Pin1 specifically catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro bonds and plays an important role in many cellular events through the effects of conformational change on the function of its biological substrates, including cell division cycle 25 C (Cdc25C), c-Jun and p53. Pin1 is overexpressed in many human cancer tissues, including breast, prostate and lung cancer. Its expression correlates with cyclin D1 levels, which contribute to cell transformation. Overexpression of Pin1 promotes tumor growth, while inhibition of Pin1 causes tumor cell apoptosis. Pin1 plays an important role in oncogenesis and therefore may serve as an effective anticancer target. Many inhibitors of Pin1 have been discovered, including several classes of designed inhibitors (alkene isosteres, reduced amides, indanyl ketones) and natural products (juglone, pepticinnamin E analogues, PiB and its derivatives obtained from a library screen). Pin1 inhibitors could be used as a novel type of anticancer drug by blocking cell cycle progression. Therefore, Pin1 represents a new diagnostic and therapeutic anticancer drug target.

The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cis-trans isomerization of p(S/T)-P bonds and might alter conformation and function of client proteins. Since the trans conformation of p(S/T)-P bonds is preferred by protein phosphatase 2A (PP2A), Pin1 may facilitate PP2A-mediated dephosphorylation. Juglone irreversibly inhibits parvulins and is often used to study the function of Pin1 in vivo. The drug prevents dephosphorylation of mitotic phosphoproteins, perhaps because they bind Pin1 and are dephosphorylated by PP2A. We show here however that juglone inhibited post-mitotic dephosphorylation and the exit of mitosis, independent of Pin1. This effect involved covalent modification of sulfhydryl groups in proteins essential for metaphase/anaphase transition. Particularly cytoplasmic proteins with a high cysteine content were vulnerable to the drug. Alkylation of sulfhydryl groups altered the conformation of such proteins, as evidenced by the disappearance of antibody epitopes on tubulin and the mitotic checkpoint component BubR1. The latter activates the anaphase-promoting complex/cyclosome, which degrades regulatory proteins, such as cyclin B1 and securins, and is required for mitotic exit. Indeed, juglone-treated cells failed to assemble a mitotic spindle, which correlated with perturbed microtubule dynamics, loss of immunodetectable tubulin, and formation of tubulin aggregates. Juglone also prevented degradation of cyclin B1, independently of the Mps1-controlled mitotic spindle checkpoint. Since juglone affected cell cycle progression at several levels, more specific drugs need to be developed for studies of Pin1 function in vivo.

DAP-kinase (DAPK) is a Ca(2+)/calmodulin regulated Ser/Thr kinase that activates a diverse range of cellular activities. It is subject to multiple layers of regulation involving both intramolecular signaling, and interactions with additional proteins, including other kinases and phosphatases. Its protein stability is modulated by at least three distinct ubiquitin-dependent systems. Like many kinases, DAPK participates in several signaling cascades, by phosphorylating additional kinases such as ZIP-kinase and protein kinase D (PKD), or Pin1, a phospho-directed peptidyl-prolyl isomerase that regulates the function of many phosphorylated proteins. Other substrate targets have more direct cellular effects; for example, phosphorylation of the myosin II regulatory chain and tropomyosin mediate some of DAPK's cytoskeletal functions, including membrane blebbing during cell death and cell motility. DAPK induces distinct death pathways of apoptosis, autophagy and programmed necrosis. Among the substrates implicated in these processes, phosphorylation of PKD, Beclin 1, and the NMDA receptor has been reported. Interestingly, not all cellular effects are mediated by DAPK's catalytic activity. For example, by virtue of protein-protein interactions alone, DAPK activates pyruvate kinase isoform M2, the microtubule affinity regulating kinases and inflammasome protein NLRP3, to promote glycolysis, influence microtubule dynamics, and enhance interleukin-1β production, respectively. In addition, a number of other substrates and interacting proteins have been identified, the physiological significance of which has not yet been established. All of these substrates, effectors and regulators together comprise the DAPK interactome. By presenting the components of the interactome network, this review will clarify both the mechanisms by which DAPK function is regulated, and by which it mediates its various cellular effects.

