Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a ubiquitin-protein ligase containing multiple WW domains. We have previously demonstrated the association between the WW domains of Nedd4 and PPxY (PY) motifs of the epithelial sodium channel (ENaC). In this paper, we report the assignment of backbone 1H alpha, 1HN, 15N, 13C', 13C alpha, and aliphatic 13C resonances of a fragment of rat Nedd4 (rNedd4) containing the two C-terminal WW domains, WW(II+III), complexed to a PY motif-containing peptide derived from the beta subunit of rat ENaC, the betaP2 peptide. The secondary structures of these two WW domains, determined from chemical shifts of 13C alpha and 13C beta resonances, are virtually identical to those of the WW domains of the Yes-associated protein YAP65 and the peptidyl-prolyl isomerase Pin1. Triple resonance experiments that detect the 1H alpha chemical shift were necessary to complete the chemical shift assignment, owing to the large number of proline residues in this fragment of rNedd4. A new experiment, which correlates sequential residues via their 15N nuclei and also detects 1H alpha chemical shifts, is introduced and its utility for the chemical shift assignment of sequential proline residues is discussed. Data collected on the WW(II+III)-betaP2 complex indicate that these WW domains have different affinities for the betaP2 peptide.

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, [Formula: see text] and apparent Michaelis constants, [Formula: see text]. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific (13)C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide (13)C-(1)H constant time HSQC spectra to determine [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

Allostery is an essential means for regulating biomolecular functions and provides unique opportunities for drug design, yet our ability to elucidate allosteric mechanisms remains limited. Here, based on extensive molecular dynamics simulations, we present an atomistic picture of the pathways mediating the allosteric regulation of the PPIase domain of Pin1 by its WW domain. Two pathways jointly propagate the action of substrate-WW binding to produce closure and rigidification of three PPIase catalytic-site loops. One pathway preexists in the apo protein, but remains dormant until substrate-WW binding completes the second. The reduction in conformational entropy and preorganization of the catalytic-site loops observed here may explain why substrate-WW binding enhances ligand affinity and catalytic activity of the PPIase domain and suggest a combination drug therapy for Pin1-related diseases. Whereas the traditional view of allostery has emphasized conformational transition, our study identifies a distinct role of conformational dynamics in eliciting allostery.

Here we show that prolyl isomerase Pin1 is involved in the Abeta production central to the pathogenesis of Alzheimer's disease. Enzyme immunoassay of brains of the Pin1-deficient mice revealed that production of Abeta40 and Abeta42 was lower than that of the wild-type mice, indicating that Pin1 promotes Abeta production in the brain. GST-Pin1 pull-down and immunoprecipitation assay revealed that Pin1 binds phosphorylated Thr668-Pro of C99. In the Pin1-/- MEF transfected with C99, Pin1 co-transfection enhanced the levels of Abeta40 and Abeta42 compared to that without Pin1 co-transfection. In COS7 cells transfected with C99, Pin1 co-transfection enhanced the generation of Abeta40 and Abeta42, and reduced the expression level of C99, facilitating the C99 turnover. Thus, Pin1 interacts with C99 and promotes its gamma-cleavage, generating Abeta40 and Abeta42. Further, GSK3 inhibitor lithium blocked Pin1 binding to C99 by decreasing Thr668 phosphorylation and attenuated Abeta generation, explaining the inhibitory effect of lithium on Abeta generation.

Two new amide isosteres of Ser-cis-Pro and Ser-trans-Pro dipeptides were designed and stereoselectively synthesized to be incorporated into potential inhibitors of the phosphorylation-dependent peptidylprolyl isomerase Pin1, an essential regulator of the cell cycle. The cis mimic, the (Z)-alkene isomer, was formed through the use of a Still-Wittig [2,3]-sigmatropic rearrangement, while the trans mimic, the (E)-alkene, was synthesized through the use of an Ireland-Claisen [3,3]-sigmatropic rearrangement. Starting from N-Boc-Ser(OBn)-N(OMe)Me, both mimics were synthesized in Boc-protected form suitable for peptide synthesis with an overall yield of 20% in 10 steps for the cis mimic and 13% in eight steps for the trans mimic.

Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for ∼100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host.

