1 has therefore been identified as a possible drug target in many diseases, including cancer and Alzheimer's. The effects of phosphorylation on Pin1 substrates, and the atomistic basis for Pin1 recognition and catalysis, are not well understood. Here, we determine the conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 using all-atom molecular dynamics simulations. We show that phosphorylation induces backbone conformational changes on the peptide substrate analogues. We also show that Pin1 recognizes specific conformations of its substrate by conformational selection. Furthermore, dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme-substrate complex when the substrate is in the transition state configuration, suggesting that these motions play significant roles during catalytic turnover. These results provide a detailed atomistic picture of the mechanism of Pin1 recognition that can be exploited for drug design purposes and further our understanding of the synergistic complexities of post-translational phosphorylation and cis-trans isomerization.     

Role of Pin2/TRF1 in telomere maintenance and cell cycle control

Telomeres are specialized structures found at the extreme ends of chromosomes, which have many functions, including preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Deregulation of telomere length has been implicated in cancer and ageing. Telomere maintenance is tightly regulated by telomerase and many other telomere-associated proteins and is also closely linked to cell cycle control, especially mitotic regulation. However, little is known about the identity and function of the signaling molecules connecting telomere maintenance and cell cycle control. Pin2/TRF1 was originally identified as a protein bound to telomeric DNA (TRF1) and as a protein involved in mitotic regulation (Pin2). Pin2/TRF1 negatively regulates telomere length and importantly, its function is tightly regulated during the cell cycle, acting as an important regulator of mitosis. Recent identification of many Pin2/TRF1 upstream regulators and downstream targets has provided important clues to understanding the dual roles of Pin2/TRF1 in telomere maintenance and cell cycle control. These results have led us to propose that Pin2/TRF1 functions as a key molecule in connecting telomere maintenance and cell cycle control.     

IGF-1 induces Pin1 expression in promoting cell cycle S-phase entry.

Insulin-like growth factor I (IGF-1) is a well-established mitogen to many different cell types and is implicated in progression of a number of human cancers, notably breast cancer. The prolyl isomerase Pin1 plays an important role in cell cycle regulation through its specific interaction with proteins that are phosphorylated at Ser/Thr-Pro motifs. Pin1 knockout mice appear to have relatively normal development yet the Pin1(-/-)mouse embryo fibroblast (MEF) cells are defective in re-entering cell cycle in response to serum stimulation after G0 arrest. Here, we report that Pin1(-/-) MEF cells display a delayed cell cycle S-phase entry in response to IGF stimulation and that IGF-1 induces Pin1 protein expression which correlates with the induction of cyclin D1 and RB phosphorylation in human breast cancer cells. The induction of Pin1 by IGF-1 is mediated via the phosphatidylinositol 3-kinase as well as the MAP kinase pathways. Treatment of PI3K inhibitor LY294002 and the MAP kinase inhibitor PD098059, but not p38 inhibitor SB203580, effectively blocks IGF-1-induced upregulation of Pin1, cyclin D1 and RB phosphorylation. Furthermore, we found that Cyclin D1 expression and RB phosphorylation are dramatically decreased in Pin1(-/-) MEF cells. Reintroducing a recombinant adenovirus encoding Pin1 into Pin1(-/-) MEF cells restores the expression of cyclin D1 and RB phosphorylation. Thus, these data suggest that the mitogenic function of IGF-1 is at least partially linked to the induction of Pin1, which in turn stimulates cyclin D1 expression and RB phosphorylation, therefore contributing to G0/G1-S transition.     

Pinning down cell signaling, cancer and Alzheimer's disease

Protein phosphorylation on certain serine or threonine residues preceding proline (Ser/Thr-Pro) is a pivitol signaling mechanism in diverse cellular processes and its deregulation can lead to human disease. However, little is known about how these phosphorylation events actually control cell signaling. Pin1 is a highly conserved enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Recent results indicate that such conformational changes following phosphorylation are a novel signaling mechanism pivotal in regulating many cellular functions. This mechanism also offers new insights into the pathogenesis and treatment of human disease, most notably cancer and Alzheimer's disease. Thus, Pin1 plays a key role in linking signal transduction to the pathogenesis of cancer and Alzheimer's disease - two major age-related diseases.     

Pin1 in Alzheimer's disease: multiple substrates, one regulatory mechanism?