The periodic formation of plant organs such as leaves and flowers gives rise to intricate patterns that have fascinated biologists and mathematicians alike for hundreds of years [1]. The plant hormone auxin plays a central role in establishing these patterns by promoting organ formation at sites where it accumulates due to its polar, cell-to-cell transport [2-6]. Although experimental evidence as well as modeling suggest that feedback from auxin to its transport direction may help specify phyllotactic patterns [7-12], the nature of this feedback remains unclear [13]. Here we reveal that polarization of the auxin efflux carrier PIN-FORMED 1 (PIN1) is regulated by the auxin response transcription factor MONOPTEROS (MP) [14]. We find that in the shoot, cell polarity patterns follow MP expression, which in turn follows auxin distribution patterns. By perturbing MP activity both globally and locally, we show that localized MP activity is necessary for the generation of polarity convergence patterns and that localized MP expression is sufficient to instruct PIN1 polarity directions non-cell autonomously, toward MP-expressing cells. By expressing MP in the epidermis of mp mutants, we further show that although MP activity in a single-cell layer is sufficient to promote polarity convergence patterns, MP in sub-epidermal tissues helps anchor these polarity patterns to the underlying cells. Overall, our findings reveal a patterning module in plants that determines organ position by orienting transport of the hormone auxin toward cells with high levels of MP-mediated auxin signaling. We propose that this feedback process acts broadly to generate periodic plant architectures.

The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate vesicle formation by activating ARF GTPases on specific membranes in animals, plants, and fungi. However, apart from the catalytic exchange activity of the SEC7 domain, the functional significance of other conserved domains is virtually unknown. Here, we show that a distinct N-terminal domain of GNOM mediates dimerization and in addition interacts heterotypically with two other conserved domains in vivo. In contrast with N-terminal dimerization, the heterotypic interaction is essential for GNOM function, as mutations abolishing this interaction inactivate the GNOM protein and compromise its membrane association. Our results suggest a general model of large ARF-GEF function in which regulated changes in protein conformation control membrane association of the exchange factor and, thus, activation of ARFs.

OBJECTIVE

Our previous work suggested an important role for the peptidyl-prolyl isomerase, Pin1, in hepatic NF-kappaB activation and liver injury during ischemia/reperfusion (I/R). In this study, we sought to determine the function of Pin1 in the injury response to hepatic I/R.

METHODS

Wild-type and Pin1(-/-) mice were subjected to partial hepatic I/R. In addition, hepatocytes and Kupffer cells were isolated from these mice.

RESULTS

Pin1(-/-) mice had reduced hepatic NF-kappaB activation and more liver injury after I/R than wild-type mice. The increased injury was not a result of enhanced inflammation as Pin1(-/-) mice had the same level of proinflammatory cytokine production and less neutrophil accumulation in the liver. The reduced NF-kappaB activation was not a result of a defect in nuclear translocation of NF-kappaB. In fact, hepatic nuclear p65 protein expression was higher in Pin1(-/-) mice than wild-type mice. This suggests that Pin1 is important for NF-kappaB-DNA binding. This effect was specific to hepatocytes as isolated Kupffer cells from wild-type and Pin1(-/-) mice were identical in their activation of NF-kappaB and production of cytokines after stimulation. In contrast, hepatocytes stimulated with TNFalpha had greatly reduced NF-kappaB activation, reduced production of the CXC chemokine, MIP-2, and increased cell death.

CONCLUSIONS

These data suggest that Pin1 is a critical regulator of NF-kappaB activation in hepatocytes and its role in these cells appears to confer direct protective effects.

