Twelve genes of the PIN family in rice were analyzed for gene and protein structures and an evolutionary relationship with reported AtPINs in Arabidopsis. Four members of <em>PIN1</em> (designated as Os<em>PIN1</em>a-d), one gene paired with AtPIN2 (OsPIN2), three members of PIN5 (OsPIN5a-c), one gene paired with AtPIN8 (OsPIN8), and three monocot-specific PINs (OsPIN9, Os<em>PIN1</em>0a, and b) were identified from the phylogenetic analysis. Tissue-specific expression patterns of nine PIN genes among them were investigated using RT-PCR and GUS reporter. The wide variations in the expression domain in different tissues of the PIN genes were observed. In general, PIN genes are up-regulated by exogenous auxin, while different responses of different PIN genes to other hormones were found.

Phosphorylation of proteins on serine or threonine residues preceding proline (Ser/Thr-Pro) is a major intracellular signaling mechanism. The phosphorylated Ser/Thr-Pro motifs in a certain subset of phosphoproteins are isomerized specifically by the peptidyl-prolyl cis-trans isomerase Pin1. This post-phosphorylation isomerization can lead to conformational changes in the substrate proteins and modulate their functions. Pin1 interacts with a number of mitotic phosphoproteins, and plays a critical role in mitotic regulation. Recent work indicates that Pin1 is overexpressed in many human cancers and plays an important role in oncogenesis. Pin1 regulates the expression of cyclin D1 by cooperating with Ras signaling and inhibiting the interaction of beta-catenin with the tumor suppressor APC and also directly stabilizing cyclin D1 protein. Furthermore, PIN1 is an E2F target gene essential for the Neu/Ras-induced transformation of mammary epithelial cells. Pin1 is also a critical regulator of the tumor suppressor p53 during DNA damage response. Given its role in cell growth control and oncogenesis, Pin1 could represent a new anti-cancer target.

Activation of Cdc2, is the universal event controlling the onset of mitosis. In higher eukaryotes, Cdc2 activity is in part regulated by inhibitory phosphorylation of Thr14 and Tyr15, catalyzed by Wee1 and Myt1, which prevents catastrophic premature entry into mitosis. In this study we defined the function of Myt1 by overexpression studies in both S. pombe and a human osteosarcoma cell line. Similar to Wee1, overexpression of human Myt1 prevented entry into mitosis in both cell types; however, Myt1 catalytic activity was not essential for the cell cycle delay observed with human cells. Myt1 expression was restricted to proliferating cells. Furthermore, we detected no major decline in Myt1 protein abundance prior to the entry into mitosis, which coincides with the loss of Myt1 activity. We localized mitotic phosphoepitopes, recognized by the monoclonal antibody MPM-2, to the C-terminal domain of Myt1. The mitotic peptidyl-prolyl isomerase, Pin1, was able to associate with this domain in a phosphorylation-dependent manner. Truncation of the C-terminal domain of Myt1 prevented its ability to induce G(2)/M phase arrest in overexpression studies in human cells and dramatically reduced its ability to phosphorylate Cdc2 in vitro. We demonstrate that the C-terminal domain of Myt1 was required for recruitment of Cdc2, and we infer that this domain lies in the cytoplasm because it can interact with and is phosphorylated by Cdc2. In conclusion, we propose that Myt1 can negatively regulate Cdc2/cyclin B1 and inhibit G(2)/M progression by two means, both of which require the C-terminal domain; first, Myt1 can bind and sequester Cdc2/cyclin B1 in the cytoplasm preventing entry into the nucleus, and, second, it can phosphorylate associated Cdc2/cyclin B1 at Thr14 and Tyr15 thus inhibiting its catalytic activity.

BACKGROUND

Histone deacetylase (HDAC) inhibitors are currently undergoing clinical evaluation as anti-cancer agents. Dietary constituents share certain properties of HDAC inhibitor drugs, including the ability to induce global histone acetylation, turn-on epigenetically-silenced genes, and trigger cell cycle arrest, apoptosis, or differentiation in cancer cells. One such example is sulforaphane (SFN), an isothiocyanate derived from the glucosinolate precursor glucoraphanin, which is abundant in broccoli. Here, we examined the time-course and reversibility of SFN-induced HDAC changes in human colon cancer cells.