Pin1 is a unique regulator, which catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified CRTC2 as a Pin1-binding protein by overexpressing Pin1 with Myc and FLAG tags in mouse livers and subsequent purification of the complex containing Pin1. The association between Pin1 and CRTC2 was observed not only in overexpression experiments but also endogenously in the mouse liver. Interestingly, Ser(136) in the nuclear localization signal of CRTC2 was shown to be involved in the association with Pin1. Pin1 overexpression in HepG2 cells attenuated forskolin-induced nuclear localization of CRTC2 and cAMP-responsive element (CRE) transcriptional activity, whereas gene knockdown of Pin1 by siRNA enhanced both. Pin1 also associated with CRTC1, leading to their cytosol localization, essentially similar to the action of CRTC2. Furthermore, it was shown that CRTC2 associated with Pin1 did not bind to CREB. Taken together, these observations indicate the association of Pin1 with CRTC2 to decrease the nuclear CBP·CRTC·CREB complex. Indeed, adenoviral gene transfer of Pin1 into diabetic mice improved hyperglycemia in conjunction with normalizing phosphoenolpyruvate carboxykinase mRNA expression levels, which is regulated by CRE transcriptional activity. In conclusion, Pin1 regulates CRE transcriptional activity, by associating with CRTC1 or CRTC2.

The phosphorylation-specific peptidyl prolyl cis/trans isomerase (PPIase) Pin1 in humans and its homologues in yeast and animal species play an important role in cell cycle regulation. These PPIases consist of an NH(2)-terminal WW domain that binds to specific phosphoserine- or phosphothreonine-proline motifs present in a subset of phosphoproteins and a COOH-terminal PPIase domain that specifically isomerizes the phosphorylated serine/threonine-proline peptide bonds. Here, we describe the isolation of MdPin1, a Pin1 homologue from the plant species apple (Malus domestica) and show that it has the same phosphorylation-specific substrate specificity and can be inhibited by juglone in vitro, as is the case for Pin1. A search in the plant expressed sequence tag data bases reveals that the Pin1-type PPIases are present in various plants, and there are multiple genes in one organism, such as soybean (Glycine max) and tomato (Lycopersicon esculentum). Furthermore, all these plant Pin1-type PPIases, including AtPin1 in Arabidopsis thaliana, do not have a WW domain, but all contain a four-amino acid insertion next to the phospho-specific recognition site of the active site. Interestingly, like Pin1, both MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a temperature-sensitive mutation in the Pin1 homologue ESS1/PTF1 gene in Saccharomyces cerevisiae. However, deleting the extra four amino acid residues abolished the ability of AtPin1 to rescue the yeast mutation under non-overexpression conditions, indicating that these extra amino acids may be important for mediating the substrate interaction of plant enzymes. Finally, expression of MdPin1 is tightly associated with cell division both during apple fruit development in vivo and during cell cultures in vitro. These results have demonstrated that phosphorylation-specific PPIases are highly conserved functionally in yeast, animal, and plant species. Furthermore, the experiments suggest that although plant Pin1-type enzymes do not have a WW domain, they may fulfill the same functions as Pin1 and its homologues do in other organisms.

We have previously demonstrated that DNA damage leads to stabilization and accumulation of Che-1, an RNA polymerase II-binding protein that plays an important role in transcriptional activation of p53 and in maintenance of the G(2)/M checkpoint. Here we show that Che-1 is down-regulated during the apoptotic process. We found that the E3 ligase HMD2 physically and functionally interacts with Che-1 and promotes its degradation via the ubiquitin-dependent proteasomal system. Furthermore, we found that in response to apoptotic stimuli Che-1 interacts with the peptidyl-prolyl isomerase Pin1 and that conformational changes generated by Pin1 are required for Che-1/HDM2 interaction. Notably, a Che-1 mutant lacking the capacity to bind Pin1 exhibits an increased half-life and this correlates with a diminished apoptosis in response to genotoxic stress. Our results establish Che-1 as a new Pin1 and HDM2 target and confirm its important role in the cellular response to DNA damage.