Recently, the involvement of PIN1, a peptidyl-prolyl cis/trans isomerase, has been reported in age-related bone homeostasis and adipogenesis. However, the role of PIN1 during odontogenic and adipogenic differentiation remains to be fully understood, particularly regarding human dental pulp stem cells (HDPSCs). Thus, in the present study, we have investigated the role of PIN1 in odontogenic and adipogenic differentiation of HDPSCs and signaling pathways possibly involved. PIN1 mRNA and protein level were upregulated in a time-dependent manner during adipogenic differentiation, increasing until 1 day of odontogenic induction and then steadily declined during odontogenic differentiation. Treatment of a known PIN1 inhibitor, juglone, significantly increased odontogenic differentiation as confirmed by alkaline phosphatase (ALP) activity, calcium deposition, and mRNAs induction of odontogenic markers [ALP, osteopontin (OPN), osteocalcin (OCN), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP-1)]. On the contrary, adipogenic differentiation was dramatically reduced upon juglone treatment, with concomitant downregulation of lipid droplet accumulation and adipogenic marker genes [peroxisome proliferation-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (AP2)]. In contrast to PIN1 inhibition, the overexpression of PIN1 via adenoviral infection (Ad-PIN1) in HDPSCs inhibited odontogenic differentiation but increased adipogenic differentiation, in which stem cell property markers such as stage-specific embryonic antigen-4 (SSEA-4) and STRO-1 were upregulated during odontogenic differentiation but downregulated in adiopogenic differentiation. Consistently, juglone-mediated inhibition of PIN1 augmented the osteogenic medium (OM)-induced activation of bone morphogenetic protein (BMP), Wnt/β-catenin, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NF-κB) pathway, which response was reversed by Ad-PIN1. Moreover, juglone blocked the adipogenic induction medium-induced activation of PPARγ, C/EBPα, C/EBPβ, ERK, and NF-κB pathways, which was rescued by Ad-PIN1 infection. In summary, the present study shows for the first time that PIN1 acts as an important modulator of odontogenic and adipogenic differentiation of HDPSCs and may have clinical implications for regenerative dentistry.

OBJECTIVE

Diabetes is a major driver of cardiovascular disease, but the underlying mechanisms remain elusive. Prolyl-isomerase Pin1 recognizes specific peptide bonds and modulates function of proteins altering cellular homoeostasis. The present study investigates Pin1 role in diabetes-induced vascular disease.

RESULTS

In human aortic endothelial cells (HAECs) exposed to high glucose, up-regulation of Pin1-induced mitochondrial translocation of pro-oxidant adaptor p66(Shc) and subsequent organelle disruption. In this setting, Pin1 recognizes Ser-116 inhibitory phosphorylation of endothelial nitric oxide synthase (eNOS) leading to eNOS-caveolin-1 interaction and reduced NO availability. Pin1 also mediates hyperglycaemia-induced nuclear translocation of NF-κB p65, triggering VCAM-1, ICAM-1, and MCP-1 expression. Indeed, gene silencing of Pin1 in HAECs suppressed p66(Shc)-dependent ROS production, restored NO release and blunted NF-kB p65 nuclear translocation. Consistently, diabetic Pin1(-/-) mice were protected against mitochondrial oxidative stress, endothelial dysfunction, and vascular inflammation. Increased expression and activity of Pin1 were also found in peripheral blood monocytes isolated from diabetic patients when compared with age-matched healthy controls. Interestingly, enough, Pin1 up-regulation was associated with impaired flow-mediated dilation, increased urinary 8-iso-prostaglandin F2α and plasma levels of adhesion molecules.

CONCLUSIONS

Pin1 drives diabetic vascular disease by causing mitochondrial oxidative stress, eNOS dysregulation as well as NF-kB-induced inflammation. These findings provide molecular insights for novel mechanism-based therapeutic strategies in patients with diabetes.

The peptidyl-prolyl isomerase Pin1 is prevalently overexpressed in human cancers and is regarded as a new diagnostic and therapeutic target. Pin1 interacts with several proteins involved in cell cycle events in a phosphorylation-dependent manner. Among them, NIMA (never in mitosis, gene A) was first identified to interact with Pin1. In this report, we found that Pin1 could interact with Nek6, one of the human NIMA-related kinases (Neks). This interaction was confirmed by GST pull-down assay, which was further confirmed by immunoprecipitation experiments, as well as immunofluorescence colocalization. We further studied Pin1 and Nek6 mRNA level in 40 pairs of hepatocellular carcinoma cases, finding significant correlations between Nek6 and Pin1 mRNA expression levels in these samples.

Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) combination safely cures fatal acute promyelocytic leukemia, but their mechanisms of action and efficacy are not fully understood. ATRA inhibits leukemia, breast, and liver cancer by targeting isomerase Pin1, a master regulator of oncogenic signaling networks. Here we show that ATO targets Pin1 and cooperates with ATRA to exert potent anticancer activity. ATO inhibits and degrades Pin1, and suppresses its oncogenic function by noncovalent binding to Pin1's active site. ATRA increases cellular ATO uptake through upregulating aquaporin-9. ATO and ATRA, at clinically safe doses, cooperatively ablate Pin1 to block numerous cancer-driving pathways and inhibit the growth of triple-negative breast cancer cells and tumor-initiating cells in cell and animal models including patient-derived orthotopic xenografts, like Pin1 knockout, which is substantiated by comprehensive protein and microRNA analyses. Thus, synergistic targeting of Pin1 by ATO and ATRA offers an attractive approach to combating breast and other cancers.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem among breast cancer patients. Previously, we have reported that TAM-resistant MCF-7 cells (TAMR-MCF-7 cells) showed increased angiogenic intensity through Pin1-dependent vascular endothelial growth factor (VEGF) production. Among six flavonoids tested in the current study, VEGF gene transcription in MCF-7 cells with stable Pin1 overexpression was inhibited most effectively by quercetin. Reporter gene assays using minimal reporters containing hypoxia response elements and activator protein-1 (AP-1) elements revealed that the activities of hypoxia inducible factor-1α (HIF-1α) and AP-1, key transcription factors for VEGF gene transcription, were suppressed by quercetin. Western blot analyses confirmed that the increased nuclear levels of c-Jun and HIF-1α in TAMR-MCF-7 cells were blocked by quercetin. Moreover, quercetin inhibited the enhanced VEGF secretion and Pin1 expression in TAMR-MCF-7 cells, which was dependent on its phosphatidyl inositol 3-kinase inhibiting effect. Chick chorioallantoic membrane assays demonstrated that the enhanced angiogenesis intensity of TAMR-MCF-7 cells was also suppressed significantly by quercetin. These results demonstrate that quercetin may have therapeutic potential for the treatment of TAM-resistant breast cancer via Pin1 inhibition.

The aim of the present study was to explore the signaling pathways associated with the effect of simvastatin on matrix metalloproteinase-2 (MMP-2)/MMP-9 expression in human leukemia K562 cells. In sharp contrast to its insignificant effect on MMP-2, simvastatin down-regulated MMP-9 protein expression and mRNA levels in K562 cells. Simvastatin-induced Pin1 down-regulation evoked NFκB/p65 degradation. Meanwhile, simvastatin induced JNK-mediated c-Jun and ATF-2 activation. Over-expression of Pin1 suppressed simvastatin-induced MMP-9 down-regulation. Treatment with SP600125 (a JNK inhibitor) or knock-down of JNK1 reduced MMP-2 expression in simvastatin-treated cells. Simvastatin enhanced the binding of c-Jun/ATF-2 with the MMP-2 promoter. Down-regulation of c-Jun or ATF-2 by siRNA revealed that c-Jun/ATF-2 activation was crucial for MMP-2 expression. Suppression of p65 activation or knock-down of Pin1 by shRNA reduced MMP-2 and MMP-9 expression in K562 cells. Over-expression of constitutively active JNK1 rescued MMP-2 expression in Pin1 shRNA-transfected cells. Simvastatin treatment also suppressed MMP-9 but not MMP-2 expression in human leukemia U937 and KU812 cells. Taken together, our data indicate that simvastatin-induced p65 instability leads to MMP-9 down-regulation in leukemia cells, while simvastatin-induced JNK1/c-Jun/ATF-2 activation maintains the MMP-2 expression underlying p65 down-regulation.

SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.

