The prolyl isomerase Pin1 functions in mitotic chromosome condensation

The prolyl isomerase Pin1 plays important roles in numerous cellular processes. Here we provide evidence that Pin1 has an important function in chromosome condensation during mitosis. We first demonstrate that the interaction of Pin1 with chromatin is greatly elevated in G2/M phase and that this correlates with the presence on chromosomes of several mitotic phosphoproteins, especially topoisomerase (Topo) IIalpha. Inducible overexpression of Pin1 was shown to result in higher M phase-specific phosphorylation, while downregulation of Pin1 by siRNA treatment reduced phosphorylation of TopoIIalpha and other mitotic proteins. Furthermore, immunodepletion of Pin1 from mitotic cell extracts prevented such extracts from inducing chromosome condensation when added to S phase nuclei. Indeed, purified Pin1 and cdc2/cyclin B kinase were by themselves sufficient to induce condensation. This reflects the ability of Pin1 to increase TopoIIalpha phosphorylation by cdc2/cyclin B in vitro, which in turn dramatically increased formation of a TopoIIalpha/Pin1/DNA complex.     

HSP25 down-regulation enhanced p53 acetylation by dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis

Heat shock proteins (HSPs) play important roles in cellular stress resistance. Previous reports had already suggested that HSP27 played multiple roles in preventing doxorubicin-induced cardiotoxicity. Although HSP25 might have biological functions similar to its human homolog HSP27, the mechanism of HSP25 is still unclear in doxorubicin-induced cardiomyocyte apoptosis. To investigate HSP25 biological function on doxorubicin-induced apoptosis, flow cytometry was employed to analyze cell apoptosis in over-expressing HSP25 H9c2 cells in presence of doxorubicin. Unexpectedly, the H9c2 cells of over-expressing HSP25 have no protective effect on doxorubicin-induced apoptosis. Moreover, no detectable interactions were detected by coimmunoprecipitation between HSP25 and cytochrome c, and HSP25 over-expression failed in preventing cytochrome c release induced by doxorubicin. However, down-regulation of endogenous HSP25 by a specific small hairpin RNA aggravates apoptosis in H9c2 cells. Subsequent studies found that HSP25, but not HSP90, HSP70, and HSP20, interacted with SIRT1. Knockdown of HSP25 decreased the interaction between SIRT1 and p53, leading to increased p53 acetylation on K379, up-regulated pro-apoptotic Bax protein expression, induced cytochrome c release, and triggered caspase-3 and caspase-9 activation. These findings indicated a novel mechanism by which HSP25 regulated p53 acetylation through dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis.     

Molecular mechanisms of the phospho-dependent prolyl cis/trans isomerase Pin1

Since its discovery 10 years ago, Pin1, a prolyl cis/trans isomerase essential for cell cycle progression, has been implicated in a large number of molecular processes related to human diseases, including cancer and Alzheimer's disease. Pin1 is made up of a WW interaction domain and a C-terminal catalytic subunit, and several high-resolution structures are available that have helped define its function. The enzymatic activity of Pin1 towards short peptides containing the pSer/Thr-Pro motif has been well documented, and we discuss the available evidence for the molecular mechanisms of its isomerase activity. We further focus on those studies that examine its cis/trans isomerase function using full-length protein substrates. The interpretation of this research has been further complicated by the observation that many of its pSer/Thr-Pro substrate motifs are located in natively unstructured regions of polypeptides, and are characterized by minor populations of the cis conformer. Finally, we review the data on the possibility of alternative modes of substrate binding and the complex role that Pin1 plays in the degradation of its substrates. After considering the available work, it seems that further analysis is required to determine whether binding or catalysis is the primary mechanism through which Pin1 affects cell cycle progression.     

A dual inhibitor against prolyl isomerase Pin1 and cyclophilin discovered by a novel real-time fluorescence detection method

Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), is a potential target molecule for cancer, infectious disease, and Alzheimer’s disease. We established a high-throughput screening method for Pin1 inhibitors, which employs a real-time fluorescence detector. This screening method identified 66 compounds that inhibit Pin1 out of 9756 compounds from structurally diverse chemical libraries. Further evaluations of surface plasmon resonance methods and a cell proliferation assay were performed. We discovered a cell-active inhibitor, TME-001 (2-(3-chloro-4-fluoro-phenyl)-isothiazol-3-one). Surprisingly, kinetic analyses revealed that TME-001 is the first compound that exhibits dual inhibition of Pin1 (IC₅₀ = 6.1 μM) and cyclophilin, another type of PPIase, (IC₅₀ = 13.7 μM). This compound does not inhibit FKBP. This finding suggests the existence of similarities of structure and reaction mechanism between Pin1 and cyclophilin, and may lead to a more complete understanding of the active sites of PPIases.     

iASPP protects the heart from ischemia injury by inhibiting p53 expression and cardiomyocyte apoptosis