PIN1, an isomerase that causes conformational changes in proteins, plays an important role in mammary epithelial cell growth both physiologically and pathologically. Thus, genetic variants in the PIN1 gene may alter protein function and cancer risk. We have previously demonstrated an association between a PIN1 promoter variant (-842G>C; rs2233678) and risk of squamous cell carcinoma of the head and neck, a finding supported by additional functional data. In the present study, we genotyped two promoter single nucleotide polymorphisms (SNPs) (-842G>C, rs2233678 and -667T>C, rs2233679) and one synonymous SNP (Gln33Gln; G>A, rs2233682) in exon 2 to evaluate their associations with risk of sporadic breast cancer in non-Hispanic white women 55 years and younger. We found that the carriers of -842C variant alleles had decreased risk of breast cancer with an adjusted odd ratio (OR) of 0.67 and 95% confidence interval (CI) of 0.50-0.90. This reduced risk was more evident in women after reproductive age of 45 (OR = 0.63, 95% CI = 0.42-0.93), ever-smokers (OR = 0.56, 95% CI = 0.36-0.88), and ever-drinkers (OR = 0.67, 95% CI = 0.45-0.99). No such associations were observed for PIN1 -667T>C and PIN1 Gln33Gln. However, the haplotypes of these three SNPs were also associated with reduced risk of breast cancer. In conclusion, the PIN1 polymorphisms may contribute to the etiology of sporadic breast cancer in non-Hispanic white women 55 years and younger. Further validation in large population-based studies is needed.

The promyelocytic leukemia (PML) protein is a tumor suppressor that has an important role in several cellular processes, including apoptosis, viral infection, DNA damage repair, cell cycle regulation, and senescence. PML is an essential component of sub-nuclear structures called PML nuclear bodies (NBs). Our laboratory has previously demonstrated that the peptidyl-prolyl cis-trans isomerase, Pin1, binds and targets PML for degradation in a phosphorylation-dependent manner. To further elucidate the mechanisms underlying Pin1-mediated PML degradation, we aimed to identify one or more factors that promote PML phosphorylation. Here we show that treatment with U0126, an inhibitor of the ERK2 upstream kinases MEK1/2, leads to an increase in PML protein accumulation and an inhibition of the interaction between Pin1 and PML in MDA-MB-231 breast cancer cells. Consistent with this observation, phosphorylated ERK2 partially co-localized with PML NBs. Although U0126 up-regulated exogenous wild-type PML levels, it did not have an effect on the steady-state level of a mutant form of PML that is defective in binding Pin1. In addition, exogenous wild-type, but not Pin1 binding-defective PML protein expression levels were decreased by overexpression of ERK2. In contrast, knockdown of ERK2 by siRNA resulted in an increase in PML protein levels and an increase in the formation of PML NBs. Using phospho-specific antibodies, we identified Ser-403 and Ser-505 as the ERK2 targets that promote Pin1-mediated PML degradation. Finally, we demonstrated that EGF induced activation of ERK and interaction between PML and phosphorylated ERK resulting in a decrease in PML protein levels. Taken together, our results support a model in which Pin1 promotes PML degradation in an ERK2-dependent manner.

A series of peptides corresponding to isolated regions of Tau (tau) protein have been synthesized and their conformations determined by (1)H NMR spectroscopy. Immunodominant peptides corresponding to tau(224-240) and a bisphosphorylated derivative in which a single Thr and a single Ser are phosphorylated at positions 231 and 235 respectively, and which are recognized by an Alzheimer's disease-specific monoclonal antibody, were the main focus of the study. The nonphosphorylated peptide adopts essentially a random coil conformation in aqueous solution, but becomes slightly more ordered into beta-type structure as the hydrophobicity of the solvent is increased by adding up to 50% trifluoroethanol (TFE). Similar trends are observed for the bisphosphorylated peptide, with a somewhat stronger tendency to form an extended structure. There is tentative NMR evidence for a small population of species containing a turn at residues 229-231 in the phosphorylated peptide, and this is strongly supported by CD spectroscopy. A proposal that the selection of a bioactive conformation from a disordered solution ensemble may be an important step (in either tubulin binding or in the formation of PHF) is supported by kinetic data on Pro isomerization. A recent study showed that Thr231 phosphorylation affected the rate of prolyl isomerization and abolished tubulin binding. This binding was restored by the action of the prolyl isomerase Pin1. In the current study, we find evidence for the existence of both trans and cis forms of tau peptides in solution but no difference in the equilibrium distribution of cis-trans isomers upon phosphorylation. Increasing hydrophobicity decreases the prevalence of cis forms and increases the major trans conformation of each of the prolines present in these molecules. We also synthesized mutant peptides containing Tyr substitutions preceding the Pro residues and found that phosphorylation of Tyr appears to have an effect on the equilibrium ratio of cis-trans isomerization and decreases the cis content.