Expression of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 is required for the accumulation of APC/C substrates crucial for DNA synthesis and mitotic entry. We show that in vivo Emi1 expression correlates with the proliferative status of the cellular compartment and that cells lacking Emi1 undergo cellular senescence. Emi1 depletion leads to strong decreases in E2F target mRNA and APC/C substrate protein abundances. However, cyclin E mRNA and cyclin E protein levels and associated kinase activities are increased. Cells lacking Emi1 undergo DNA damage, likely explained by replication stress upon deregulated cyclin E- and A-associated kinase activities. Inhibition of ATM kinase prevents induction of senescence, implying that senescence is a consequence of DNA damage. Surprisingly, no senescence or no extensive amount of senescence is evident upon depletion of the Emi1-stabilizing factor Evi5 or Pin1, respectively. Our data suggest that maintenance of a protein stabilization/mRNA expression positive-feedback circuit fueled by Emi1 is required for accurate cell cycle progression, maintenance of DNA integrity, and prevention of cellular senescence.

PIN1, a peptidyl-prolyl-isomerase, binds a specific motif comprising a phosphorylated serine or threonine preceding a proline (p-Ser/Thr-Pro) residue in proteins. Through cis-trans isomerization, it induces conformational changes and modulates functions of many proteins that are involved in cell cycle progression, cell proliferation, and oncogenesis. PIN1 is overexpressed in hepatocellular carcinomas (HCC) and contributes to hepatocarcinogenesis. We investigated the role of PIN1 and the significance of its interaction with the inhibitor of apoptosis protein survivin in evading apoptosis in HCC cells. Using cell line and xenograft models, we determined that PIN1 overexpression inhibits apoptosis through suppression of caspase-3 and caspase-9 activity. In addition, down-regulation of survivin in PIN1-overexpressing cells attenuated the antiapoptotic effect induced by PIN1, suggesting that the inhibition of apoptosis is mediated through PIN1-survivin interaction. Coimmunoprecipitation assays showed that PIN1 interacted with survivin via the phosphorylated Thr34-Pro35 motif and enhanced binding among survivin phosphorylated at Thr34, hepatitis B X-interacting protein (HBXIP), and pro-caspase-9. Taken together, these results suggest that the inhibition of apoptosis by PIN1 in HCC cells is mediated through modulation of the antiapoptotic function of survivin by increasing its binding to pro-caspase-9 via HBXIP. Such functional interaction between PIN1 and survivin may therefore play an important role in hepatocarcinogenesis and chemoresistance.

Unlike several tumor suppressor genes, whose inactivation is due to deletions or truncating mutations, TP53 is most frequently hit by missense mutations in its DNA binding domain.

Tumor suppressor p53 is essential for checkpoint control in response to a variety of genotoxic stresses. DNA damage leads to phosphorylation on the Ser/Thr-Pro motifs of p53, which facilitates interaction with Pin1, a pSer/pThr-Pro-specific peptidyl prolyl isomerase. Pin1 is required for the timely activation of p53, resulting in apoptosis or cell cycle arrest. To investigate the physiological relationship between Pin1 and p53, we created Pin1-/-p53-/- mice. These p53-deficient mice spontaneously developed lymphomas, mainly of thymic origin, as well as generalized lymphoma infiltration into other organs, including the liver, kidneys and lungs. Ablation of Pin1, in addition to p53, accelerated the thymic hyperplasia, but the thymocytes in these Pin1-/-p53-/- mice did not infiltrate other organs. The thymocytes in 12-week-old Pin1-/-p53-/- mice were CD4(-)CD8(-) (double negative) and had significantly higher levels of the intracellular form of Notch1 (NIC) than the thymocytes of p53-/- or wild-type mice. Presenilin-1, a cleavage enzyme for NIC generation from full-length Notch1 was increased in the thymocytes of Pin1-/-p53-/- mice. Pin1 depletion also inhibited the degradation of NIC by proteasomes. These results suggest that both Pin1 and p53 control the normal proliferation and differentiation of thymocytes by regulating the NIC level.

Fragment density functional theory (DFT) calculation of NMR chemical shifts for several proteins (Trp-cage, Pin1 WW domain, the third IgG-binding domain of Protein G (GB3) and human ubiquitin) has been carried out. The present study is based on a recently developed automatic fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) approach but the solvent effects are included by using the PB (Poisson-Boltzmann) model. Our calculated chemical shifts of (1)H and (13)C for these four proteins are in excellent agreement with experimentally measured values and represent clear improvement over that from the gas phase calculation. However, although the inclusion of the solvent effect also improves the computed chemical shifts of (15)N, the results do not agree with experimental values as well as (1)H and (13)C. Our study also demonstrates that AF-QM/MM calculated results accurately reproduce the separation of α-helical and β-sheet chemical shifts for (13)C(α) atoms in proteins, and using the (1)H chemical shift to discriminate the native structure of proteins from decoys is quite remarkable.