RESULTS

Cells underwent progressive G2/M arrest over the period 6-72 h after SFN treatment, during which time HDAC activity increased in the vehicle-treated controls but not in SFN-treated cells. There was a time-dependent loss of class I and selected class II HDAC proteins, with HDAC3 depletion detected ahead of other HDACs. Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment. Among the protein partners implicated in the HDAC3 turnover mechanism, silencing mediator for retinoid and thyroid hormone receptors (SMRT) was phosphorylated in the nucleus within 6 h of SFN treatment, as was HDAC3 itself. Co-immunoprecipitation assays revealed SFN-induced dissociation of HDAC3/SMRT complexes coinciding with increased binding of HDAC3 to 14-3-3 and peptidyl-prolyl cis/trans isomerase 1 (Pin1). Pin1 knockdown blocked the SFN-induced loss of HDAC3. Finally, SFN treatment for 6 or 24 h followed by SFN removal from the culture media led to complete recovery of HDAC activity and HDAC protein expression, during which time cells were released from G2/M arrest.

CONCLUSIONS

The current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor complex and enhanced binding of HDAC3 to 14-3-3 or Pin1. In the cytoplasm, release of HDAC3 from 14-3-3 followed by nuclear import is postulated to compete with a Pin1 pathway that directs HDAC3 for degradation. The latter pathway predominates in colon cancer cells exposed continuously to SFN, whereas the former pathway is likely to be favored when SFN has been removed within 24 h, allowing recovery from cell cycle arrest.

The plant hormones strigolactones and smoke-derived karrikins are butenolide signals that control distinct aspects of plant development. Perception of both molecules in Arabidopsis thaliana requires the F-box protein MORE AXILLARY GROWTH2 (MAX2). Recent studies suggest that the homologous SUPPRESSOR OF MAX2 1 (SMAX1) in Arabidopsis and DWARF53 (D53) in rice (Oryza sativa) are downstream targets of MAX2. Through an extensive analysis of loss-of-function mutants, we demonstrate that the Arabidopsis SMAX1-LIKE genes SMXL6, SMXL7, and SMXL8 are co-orthologs of rice D53 that promote shoot branching. SMXL7 is degraded rapidly after treatment with the synthetic strigolactone mixture rac-GR24. Like D53, SMXL7 degradation is MAX2- and D14-dependent and can be prevented by deletion of a putative P-loop. Loss of SMXL6,7,8 suppresses several other strigolactone-related phenotypes in max2, including increased auxin transport and PIN1 accumulation, and increased lateral root density. Although only SMAX1 regulates germination and hypocotyl elongation, SMAX1 and SMXL6,7,8 have complementary roles in the control of leaf morphology. Our data indicate that SMAX1 and SMXL6,7,8 repress karrikin and strigolactone signaling, respectively, and suggest that all MAX2-dependent growth effects are mediated by degradation of SMAX1/SMXL proteins. We propose that functional diversification within the SMXL family enabled responses to different butenolide signals through a shared regulatory mechanism.

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle-tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.

Protein serine-threonine kinase casein kinase II (CK2) is involved in a myriad of cellular processes including cell growth and proliferation through its phosphorylation of hundreds of substrates, yet how CK2 function is regulated is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344). We use protein semisynthesis to show that phosphorylation of Thr344 increases the cellular stability of CK2α by strengthening its interaction with Pin1, whereas glycosylation of Ser347 seems to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with site-specifically phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that the kinase substrate selectivity of CK2 is modulated by these specific post-translational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.

To identify molecular mechanisms controlling vein patterns, we analyzed scarface (sfc) mutants. sfc cotyledon and leaf veins are largely fragmented, unlike the interconnected networks in wild-type plants. SFC encodes an ADP ribosylation factor GTPase activating protein (ARF-GAP), a class with well-established roles in vesicle trafficking regulation. Quadruple mutants of SCF and three homologs (ARF-GAP DOMAIN1, 2, and 4) showed a modestly enhanced vascular phenotype. Genetic interactions between sfc and pinoid and between sfc and gnom suggest a possible function for SFC in trafficking of auxin efflux regulators. Genetic analyses also revealed interaction with cotyledon vascular pattern2, suggesting that lipid-based signals may underlie some SFC ARF-GAP functions. To assess possible roles for SFC in auxin transport, we analyzed sfc roots, which showed exaggerated responses to exogenous auxin and higher auxin transport capacity. To determine whether PIN1 intracellular trafficking was affected, we analyzed PIN1:green fluorescent protein (GFP) dynamics using confocal microscopy in sfc roots. We found normal PIN1:GFP localization at the apical membrane of root cells, but treatment with brefeldin A resulted in PIN1 accumulating in smaller and more numerous compartments than in the wild type. These data suggest that SFC is required for normal intracellular transport of PIN1 from the plasma membrane to the endosome.

Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors. The structures of the bound high-affinity peptides identify a type-I beta-turn conformation for Pin1 prolyl peptide isomerase domain-peptide binding and an extensive molecular interface for high-affinity recognition. Moreover, these structures suggest chemical elements that may further improve the affinity and pharmacological properties of future peptide-based Pin inhibitors. Finally, an intramolecular hydrogen bond observed in both peptide complexes mimics the cyclic conformation of FK506 and rapamycin. Both FK506 and rapamycin are clinically important inhibitors of other peptidyl-prolyl cis-trans isomerases. This comparative discovery suggests that a cyclic peptide polyketide bridge, like that found in FK506 and rapamycin or a similar linkage, may significantly improve the binding affinity of structure-based Pin1 inhibitors.

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML-RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.

Proline-directed phosphorylation is a posttranslational modification that is instrumental in regulating signaling from the plasma membrane to the nucleus, and its dysregulation contributes to cancer development. Protein interacting with never in mitosis A1 (Pin1), which is overexpressed in many types of cancer, isomerizes specific phosphorylated Ser/Thr-Pro bonds in many substrate proteins, including glycolytic enzyme, protein kinases, protein phosphatases, methyltransferase, lipid kinase, ubiquitin E3 ligase, DNA endonuclease, RNA polymerase, and transcription activators and regulators. This Pin1-mediated isomerization alters the structures and activities of these proteins, thereby regulating cell metabolism, cell mobility, cell cycle progression, cell proliferation, cell survival, apoptosis and tumor development.

The C-terminal domain of the RNA polymerase (RNAP) II largest subunit (CTD) plays a critical role in coordinating multiple events in pre-mRNA transcription and processing. Previously we reported that the peptidyl prolyl isomerase Pin1 modulates RNAP II function during the cell cycle. Here we provide evidence that Pin1 affects multiple aspects of RNAP II function via its regulation of CTD phosphorylation. Using chromatin immunoprecipitation (ChIP) assays with CTD phospho-specific antibodies, we confirm that RNAP II displays a dynamic association with specific genes during the cell cycle, preferentially associating with transcribed genes in S phase, while disassociating in M phase in a matter that correlates with changes in CTD phosphorylation. Using inducible Pin1 cell lines, we show that Pin1 overexpression is sufficient to release RNAP II from chromatin, which then accumulates in a hyperphosphorylated form in nuclear speckle-associated structures. In vitro transcription assays show that Pin1 inhibits transcription in nuclear extract, while an inactive Pin1 mutant in fact stimulates it. Several assays indicate that the inhibition largely reflects Pin1 activity during transcription initiation and not elongation, suggesting that Pin1 modulates CTD phosphorylation, and RNAP II activity, during an early stage of the transcription cycle.

OBJECTIVE

The peptidyl prolyl isomerase Pin1 frequently is overexpressed in hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) is the most common etiologic agent in HCC, and its encoded X-protein (HBx) is oncogenic and possesses a serine-proline motif that may bind Pin1. The role of Pin1 in hepatocarcinogenesis, particularly in HBV-related HCC, was investigated.

METHODS

Immunohistochemical staining was performed to evaluate the prevalence of Pin1 overexpression in HCCs of different etiologies. Glutathione S-transferase pull-down and co-immunoprecipitation experiments were used to validate the physical interaction between Pin1 and HBx. Reporter assay, cell proliferation assay, and xenotransplantation experiments were used to show the functional consequence and importance of Pin1-HBx interaction in hepatocarcinogenesis.

RESULTS

We showed preferential Pin1 overexpression in HBV-related tumors and confirmed the interaction between Pin1 and HBx at the specific serine-proline motif. Pin1 overexpression increased the protein stability of HBx. Furthermore, HBx-mediated transactivation was enhanced by co-expression of Pin1. HepG2 expressing Pin1 and HBx showed a synergistic increase in cellular proliferation, as compared with cells expressing Pin1 or HBx alone. Furthermore, concomitant expression of Pin1 and HBx in the nontumorigenic human hepatocyte cell line MIHA led to a synergistic increase in tumor growth. Finally, in Hep3B cells with suppressed Pin1 expression, HBx-enhanced tumor growth in nude mice was abrogated.

CONCLUSIONS

Pin1 binds HBx to enhance hepatocarcinogenesis in HBV-infected hepatocytes. The discovery of an interaction between Pin1 and HBx will further our understanding of the molecular pathogenic mechanism of HBV-related HCC in human beings.