In Arabidopsis thaliana, lateral roots (LRs) initiate from anticlinal cell divisions of pericycle founder cells. The formation of LR primordia is regulated antagonistically by the phytohormones cytokinin and auxin. It has previously been shown that cytokinin has an inhibitory effect on the patterning events occurring during LR formation. However, the molecular players involved in cytokinin repression are still unknown. In a similar manner to protoxylem formation in Arabidopsis roots, in which AHP6 (ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6) acts as a cytokinin inhibitor, we reveal that AHP6 also functions as a cytokinin repressor during early stages of LR development. We show that AHP6 is expressed at different developmental stages during LR formation and is required for the correct orientation of cell divisions at the onset of LR development. Moreover, we demonstrate that AHP6 influences the localization of the auxin efflux carrier PIN1, which is necessary for patterning the LR primordia. In summary, we show that the inhibition of cytokinin signaling through AHP6 is required to establish the correct pattern during LR initiation.

Using the human Pin1 WW domain (hPin1 WW), we show that replacement of two nearest neighbor non-hydrogen-bonded residues on adjacent beta-strands with tryptophan (Trp) residues increases beta-sheet thermodynamic stability by 4.8 kJ mol(-1) at physiological temperature. One-dimensional NMR studies confirmed that introduction of the Trp-Trp pair does not globally perturb the structure of the triple-stranded beta-sheet, while circular dichroism studies suggest that the engineered cross-strand Trp-Trp pair adopts a side-chain conformation similar to that first reported for a designed "Trp-zipper" beta-hairpin peptide, wherein the indole side chains stack perpendicular to each other. Even though the mutated side chains in wild-type hPin1 WW are not conserved among WW domains and compose the beta-sheet surface opposite to that responsible for ligand binding, introduction of the cross-strand Trp-Trp pair effectively eliminates hPin1 WW function as assessed by the loss of binding affinity toward a natural peptide ligand. Maximizing both thermodynamic stability and the domain function of hPin1 WW by the above mentioned approach appears to be difficult, analogous to the situation with loop 1 optimization explored previously. That introduction of a non-hydrogen-bonded cross-strand Trp-Trp pair within the hPin1 WW domain eliminates function may provide a rationale for why this energetically favorable pairwise interaction has not yet been identified in WW domains or any other biologically evolved protein with known three-dimensional structure.

Esophageal carcinoma is one of the most lethal tumors, and identification of prognostic factors for patients with this disease is important. Propyl isomerase Pin1 is overexpressed in some human cancers and thought to be an important regulator of cyclinD1. However, the relationships between Pin1 expression and clinicopathologic features in patients with esophageal squamous cell carcinoma (SCC) have not been explored. Here, we investigated the role of Pin1 in association with cyclinD1 in esophageal SCC progression and its clinicopathological significance. The expressions of Pin1 and cyclinD1 were examined immunohistochemically in surgical specimens from 119 esophageal SCC patients. The expression levels of Pin1 and cyclinD1 in 6 esophageal SCC-derived cell lines were compared with those in an immortalized human esophageal cell line by western blotting. Pin1 overexpression was correlated with lymph node metastasis (P=0.0384), and its expression was related to cyclinD1 expression. Pin1 expression was correlated with poor prognosis in esophageal SCC patients (P=0.0044), and found to be an independent prognostic factor (P=0.0277). Pin1 was overexpressed in 5 of 6 esophageal SCC-derived cell lines compared with immortalized esophageal keratinocytes. Moreover, the Pin1 level was correlated with the cyclinD1 level in 4 of the 6 cell lines. In conclusion, Pin1 expression is correlated with cyclinD1 expression and may be a useful prognostic factor for esophageal SCC.

BACKGROUND

Theauxin efflux carrier PIN1 is a key mediator of polar auxin transport in developing plant tissues. This is why factors that are supposed to be involved in auxin distribution are frequently tested in the regulation of PIN1 expression. As a result, diverse aspects of PIN1 expression are dispersed across dozens of papers entirely devoted to other specific topics related to the auxin pathway. Integration of these puzzle pieces about PIN1 expression revealed that, along with a recurring pattern, some features of PIN1 expression varied from article to article. To determine if this uncertainty is related to the specific foci of articles or has a basis in the variability of PIN1 gene activity, we performed a comprehensive 3D analysis of PIN1 expression patterns in Arabidopsis thaliana roots.