Root hairs may play a critical role in nutrient acquisition of plants grown under elevated CO(2) . This study investigated how elevated CO(2) enhanced the development of root hairs in Arabidopsis thaliana (L.) Heynh. The plants under elevated CO(2) (800 µL L(-1)) had denser and longer root hairs, and more H-positioned cells in root epidermis than those under ambient CO(2) (350 µL L(-1)). The elevated CO(2) increased auxin production in roots. Under elevated CO(2) , application of either 1-naphthoxyacetic acid (1-NOA) or N-1-naphthylphthalamic acid (NPA) blocked the enhanced development of root hairs. The opposite was true when the plants under ambient CO(2) were treated with 1-naphthylacetic acid (NAA), an auxin analogue. Furthermore, the elevated CO(2) did not enhance the development of root hairs in auxin-response mutants, axr1-3, and auxin-transporter mutants, axr4-1, aux1-7 and pin1-1. Both elevated CO(2) and NAA application increased expressions of caprice, triptychon and rho-related protein from plants 2, and decreased expressions of werewolf, GLABRA2, GLABRA3 and the transparent testa glabra 1, genes related to root-hair development, while 1-NOA and NPA application had an opposite effect. Our study suggests that elevated CO(2) enhanced the development of root hairs in Arabidopsis via the well-characterized auxin signalling and transport that modulate the initiation of root hairs and the expression of its specific genes.

The peptidyl-prolyl isomerase Pin1 is over-expressed in several cancer tissues is a potential prognostic marker in prostate cancer, and Pin1 ablation can suppress tumorigenesis in breast and prostate cancers. Pin1 can co-operate with activated ErbB2 or Ras to enhance tumorigenesis. It does so by regulating the activity of proteins that are essential for gene expression and cell proliferation. Several targets of Pin1 such as c-Myc, the Androgen Receptor, Estrogen Receptor-alpha, Cyclin D1, Cyclin E, p53, RAF kinase and NCOA3 are deregulated in cancer. At the posttranscriptional level, emerging evidence indicates that Pin1 also regulates mRNA decay of histone mRNAs, GM-CSF, Pth, and TGFβ mRNAs by interacting with the histone mRNA specific protein SLBP, and the ARE-binding proteins AUF1 and KSRP, respectively. To understand how Pin1 may affect mRNA abundance on a genome-wide scale in mammalian cells, we used RNAi along with DNA microarrays to identify genes whose abundance is significantly altered in response to a Pin1 knockdown. Functional scoring of differentially expressed genes showed that Pin1 gene targets control cell adhesion, leukocyte migration, the phosphatidylinositol signaling system and DNA replication. Several mRNAs whose abundance was significantly altered by Pin1 knockdown contained AU-rich element (ARE) sequences in their 3' untranslated regions. We identified HuR and AUF1 as Pin1 interacting ARE-binding proteins in vivo. Pin1 was also found to stabilize all core histone mRNAs in this study, thereby validating our results from a previously published study. Statistical analysis suggests that Pin1 may target the decay of essential mRNAs that are inherently unstable and have short to medium half-lives. Thus, this study shows that an important biological role of Pin1 is to regulate mRNA abundance and stability by interacting with specific RNA-binding proteins that may play a role in cancer progression.