Currently, there remains a great need to elucidate the molecular mechanism of acute myocardial infarction in order to facilitate the development of novel therapy. Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is a member of the ASPP family proteins and an evolutionarily preserved inhibitor of p53 that is involved in many cellular processes, including apoptosis of cancer cells. The purpose of this study was to investigate the possible role of iASPP in acute myocardial infarction. The protein level of iASPP was markedly reduced in the ischemic hearts in vivo and hydrogen peroxide-exposed cardiomyocytes in vitro. Overexpression of iASPP reduced the infarct size and cardiomyocyte apoptosis of mice subjected to 24 h of coronary artery ligation. Echocardiography showed that cardiac function was improved as indicated by the increase in ejection fraction and fractional shortening. In contrast, knockdown of iASPP exacerbated cardiac injury as manifested by impaired cardiac function, increased infarct size, and apoptosis rate. Mechanistically, overexpression of iASPP inhibited, while knockdown of iASPP increased the expressions of p53 and Bax, the key regulators of apoptosis. Taken together, our results suggested that iASPP is an important regulator of cardiomyocyte apoptosis, which represents a potential target in the therapy of myocardial infarction.     

The prolyl isomerase Pin1 modulates development of CD8+ cDC in mice

Background

Pin1 has previously been described to regulate cells that participate in both innate and adaptive immunity. Thus far, however, no role for Pin1 has been described in modulating conventional dendritic cells, innate antigen presenting cells that potently activate naïve T cells, thereby bridging innate and adaptive immune responses.

Methodology/Principal Findings

When challenged with LPS, Pin1-null mice failed to accumulate spleen conventional dendritic cells (cDC). Analysis of steady-state spleen DC populations revealed that Pin1-null mice had fewer CD8+ cDC. This defect was recapitulated by culturing Pin1-null bone marrow with the DC-instructive cytokine Flt3 Ligand. Additionally, injection of Flt3 Ligand for 9 days failed to induce robust expansion of CD8+ cDC in Pin1-null mice. Upon infection with Listeria monocytogenes, Pin1-null mice were defective in stimulating proliferation of adoptively transferred WT CD8+ T cells, suggesting that decreases in Pin1 null CD8+ cDC may affect T cell responses to infection in vivo. Finally, upon analyzing expression of proteins involved in DC development, elevated expression of PU.1 was detected in Pin1-null cells, which resulted from an increase in PU.1 protein half-life.

Conclusions/Significance

We have identified a novel role for Pin1 as a modulator of CD8+ cDC development. Consistent with reduced numbers of CD8+ cDC in Pin1-null mice, we find that the absence of Pin1 impairs CD8+ T cell proliferation in response to infection with Listeria monocytogenes. These data suggest that, via regulation of CD8+ cDC production, Pin1 may serve as an important modulator of adaptive immunity.     

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.     

A critical step for JNK activation: isomerization by the prolyl isomerase Pin1

c-Jun N-terminal kinase (JNK) is activated by dual phosphorylation of both threonine and tyrosine residues in the phosphorylation loop of the protein in response to several stress factors. However, the precise molecular mechanisms for activation after phosphorylation remain elusive. Here we show that Pin1, a peptidyl-prolyl isomerase, has a key role in the JNK1 activation process by modulating a phospho-Thr-Pro motif in the phosphorylation loop. Pin1 overexpression in human breast cancer cell lines correlates with increased JNK activity. In addition, small interfering RNA (siRNA) analyses showed that knockdown of Pin1 in a human breast cancer cell line decreased JNK1 activity. Pin1 associates with JNK1, and then catalyzes prolyl isomerization of the phospho-Thr-Pro motif in JNK1 from trans- to cis-conformation. Furthermore, Pin1 enhances the association of JNK1 with its substrates. As a result, Pin1(-/-) cells are defective in JNK activation and resistant to oxidative stress. These results provide novel insights that, following stress-induced phosphorylation of Thr in the Thr-Pro motif of JNK1, JNK1 associates with Pin1 and undergoes conformational changes to promote the binding of JNK1 to its substrates, resulting in cellular responses from extracellular signals.     

Death-associated protein kinase 1 phosphorylates Pin1 and inhibits its prolyl isomerase activity and cellular function

Pin1 is a phospho-specific prolyl isomerase that regulates numerous key signaling molecules and whose deregulation contributes to disease notably cancer. However, since prolyl isomerases are often believed to be constitutively active, little is known whether and how Pin1 catalytic activity is regulated. Here, we identify death-associated protein kinase 1 (DAPK1), a known tumor suppressor, as a kinase responsible for phosphorylation of Pin1 on Ser71 in the catalytic active site. Such phosphorylation fully inactivates Pin1 catalytic activity and inhibits its nuclear location. Moreover, DAPK1 inhibits the ability of Pin1 to induce centrosome amplification and cell transformation. Finally, Pin1 pSer71 levels are positively correlated with DAPK1 levels and negatively with centrosome amplification in human breast cancer. Thus, phosphorylation of Pin1 Ser71 by DAPK1 inhibits its catalytic activity and cellular function, providing strong evidence for an essential role of the Pin1 enzymatic activity for its cellular function.     