Presence of neuritic plaques and neurofibrillary tangles in the brain are two neuropathological hallmarks of Alzheimer's disease (AD), although the molecular basis of their coexistence remains elusive. The neurofibrillary tangles are composed of microtubule binding protein Tau, whereas neuritic plaques consist of amyloid-beta peptides derived from amyloid precursor protein (APP). Recently, the peptidyl-prolyl cis/trans isomerase Pin1 has been identified to regulate the function of certain proteins after phosphorylation and to play an important role in cell cycle regulation and cancer development. New data indicate that Pin1 also regulates the function and processing of Tau and APP, respectively, and is important for protecting against age-dependent neurodegeneration. Furthermore, Pin1 is the only gene known so far that, when deleted in mice, can cause both Tau and Abeta-related pathologies in an age-dependent manner, resembling many aspects of human Alzheimer's disease. Moreover, in the human AD brain Pin1 is downregulated or inhibited by oxidative modifications and/or genetic changes. These results suggest that Pin1 deregulation may provide a link between formation of tangles and plaques in AD.     

Prolyl isomerase Pin1 as a molecular switch to determine the fate of phosphoproteins

Pin1 is a highly conserved enzyme that only isomerizes specific phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Such conformational changes represent a novel and tightly controlled signaling mechanism regulating a spectrum of protein activities in physiology and disease; often through phosphorylation-dependent, ubiquitin-mediated proteasomal degradation. In this review, we summarize recent advances in elucidating the role and regulation of Pin1 in controlling protein stability. We also propose a mechanism by which Pin1 functions as a molecular switch to control the fates of phosphoproteins. We finally stress the need to develop tools to visualize directly Pin1-catalyzed protein conformational changes as a way to determine their roles in the development and treatment of human diseases.     

The peptidyl prolyl cis/trans isomerase Pin1 downregulates the Inhibitor of Apoptosis Protein Survivin

The peptidyl prolyl cis-trans isomerase Pin1 and the Inhibitor of Apoptosis Protein (IAP) Survivin are two major proteins involved in cancer. They both modulate apoptosis, mitosis, centrosome duplication and neuronal development but until now no functional relationship has been reported between these two proteins. We tested Pin1-induced regulation of Survivin in neuroblastoma cells. Pin1 overexpression in SY5Y neuroblastoma cells decreased Survivin levels. Immunocytochemical studies indicated that they partially co-localized in interphase and mitotic cells. Co-immunoprecipitation further demonstrates the existence of a Pin1/Survivin complex. Pin1-induced effect on Survivin was confirmed in COS cells. RT-PCR and mutagenesis experiments suggested that this Pin1-induced decrease of Survivin occurred at the protein level. Survivin downregulation depended on the binding ability of Pin1 but was not related to the single Thr-Pro site, suggesting an indirect relationship into a protein complex. Finally, this functional regulation of Survivin by Pin1 is reciprocal since Pin1 silencing led to an increase in Survivin levels. The characterization of this functional relationship between Pin1 and Survivin might help to better understand mitosis control and cancer mechanisms.     

The F-box protein FBX4 targets PIN2/TRF1 for ubiquitin-mediated degradation and regulates telomere maintenance

Pin2/TRF1 was identified previously as both a protein (TRF1) that binds to telomeric DNA repeats and as a protein (Pin2) that associates with the kinase NIMA and suppresses its mitosis inducing activity. Pin2/TRF1 negatively regulates telomere length and also plays a critical role in cell cycle checkpoint control. Pin2/TRF1 is down-regulated in many human cancers and may be degraded by the ubiquitin-proteasome pathway, but components of the pathway involved in Pin2/TRF1 turnover have not been elucidated. By using the two-hybrid system, we recently identified Pin2/TRF1-interacting proteins, PinX1-4, and we demonstrated that PinX1 is a conserved telomerase inhibitor and a putative tumor suppressor. Here we report the characterization of PinX3. PinX3 was later found to be identical to Fbx4, a member of the F-box family of proteins, which function as substrate-specific adaptors of Cul1-based ubiquitin ligases. Fbx4 interacts with both Pin2 and TRF1 isoforms and promotes their ubiquitination in vitro and in vivo. Moreover, overexpression of Fbx4 reduces endogenous Pin2/TRF1 protein levels and causes progressive telomere elongation in human cells. In contrast, inhibition of Fbx4 by RNA interference stabilizes Pin2/TRF1 and promotes telomere shortening, thereby impairing cell growth. These results demonstrate that Fbx4 is a central regulator of Pin2/TRF1 protein abundance and that alterations in the stability of Pin2/TRF1 can have a dramatic impact on telomere length. Thus, Fbx4 may play a critical role in telomere maintenance.     