RESULTS

We provide here a detailed map of PIN1 expression in the primary root, in the lateral root primordia and at the root-shoot junction. The variability in PIN1 expression pattern observed in individual roots may occur due to differences in auxin distribution between plants. To simulate this effect, we analysed PIN1 expression in the roots from wild type seedlings treated with different IAA concentrations and pin mutants. Most changes in PIN1 expression after exogenous IAA treatment and in pin mutants were also recorded in wild type but with lower frequency and intensity. Comparative studies of exogenous auxin effects on PIN1pro:GUS and PIN1pro:PIN1-GFP plants indicated that a positive auxin effect is explicit at the level of PIN1 promoter activity, whereas the inhibitory effect relates to post-transcriptional regulation.

CONCLUSIONS

Our results suggest that the PIN1 expression pattern in the root meristem accurately reflects changes in auxin content. This explains the variability of PIN1 expression in the individual roots and makes PIN1 a good marker for studying root meristem activity.

Neurofibrillary tangles (aggregates of cytoskeletal Tau protein) and senile plaques (aggregates mainly formed by amyloid β peptide) are two landmark lesions in Alzheimer׳s disease. Some researchers have proposed tangles, whereas others have proposed plaques, as primary lesions. For a long time, these were thought of as independent mechanisms. However, experimental evidence suggests that both lesions are intimately related. We review here some molecular pathways linking amyloid β and Tau toxicities involving, among others, glycogen synthase kinase 3β, p38, Pin1, cyclin-dependent kinase 5, and regulator of calcineurin 1. Understanding amyloid β and Tau toxicities as part of a common pathophysiological mechanism may help to find molecular targets to prevent or even treat the disease.

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

Plant vascular tissues are essential for the existence of land plants. Many studies of transcriptional regulation and cell-cell communication have revealed the process underlying the development of vascular tissues from vascular initial cells. However, the initiation of vascular cell differentiation is still a mystery. Here, we report that LONESOME HIGHWAY (LHW), which encodes a bHLH transcription factor, is expressed in pericycle-vascular mother cells at the globular embryo stage and is required for proper asymmetric cell division to generate vascular initial cells. In addition, ectopic expression of LHW elicits an ectopic auxin response. Moreover, LHW is required for the correct expression patterns of components related to auxin flow, such as PIN-FORMED 1 (PIN1), MONOPTEROS (MP) and ATHB-8, and ATHB-8 partially rescues the vascular defects of lhw. These results suggest that LHW functions as a key regulator to initiate vascular cell differentiation in association with auxin regulation.

Cyclin-dependent kinases regulate the cell cycle and transcription in higher eukaryotes. We have determined the crystal structure of the transcription kinase Cdk13 and its Cyclin K subunit at 2.0 Å resolution. Cdk13 contains a C-terminal extension helix composed of a polybasic cluster and a DCHEL motif that interacts with the bound ATP. Cdk13/CycK phosphorylates both Ser5 and Ser2 of the RNA polymerase II C-terminal domain (CTD) with a preference for Ser7 pre-phosphorylations at a C-terminal position. The peptidyl-prolyl isomerase Pin1 does not change the phosphorylation specificities of Cdk9, Cdk12, and Cdk13 but interacts with the phosphorylated CTD through its WW domain. Using recombinant proteins, we find that flavopiridol inhibits Cdk7 more potently than it does Cdk13. Gene expression changes after knockdown of Cdk13 or Cdk12 are markedly different, with enrichment of growth signaling pathways for Cdk13-dependent genes. Together, our results provide insights into the structure, function, and activity of human Cdk13/CycK.

Pin1, a conserved eukaryotic peptidyl-prolyl cis/trans isomerase, has important roles in cellular regulation. Because of its activity to switch the conformation of peptidyl-proline bonds in polypeptide chains, Pin1 operates as a binary switch, often in fate-determining pathways. Pin1 activity is usually controlled by substrate phosphorylation, but how Pin1 switches protein fates has been unclear. Here we show that Pin1 controls the degree of substrate ubiquitylation and thereby protein functions. We found that yeast Pin1 (Ess1) is essential for viability because it controls the NF-kappaB-related Spt23 transcription factor involved in unsaturated fatty-acid synthesis. High Pin1 activity results in low ubiquitylation of Spt23, which triggers Spt23 precursor processing and hence transcription factor activation. By contrast, decreased Pin1 activity leads to robust Spt23 polyubiquitylation and subsequent proteasomal degradation. Inhibition of Pin1 in mammalian cells changes the ubiquitylation status of the tumour suppressor protein p53 from oligoubiquitylation, which is known to trigger nuclear export, to polyubiquitylation, which causes nuclear p53 degradation. This suggests that the Pin1 activity is often translated into a fate-determining ubiquitylation switch, and that Pin1 may affect the degree of substrate ubiquitylation in other pathways as well.