Naturally occurring phenethyl isothiocyanate (PEITC) selectively inhibits growth of cancer cells by causing apoptosis, but the mechanism of cell death induction is not fully understood. We now show, for the first time, that growth factor adapter protein p66(Shc) is indispensable for PEITC-induced apoptosis. Mouse embryonic fibroblasts derived from p66(Shc) knockout mice were significantly more resistant to PEITC-mediated growth inhibition, cytoplasmic histone-associated apoptotic DNA fragmentation, and caspase-3 activation compared with wild-type fibroblasts. The PEITC treatment resulted in induction as well as increased Ser(36) phosphorylation of p66(Shc) in PC-3 and LNCaP human prostate cancer cells. Knockdown of p66(Shc) protein conferred significant protection against PEITC-mediated cytoplasmic histone-associated DNA fragmentation as well as production of reactive oxygen species in both PC-3 and LNCaP cells. The PEITC-treated PC-3 and LNCaP cells exhibited increased binding of p66(Shc) with prolyl isomerase Pin1, a protein implicated in translocation of p66(Shc) to mitochondria. Consistent with these results, treatment of PC-3 cells with PEITC resulted in translocation of p66(Shc) to the mitochondria as judged by immunoblotting using cytosolic and mitochondrial fractions and immunofluorescence microscopy. Growth suppression and apoptosis induction in tumor xenografts in vivo by oral administration of PEITC to the PC-3 tumor-bearing male athymic mice were accompanied by statistically significant increase in the level of Ser(36)-phosphorylated p66(Shc). Collectively, these results provide novel insight into the critical role of p66(Shc) in regulation of PEITC-induced apoptotic cell death in human prostate cancer cells.

Our understanding of phyllotaxis is still largely based on surgical and pharmacological experiments carried out before 1970. Recent experiments implicate the plant hormone auxin in the regulation of phyllotaxis. A recent paper shows how the polar auxin transport mutant, pin1-1, which fails to make flowers, affects the expression of well known meristem genes. This work opens the door for the genetic analysis of phyllotaxis.

Phyllotaxy describes the geometric arrangement of leaves and is important for plant productivity. Auxin is well known to regulate phyllotactic patterns via PIN1-dependent auxin polar transport, and studies of maize (Zea mays) aberrant phyllotaxy1 (abph1) mutants suggest the importance of auxin and cytokinin signaling for control of phyllotaxy. However, whether additional regulators control these patterns is poorly understood. Here, we report a new dominant maize mutant, Aberrant phyllotaxy2 (Abph2), in which the shoot meristems are enlarged and the phyllotactic pattern switches from alternate to decussate. Map-based cloning revealed that the Abph2 mutation was caused by transposition of a glutaredoxin gene, MALE STERILE CONVERTED ANTHER1 (MSCA1), which gained an altered expression pattern in Abph2 mutant embryos. msca1 loss-of-function mutants have reduced meristem size and revealed a novel function of glutaredoxins in meristem growth. In addition, MSCA1 interacts with a TGA transcription factor, FASCIATED EAR4, suggesting a novel regulatory module for regulating shoot meristem size.

Endocrine therapies that inhibit estrogen receptor (ER)-α signaling are the most common and effective treatment for ER-α-positive breast cancer. However, the use of these agents is limited by the frequent development of resistance. The aim of this study was to elucidate the mechanisms by which downregulation of CDK10 expression confers resistance to tamoxifen in breast cancer. Here, we show that peptidyl-prolyl isomerase Pin1 downregulates CDK10 protein as a result of its interaction with and ubiquitination of CDK10, thereby affecting CDK10-dependent Raf-1 phosphorylation (S338). Pin1(-/-) mouse embryonic fibroblasts (MEFs) show higher CDK10 expression than Pin1(+/+) MEFs, whereas CDK10 protein was downregulated in the rescued Pin1(-/-) MEFs after reexpression of Pin1. Pin1 silencing in SKBR-3 and MCF7 cells increased the CDK10 expression. In human tamoxifen-resistant breast cancer and tamoxifen-resistant MCF7 cells, immunohistochemical staining and immunoblotting analysis shows an inverse correlation between the expression of CDK10 and the degree of tamoxifen resistance. There was also a positive correlation between the high level of P-Raf-1 (Ser338) and Pin1 in human tamoxifen-resistant breast cancer and tamoxifen-resistant MCF7 (TAMR-MCF7) cells. Importantly, 4-OH tamoxifen (4-OHT), when used in combination with overexpressed CDK10 or Raf-1 inhibitor, increased cleaved PARP and DNA fragmentation to inhibit cologenic growth of MCF7 cells and Tamoxifen-resistant MCF7 cells, respectively. On the basis of these findings, we suggest that the Pin1-mediated CDK10 ubiquitination is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.