Pin1 inhibition activates cyclin D and produces neurodegenerative pathology

Abnormal cell cycle events are increasingly becoming important attributes of neurodegenerative pathology. Pin1 is a crucial target of neurodegeneration in relation to its functions regarding these abnormal cell cycle events in neurons. Pin1 is majorly involved in many aspects of cell cycle regulation and it has also been suggested to have a neuroprotective function against neurodegenerative pathologies. Oxidative dysregulation of Pin1 affects not only normal tau regulation, eventually causing tangle formation, but also cell cycle regulation in neurons. Presence of cell cycle proteins has been shown in many neurodegenerative diseases. Importantly, many of these proteins have physical interactions with Pin1. Hence, understanding Pin1's role in abnormal cell cycle re-entry is critical in terms of finding new approaches for the future therapeutic options treating neurodegenerative pathologies. Here, we show that inhibition of Pin1 by its selective inhibitor juglone leads to up-regulation of cyclinD1, phospho-tau, and caspase 3, producing apoptosis in cultured rat hippocampal neurons. We also observed axonal retraction with a change in sub-cellular localizations of cyclins. Therefore, Pin1 dysregulation, in relation to its role in cell cycle regulation in neurons, may have profound effects in the progression of neurodegenerative pathology, making it a possible crucial target behind many neurodegenerative diseases.     

Pin1 mediates neural-specific activation of the mitochondrial apoptotic machinery

Apoptosis of neurons plays fundamental roles in brain development and disease. Although neurons share with other cell types components of the mitochondrial apoptotic machinery, how this machinery is specifically activated in neurons remains poorly understood. Remarkably, phosphorylation of the BH3-only protein BIMEL at Ser65 triggers apoptosis in neurons but suppresses cell death in non-neural cells. Here, we report that the prolyl isomerase Pin1 interacts with Ser65-phosphorylated BIMEL in neurons. Pin1 is enriched at the mitochondrial membrane in neurons, where it forms a physical complex with the neuron-specific JNK scaffold protein JIP3. Activation of JNK signaling induces the dissociation of Pin1 from JIP3 and concomitantly promotes Pin1 binding to phosphorylated BIMEL. The interaction of Pin1 with phosphorylated BIMEL stabilizes BIMEL and thereby activates neuronal apoptosis. These findings define a neural-specific mechanism of cell death whereby Pin1 couples phosphorylation of BH3-only proteins to activation of the mitochondrial apoptotic machinery.     

Trypanosomatid Pin1‐Type Peptidyl‐Prolyl Isomerase Is Cytosolic and Not Essential for Cell Proliferation

Pin1‐type peptidyl‐prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)‐Pro residues, regulating various cellular events. Previously, we reported a Pin1‐type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1‐like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei. In addition, RNAi‐mediated downregulation of TbPin1 in Trypanosoma brucei did not abolish cell proliferation. Using yeast two‐hybrid assay, we identified a MORN domain‐containing protein as putative Pin1‐binding partners. These data suggest that Pin1‐mediated signaling mechanism plays a different role in protozoan parasites.     

Mitotic Regulation of SEPT9 Protein by Cyclin-dependent Kinase 1 (Cdk1) and Pin1 Protein Is Important for the Completion of Cytokinesis

Precise cell division is essential for multicellular development, and defects in this process have been linked to cancer. Septins are a family of proteins that are required for mammalian cell division, but their function and mode of regulation during this process are poorly understood. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) phosphorylates septin 9 (SEPT9) upon mitotic entry, and this phosphorylation controls association with the proline isomerase, Pin1. Both SEPT9 and Pin1 are critical for mediating the final separation of daughter cells. Expression of mutant SEPT9 that is defective in Pin1 binding was unable to rescue cytokinesis defects caused by SEPT9 depletion but rather induced dominant-negative defects in cytokinesis. However, unlike SEPT9 depletion, Pin1 was not required for the accumulation of the exocyst complex at the midbody. These results suggest that SEPT9 plays multiple roles in abscission, one of which is regulated by the action of Cdk1 and Pin1.     

Retinoblastoma tumor-suppressor protein phosphorylation and inactivation depend on direct interaction with Pin1

Inactivation of the retinoblastoma protein (pRb) by phosphorylation triggers uncontrolled cell proliferation. Accordingly, activation of cyclin-dependent kinase (CDK)/cyclin complexes or downregulation of CDK inhibitors appears as a common event in human cancer. Here we show that Pin1 (protein interacting with NIMA (never in mitosis A)-1), a peptidylprolyl isomerase involved in the control of protein phosphorylation, is an essential mediator for inactivation of the pRb. Our results indicate that Pin1 controls cell proliferation by altering pRb phosphorylation without affecting CDK and protein phosphatase 1 and 2 activity. We demonstrated that Pin1 regulates tumor cell proliferation through direct interaction with the spacer domain of the pRb protein, and allows the interaction between CDK/cyclin complexes and pRb in mid/late G1. Phosphorylation of pRb Ser 608/612 is the crucial motif for Pin1 binding. We propose that Pin1 selectively boosts the switch from hypo- to hyper-phosphorylation of pRb in tumor cells. In addition, we demonstrate that the CDK pathway is responsible for the interaction of Pin1 and pRb. Prospectively, our findings therefore suggest that the synergism among CDK and Pin1 inhibitors holds great promise for targeted pharmacological treatment of cancer patients, with the possibility of reaching high effectiveness at tolerated doses.