Hormones, such as auxin and cytokinin, are involved in the complex molecular network that regulates the coordinated development of plant organs. Genes controlling ovule patterning have been identified and studied in detail; however, the roles of auxin and cytokinin in ovule development are largely unknown. Here we show that key cytokinin pathway genes, such as isopentenyltransferase and cytokinin receptors, are expressed during ovule development. Also, in a cre1-12 ahk2-2 ahk3-3 triple mutant with severely reduced cytokinin perception, expression of the auxin efflux facilitator PIN-FORMED 1 (PIN1) was severely reduced. In sporocyteless/nozzle (spl/nzz) mutants, which show a similar phenotype to the cre1-12 ahk2-2 ahk3-3 triple mutant, PIN1 expression is also reduced. Treatment with the exogenous cytokinin N(6)-benzylaminopurine also altered both auxin distribution and patterning of the ovule; this process required the homeodomain transcription factor BELL1 (BEL1). Thus, this article shows that cytokinin regulates ovule development through the regulation of PIN1. Furthermore, the transcription factors BEL1 and SPL/NZZ, previously described as key regulators of ovule development, are needed for the auxin and cytokinin signaling pathways for the correct patterning of the ovule.

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.

Protein phosphorylation on serine or threonine residues preceding proline (Ser/Thr-Pro) plays an essential role for regulating various cellular processes, including cell cycle progression. Although phosphorylation has been proposed to regulate the function of a protein by inducing conformational changes, much less is known about what phosphate additions actually do and how the functions of phosphoproteins are coordinated. Proline is important for determining protein structure because it exists in cis or trans conformation and can put kinks into a polypeptide chain. We have shown that phosphorylation on Ser/Thr-Pro motifs reduces the cis/trans isomerization rate of Ser/Thr-Pro bonds. At the same time, proteins containing phosphorylated Ser/Thr-Pro motifs are substrates for the prolyl isomerase Pin1. The WW domain of Pin1 acts as a phosphoserine/threonine-binding module binding a defined subset of mitosis-specific phosphoproteins, such as Cdc25 and tau. These interactions target the enzymatic activity of Pin1 close to its substrates. In contrast to other prolyl isomerases (peptidyl-prolyl isomerases, PPlases), Pin1 has an extremely high degree of substrate specificity, specifically isomerizing phosphorylated Ser/Thr-Pro bonds. Therefore, Pin1 binds and regulates the function of a defined subset of phosphoproteins. Furthermore, inhibiting Pin1 function is lethal for dividing cells. Interestingly, Pin1, which can restore the biological function of phosphorylated tau, is sequestered in the neurofibrillary tangles in Alzheimer's brains. Thus, we have proposed a novel signaling regulatory mechanism, where protein phosphorylation creates binding sites for Pin1, which can then latch on to and isomerize the phosphorylated Ser/Thr-Pro peptide bond. In turn, this may change the shape of the protein, regulating its activity, dephosphorylation, degradation or location in the cell. This new post-phosphorylation regulatory mechanism appears to play an important role in normal cell function, such as mitotic progression, and in the pathogenesis of some human pathologies, such as Alzheimer's disease.

Promyelocytic leukemia protein (PML) is an important regulator due to its role in numerous cellular processes including apoptosis, viral infection, senescence, DNA damage repair, and cell cycle regulation. Despite the role of PML in many cellular functions, little is known about the regulation of PML itself. We show that PML stability is regulated through interaction with the peptidyl-prolyl cis-trans isomerase Pin1. This interaction is mediated through four serine-proline motifs in the C terminus of PML. Binding to Pin1 results in degradation of PML in a phosphorylation-dependent manner. Furthermore, our data indicate that sumoylation of PML blocks the interaction, thus preventing degradation of PML by this pathway. Functionally, we show that in the MDA-MB-231 breast cancer cell line modulating levels of Pin1 affects steady-state levels of PML. Furthermore, degradation of PML due to Pin1 acts both to protect these cells from hydrogen peroxide-induced death and to increase the rate of proliferation. Taken together, our work defines a novel mechanism by which sumoylation of PML prevents Pin1-dependent degradation. This interaction likely occurs in numerous cell lines and may be a pathway for oncogenic transformation.

Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.