The plant hormone auxin plays a role in virtually every aspect of plant growth and development. Temporal and spatial distribution of auxin largely depends on the dynamic expression and subcellular localization of the PIN auxin-efflux carrier proteins. We show here that the Arabidopsis thaliana JAGGED LATERAL ORGAN (JLO) gene, a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) gene family, is required for coordinated cell division during embryogenesis. JLO promotes expression of several PINFORMED (PIN) genes during embryonic and root development. Inducible JLO misexpression reveals that JLO activity is sufficient for rapid and high level PIN1 and PIN3 transcription. Genes of the PLETHORA (PLT) family respond to auxin and direct PIN expression, but PLT genes were severely underexpressed in jlo mutants. JLO controls embryonic patterning together with the auxin dependent MONOPTEROS/BODENLOS pathway, but is itself only mildly auxin inducible. We further show that all known auxin responses in the root require JLO activity. We thereby identify JLO as a central regulator of auxin distribution and signaling throughout plant development.

In normal leaf development, a two-dimensional pattern of leaf veins is known to form by differentiation of vascular cells from ground meristem cells in a manner that is regulated by the polar flow of auxin. However, the mechanisms regulating the distribution of auxin in the leaf primordium are largely unknown. Here we show that vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), VAM3 and VTI11, are required for the formation of the leaf vascular network in a dosage-dependent manner. This is the first report to show that the pre-vacuolar compartment (PVC)-vacuole traffic pathway is required for the formation of the leaf vascular network. vam3-4, a VAM3-defective mutant, was found to have an immature vascular network. An analysis of the DR5 reporter in vam3-4 indicated that VAM3 is involved in the proper pattern formation of auxin maxima in the leaf primordium. This suggests that the immature vascular network in vam3-4 was mainly determined at the stage of procambium formation in the leaf primordium. The abnormal distribution of auxin maxima was caused by the non-polarized localization of the auxin efflux carrier PIN1 (PIN-FORMED 1) in leaf primordium cells. VAM3 is the first key protein which is required for the proper localization of PIN1 in leaf cells. Finally, we found that PIN1 proteins were constitutively transported to vacuoles in leaf and roots cells. Our findings demonstrate that the PVC-vacuole pathway is required for the formation of auxin maxima, which regulates the polar localization of PIN1, which, in turn, is required for the formation of the leaf vascular network.

MicroRNA (miRNA) 200s regulate E-cadherin by directly targeting ZEB1/ZEB2, which are transcriptional repressors of E-cadherin. Decreased expression of E-cadherin results in cancer cells losing interaction with the extracellular matrix and detaching from the primary tumor. Normally, cells will undergo anoikis after losing interaction with the extracellular matrix. Cancer cells must, therefore, possess the ability to resist anoikis during the process of metastasis. Here we show that miRNA-200b regulates anoikis by directly targeting the 3' UTR of Pin1 mRNA and regulating Pin1 expression at the translational level. We found that down-regulation of miRNA-200b promotes cancer cells survival during metastasis, and the homeless state of these cells resulted in decreased expression of miRNA-200b in the MCF-7 cell line. We also found that expression of miRNA-200b is down-regulated in human breast cancer during lymph node metastasis, which has a significant negative correlation with Pin1 expression. Two members of the ETS (E-26) family (PEA3 and ELK-1) regulate the expression of miRNA-200b. PEA3 promotes the expression of miRNA-200b, and ELK-1 is a transcriptional repressor of miRNA-200b. In addition, miRNA-200b regulates the activity of PEA3 and ELK-1 via the Pin1-pERK pathway and forms self-regulated feedback loops. This study characterizes the role of miRNA-200b in the regulation of anoikis and demonstrates the regulation of its own expression in the process of metastasis.