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.     

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.     

Bora在细胞功能调控中的作用和机制研究进展

Aurora蛋白激酶A及Polo样蛋白激酶1（PLK在）作为重要的细胞周期调节蛋白可参与调控纺锤体组装、有丝分裂等细胞进程,但其激活机制及在有丝分裂中的作用机制仍然不是很清楚。Bora作为Aurora蛋白激酶A的结合蛋白,在果蝇和脊椎动物中功能高度保守,其主要通过结合Aurora蛋白激酶A从而调节Aurora蛋白激酶A的活性、促进PLK1的磷酸化、调节纺锤体的组装以及调控细胞周期进程等。随着对Bora研究的深入,人们对AuroraA和PLK1的激活机制以及Bora、Aurora蛋白激酶A、PLK1三者对细胞的调控也有了进一步的认识。主要综述Bora在细胞功能调控中的作用和研究机制。     

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.     

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.     

PP2A function toward mitotic kinases and substrates during the cell cycle

To maintain cellular homeostasis against the demands of the extracellular environment, a precise regulation of kinases and phosphatases is essential. In cell cycle regulation mechanisms, activation of the cyclin-dependent kinase (CDK1) and cyclin B complex (CDK1:cyclin B) causes a remarkable change in protein phosphorylation. Activation of CDK1:cyclin B is regulated by two auto-amplification loops-CDK1:cyclin B activates Cdc25, its own activating phosphatase, and inhibits Wee1, its own inhibiting kinase. Recent biological evidence has revealed that the inhibition of its counteracting phosphatase activity also occurs, and it is parallel to CDK1:cyclin B activation during mitosis. Phosphatase regulation of mitotic kinases and their substrates is essential to ensure that the progression of the cell cycle is ordered. Outlining how the mutual control of kinases and phosphatases governs the localization and timing of cell division will give us a new understanding about cell cycle regulation. [BMB Reports 2013; 46(6): 289-294]     

hDaxx的克隆及其与Pin1之间的相互作用

克隆了hDaxx全长的cDNA,并证实hDaxx与肽基脯氨酰异构酶Pin1之间存在相互作用,它们共定位于细胞核内。同时发现它们能够协同激活p53的转录活性,揭示Pin1可能在hDaxx调节细胞凋亡的过程中发挥了重要作用。     

Synthesis and Pin1 inhibitory activity of thiazole derivatives

Pin1 (Protein interacting with NIMA1) is a peptidyl prolyl cis–trans isomerase (PPIase) which specifically catalyze the conformational conversion of the amide bond of pSer/Thr-Pro motifs in its substrate proteins and is a novel promising anticancer target. A series of new thiazole derivatives were designed and synthesized, and their inhibitory activities were measured against human Pin1 using a protease-coupled enzyme assay. Of all the tested compounds, a number of thiazole derivatives bearing an oxalic acid group at 4-position were found to be potent Pin1 inhibitors with IC₅₀ values at low micromolar level. The detailed structure–activity relationships were analyzed and the binding features of compound 10b (IC₅₀ 5.38 μM) was predicted using CDOCKER program. The results of this research would provide informative guidance for further optimizing thiazole derivatives as potent Pin1 inhibitors.     

Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

Ultraviolet A (UVA) radiation (λ = 320–400 nm) is considered a major cause of human skin cancer. Pin1, a peptidyl prolyl isomerase, is overexpressed in most types of cancer tissues and plays an important role in cell proliferation and transformation. Here, we demonstrated that Pin1 expression was enhanced by low energy UVA (300–900 mJ/cm²) irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. Exposure of epidermal cells to UVA radiation increased cell proliferation and cyclin D1 expression, and these changes were blocked by Pin1 inhibition. UVA irradiation also increased activator protein-1 (AP-1) minimal reporter activity and nuclear levels of c-Jun, but not c-Fos, in a Pin1-dependent manner. The increases in Pin1 expression and in AP-1 reporter activity in response to UVA were abolished by N-acetylcysteine (NAC) treatment. Finally, we found that pre-exposure of JB6 C141 cells to UVA potentiated EGF-inducible, anchorage-independent growth, and this effect was significantly suppressed by Pin1inhibition or by NAC.     

PP2R1A逆转录病毒的构建及对细胞周期的影响

目的：构建重组PP2R1A基因的逆转录病毒感染HEKTER细胞,观察其定位,验证表达,研究过表达PP2R1A对细胞生长及周期的影响。方法：逆转录病毒载体pMIG-Flag-PP2R1A-IRES-GFP与Pcll0A1瞬时共转染293T细胞,收集病毒感染HEKTER细胞,在荧光显微镜下观察定位,标记荧光单克隆。挑取不同表达强度单克隆做western验证PP2R1A蛋白表达。运用流式细胞分析、体外创伤试验及生长曲线试验研究单克隆细胞的增殖及周期。结果：获得了过表达PP2R1A的单克隆细胞株,PP2R1A在细胞内广泛表达,结合western及细胞试验证实PP2R1A高表达阻滞细胞周期并减慢细胞生长。结论：PP2R1A是丝苏氨酸蛋白磷酸酶PP2A的结构A亚基的a亚型,在细胞内广泛表达。本文成功构建了表达PP2R1A的细胞株,研究发现PP2R1A高表达会影响细胞生长及细胞周期,减缓了细胞增殖。为进一步深入研究PP2R1A对PP2A全酶活性及功能、细胞转化的影响奠定了重要的实验基础。     

Synthesis and biological evaluation of pyrimidine derivatives as novel human Pin1 inhibitors

Pin1 (Protein interacting with NIMA1) is a cis–trans isomerase and promotes the amide bond rotation of phosphoSer/Thr-Pro motifs in its substrates. Inhibition of Pin1 might be a novel strategy for developing anticancer agents. Herein, a series of pyrimidine derivatives were synthesized and their Pin1 inhibitory activities were evaluated. Among them, four compounds (2a, 2f, 2h and 2l) displayed potent inhibitory activities against Pin1 with IC₅₀ values lower than 3?µM. This series of pyrimidine-based inhibitors presented time-dependent inhibition against Pin1. The structure–activity relationships on the 2-, 4- and 5-positions of the pyrimidine ring were analyzed in details, which would facilitate further exploration of new Pin1 inhibitors.     

Enzyme-linked enzyme-binding assay for Pin1 WW domain ligands

Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif. An assay we call an enzyme-linked enzyme-binding assay (ELEBA) was developed to measure the K_d of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescent detection of ligands that block the association of the WW domain with immobilized ligand. The peptide ligands were derived from the cell cycle regulatory phosphatase, Cdc25c, residues 45-50. The K_d values for Fmoc-VPRpTPVGGGK-NH₂ and Ac-VPRpTPV-NH₂ were determined to be 36 ± 4 and 110 ± 30 μM, respectively. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain, even in the presence of the catalytic domain. This method may be applied to any dual specificity, multidomain protein.