Adriamycin (ADR) is a chemotherapeutic for the treatment of solid tumors. This quinone-containing anthracycline is well known to produce large amounts of reactive oxygen species (ROS) in vivo. A common complaint of patients undergoing long-term treatment with ADR is somnolence, often referred to as "chemobrain." While ADR itself does not cross the blood brain barrier (BBB), we recently showed that ADR administration causes a peripheral increase in tumor necrosis factor alpha (TNF-alpha), which migrates across the BBB and leads to inflammation and oxidative stress in brain, most likely contributing to the observed decline in cognition. In the current study, we measured levels of the antioxidant glutathione (GSH) in brains of mice injected intraparitoneally (i.p.) with ADR, as well as the levels and activities of several enzymes involved in brain GSH metabolism. We observed significantly decreased GSH levels, as well as altered GSH/GSSG ratio in brains of ADR treated mice relative to saline-treated controls. Also observed in brains of ADR treated mice were increased levels of glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). We also observed increased activity of GPx, but a significant reduction in GST and GR activity in mice brain, 72 h post i.p. injection of ADR (20 mg/kg body weight). Furthermore, we used redox proteomics to identify specific proteins that are oxidized and/or have differential levels in mice brains as a result of a single i.p. injection of ADR. Visinin like protein 1 (VLP1), peptidyl prolyl isomerase 1 (Pin1), and syntaxin 1 (SYNT1) showed differential levels in ADR treated mice relative to saline-treated controls. Triose phosphate isomerase (TPI), enolase, and peroxiredoxin 1 (PRX-1) showed significantly increased specific carbonylation in ADR treated mice brain. These results further support the notion ADR induces oxidative stress in brain despite not crossing the BBB, and that antioxidant intervention may prevent ADR-induced cognitive dysfunction.

In response to intense stress, the tumor protein p53 (p53) tumor suppressor rapidly mounts a direct mitochondrial death program that precedes transcription-mediated apoptosis. By eliminating severely damaged cells, this pathway contributes to tumor suppression as well as to cancer cell killing induced by both genotoxic drugs and non-genotoxic p53-reactivating molecules. Here we have explored the role had in this pathway by the prolyl-isomerase Pin1 (peptidylprolyl cis/trans isomerase, NIMA-interacting 1), a crucial transducer of p53's phosphorylation into conformational changes unleashing its pro-apoptotic activity. We show that Pin1 promotes stress-induced localization of p53 to mitochondria both in vitro and in vivo. In particular, we demonstrate that upon stress-induced phosphorylation of p53 on Ser46 by homeodomain interacting protein kinase 2, Pin1 stimulates its mitochondrial trafficking signal, that is, monoubiquitination. This pathway is induced also by the p53-activating molecule RITA, and we demonstrate the strong requirement of Pin1 for the induction of mitochondrial apoptosis by this compound. These findings have significant implications for treatment of p53-expressing tumors and for prospective use of p53-activating compounds in clinics.

Short-Root (SHR) is a well-characterized regulator of radial patterning and indeterminacy of the Arabidopsis (Arabidopsis thaliana) primary root. However, its role during the elaboration of root system architecture remains unclear. We report that the indeterminate wild-type Arabidopsis root system was transformed into a determinate root system in the shr mutant when growing in soil or agar. The root growth behavior of the shr mutant results from its primary root apical meristem failing to initiate cell division following germination. The inability of shr to reactivate mitotic activity in the root apical meristem is associated with the progressive reduction in the abundance of auxin efflux carriers, PIN-FORMED1 (PIN1), PIN2, PIN3, PIN4, and PIN7. The loss of primary root growth in shr is compensated by the activation of anchor root primordia, whose tissues are radially patterned like the wild type. However, SHR function is not restricted to the primary root but is also required for the initiation and patterning of lateral root primordia. In addition, SHR is necessary to maintain the indeterminate growth of lateral and anchor roots. We conclude that SHR regulates a wide array of Arabidopsis root-related developmental processes.

OBJECTIVE

The peptidyl-prolyl isomrase Pin1 plays a catalytic role in oncogenesis in solid cancers, including prostate cancer. In the present study, we sought to determine the potential of Pin1-targeted gene silencing in inhibiting cellular growth and tumorigenicity in prostate cancer.

METHODS

A retrovirus-mediated RNA interference targeting Pin1 was expressed in PC3 and LNCaP cells, and cell growth and several transformed properties were investigated.

RESULTS

The stable expression of Pin1-specific small interfering RNA constructs in PC3 and LNCaP cells significantly reduced cellular proliferation, colony formation, migration, and invasion but strongly enhanced the apoptotic response induced by serum depletion or treatment with anticancer agents. Furthermore, Pin1 depletion significantly suppressed tumorigenic potential in athymic mice, resulting in the inhibition of both tumor growth and angiogeneisis.

CONCLUSIONS

These results strongly suggest that Pin1 plays an important role not only in tumorigenesis but also in the maintenance of the transformed phenotype in prostate cancer cells. Hence, Pin1 may serve as a promising therapeutic target, particularly for recurrent prostate tumors.