Binding and regulation of the transcription factor NFAT by the peptidyl prolyl cis-trans isomerase Pin1

Nuclear factor of activated T cells (NFAT) plays a key role in T cell activation. The activation of NFAT involves calcium- and calcineurin-dependent dephosphorylation and nuclear translocation from the cytoplasm, a process that is opposed by protein kinases. We show here that the peptidyl prolyl cis-trans isomerase Pin1 interacts specifically with the phosphorylated form of NFAT. The NFAT-Pin1 interaction is mediated through the WW domain of Pin1 and the serine-proline-rich domains of NFAT. Furthermore, binding of Pin1 to NFAT inhibits the calcineurin-mediated dephosphorylation of NFAT in vitro, and overexpression of Pin1 in T cells inhibits calcium-dependent activation of NFAT in vivo. These results suggest a possible role for Pin1 in the regulation of NFAT in T cells.     

Thermodynamics of phosphopeptide binding to the human peptidyl prolyl cis/trans isomerase Pin1

Proteins containing phosphorylated Ser/Thr-Pro motifs play key roles in numerous regulatory processes in the cell. The peptidyl prolyl cis/trans isomerase Pin1 specifically catalyzes the conformational transition of phosphorylated Ser/Thr-Pro motifs. Here we report the direct analysis of the thermodynamic properties of the interaction of the PPIase Pin1 with its substrate-analogue inhibitor Ac-Phe-D-Thr(PO3H2)-Pip-Nal-Gln-NH2 specifically targeted to the PPIase active site based on the combination of isothermal titration calorimetry and studies on inhibition of enzymatic activity of wt Pin1 and active site variants. Determination of the thermodynamic parameters revealed an enthalpically and entropically favored interaction characterized by binding enthalpy deltaH(ITC) of -6.3 +/- 0.1 kcal mol(-1) and a TdeltaS(ITC) of 4.1 +/- 0.1 kcal mol(-1). The resulting dissociation constant KD for binding of the peptidic inhibitor with 1.8 x 10(-8) M resembles the dissociation constant of a Pin1 substrate in the transition state, suggesting a transition state analogue conformation of the bound inhibitor. The strongly decreased affinity of Pin1 for ligand at increasing ionic strength implicates that the potential of bidentate binding of a substrate protein by the PPIase and the WW domain of Pin1 may be required to deploy improved efficiency and specificity of Pin1 under conditions of physiological ionic strength.     

Molecular basis for an ancient partnership between prolyl isomerase Pin1 and phosphatase inhibitor-2

Pin1 is a prolyl isomerase that recognizes phosphorylated Ser/Thr-Pro sites, and phosphatase inhibitor-2 (I-2) is phosphorylated during mitosis at a PSpTP site that is expected to be a Pin1 substrate. However, we previously discovered I-2, but not phospho-I-2, bound to Pin1 as an allosteric modifier of Pin1 substrate specificity [Li, M., et al. (2008) Biochemistry 47, 292]. Here, we use binding assays and NMR spectroscopy to map the interactions on Pin1 and I-2 to elucidate the organization of this complex. Despite having sequences that are ～50% identical, human, Xenopus, and Drosophila I-2 proteins all exhibited identical, saturable binding to GST-Pin1 with K(0.5) values of 0.3 μM. The (1)H-(15)N heteronuclear single-quantum coherence spectra for both the WW domain and isomerase domain of Pin1 showed distinctive shifts upon addition of I-2. Conversely, as shown by NMR spectroscopy, specific regions of I-2 were affected by addition of Pin1. A single-residue I68A substitution in I-2 weakened binding to Pin1 by half and essentially eliminated binding to the isolated WW domain. On the other hand, truncation of I-2 to residue 152 had a minimal effect on binding to the WW domain but eliminated binding to the isomerase domain. Size exclusion chromatography revealed that wild-type I-2 and Pin1 formed a large (>300 kDa) complex and I-2(I68A) formed a complex of half the size that we propose are a heterotetramer and a heterodimer, respectively. Pin1 and I-2 are conserved among eukaryotes from yeast to humans, and we propose they make up an ancient partnership that provides a means for regulating Pin1 specificity and function.     

p53 regulation orchestrates the TGF-beta response

Among its multiple functions, p53 is a critical regulator of TGF-beta responses. Sasai et al. (2008) now identify a new p53 inhibitory protein, XFDL156. During embryonic development, this factor is expressed in the ectoderm germ layer and maintains the pluripotency of ectodermal cells by inhibiting TGF-beta target genes that promote mesoderm specification.     

p53 acetylation enhances Taxol-induced apoptosis in human cancer cells

Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and β-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.