Tumor suppression by the p53 protein largely depends on the elimination of damaged cells by apoptosis. Mutations in the polyproline region (PPR) of p53 impair its apoptotic function. Deletion of the PPR renders p53 more sensitive to inhibition by Mdm2 via an unknown mechanism. We have explored the mechanism by which the PPR modulates the p53/Mdm2 loop. Proline 82 of p53 was identified to be essential for its interaction with the checkpoint kinase 2 (Chk2) and consequent phosphorylation of p53 on serine 20, following DNA damage. These physical and functional interactions are regulated by Pin1 through cis-trans isomerization of proline 82. Our study unravels the pathway by which Pin1 activates p53 in response to DNA damage and explains how Pin1 protects p53 from Mdm2. Further, we propose a role for Pin1-dependent induction of p53 conformational change as a mechanism responsible for the enhanced interaction between p53 and Chk2 following DNA damage. Importantly, our findings elucidate the selection for mutations in the Pin1 target Thr81/Pro82 motif within the PPR of p53 in human cancer.

Nuclear protein peptidyl-prolyl isomerase Pin1-mediated prolyl isomerization is an essential and novel regulatory mechanism for protein phosphorylation. Therefore, tight regulation of Pin1 localization and catalytic activity is crucial for its normal nuclear functions. Pin1 is commonly dysregulated during oncogenesis and likely contributes to these pathologies; however, the mechanism(s) by which Pin1 catalytic activity and nuclear localization are increased is unknown. Here we demonstrate that mixed-lineage kinase 3 (MLK3), a MAP3K family member, phosphorylates Pin1 on a Ser138 site to increase its catalytic activity and nuclear translocation. This phosphorylation event drives the cell cycle and promotes cyclin D1 stability and centrosome amplification. Notably, Pin1 pSer138 is significantly up-regulated in breast tumors and is localized in the nucleus. These findings collectively suggest that the MLK3-Pin1 signaling cascade plays a critical role in regulating the cell cycle, centrosome numbers, and oncogenesis.

The cytosolic fraction of the tumor suppressor p53 activates the apoptotic effector protein BAX to trigger apoptosis. Here we report that p53 activates BAX through a mechanism different from that associated with activation by BH3 only proteins (BIM and BID). We observed that cis-trans isomerization of proline 47 (Pro47) within p53, an inherently rare molecular event, was required for BAX activation. The prolyl isomerase Pin1 enhanced p53-dependent BAX activation by catalyzing cis-trans interconversion of p53 Pro47. Our results reveal a signaling mechanism whereby proline cis-trans isomerization in one protein triggers conformational and functional changes in a downstream signaling partner. Activation of BAX through the concerted action of cytosolic p53 and Pin1 may integrate cell stress signals to induce a direct apoptotic response.

The HTLV Tax protein is crucial for viral replication and malignant transformation. We investigated the possible role of peptidyl prolyl isomerase Pin1 in the positive regulation of the human T-cell leukemia virus type 1 Tax. Pin1 is highly expressed in adult T-cell leukemia (ATL) cells expressing Tax protein and forced expression of Pin1 in turn increases the Tax protein expression. Pin1 prolonged the protein half-life of Tax by suppressing the ubiquitination and subsequent lysosomal degradation of Tax. Pin1 interacts with phosphorylated Tax on its Ser160-Pro motif at the mitotic phase. Finally, we found that Pin1 plays a supporting role in Tax-mediated cell transformation. Our current study demonstrates an important role for Pin1 in the post-translational regulation of Tax and suggests that the targeting of Pin1 may offer a new insight into the pathogenesis of HTLV-1 related diseases.

The phospho-Ser/Thr-Pro specific prolyl-isomerase Pin1 has been implicated in multiple aspects of cell cycle regulation. It has been suggested that Pin1 function is required for both normal mitotic progression and reentry into the cell cycle from quiescence. In support of this hypothesis, numerous key regulators of G1 and mitosis have been identified as Pin1 interacting proteins. However, the cellular consequence of Pin1 binding to these proteins has rarely been rigorously characterized. In this review we focus on the role of Pin1 and its binding proteins in cell cycle regulation and the potential value of Pin1 as a therapeutic target.

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

Pin1 is a modular enzyme that accelerates the cis-trans isomerization of phosphorylated-Ser/Thr-Pro (pS/T-P) motifs found in numerous signaling proteins regulating cell growth and neuronal survival. We have used NMR to investigate the interaction of Pin1 with three related ligands that include a pS-P substrate peptide, and two pS-P substrate analogue inhibitors locked in the cis and trans conformations. Specifically, we compared the ligand binding modes and binding-induced changes in Pin1 side-chain flexibility. The cis and trans binding modes differ, and produce different mobility in Pin1. The cis-locked inhibitor and substrate produced a loss of side-chain flexibility along an internal conduit of conserved hydrophobic residues, connecting the domain interface with the isomerase active site. The trans-locked inhibitor produces a weaker conduit response. Thus, the conduit response is stereoselective. We further show interactions between the peptidyl-prolyl isomerase and Trp-Trp (WW) domains amplify the conduit response, and alter binding properties at the remote peptidyl-prolyl isomerase active site. These results suggest that specific input conformations can gate dynamic changes that support intraprotein communication. Such gating may help control the propagation of chemical signals by Pin1, and other modular signaling proteins.

The polo-like kinase (Plk) family has been shown to have an important role in the regulation of the cell-division cycle, especially in organization of the spindle structure, in species from fungi to humans. Recent reports have demonstrated that in mammalian cells Plk is associated with components of the anaphase-promoting complex and a peptidyl-prolyl isomerase, Pin1. To characterize a putative Plk-containing complex, we fractionated mitotic cell lysates on a gel-filtration column. The Plk complex was eluted from the column at molecular sizes ranging from 669 to 2500 kDa in the presence of detergent and high concentrations of salt. Specific associations of Plk with alpha-, beta- and gamma-tubulins in both interphase and mitotic cells were shown by reciprocal immunoprecipitations and immunoblottings and were independent of the microtubule polymerization state, whereas binding assays in vitro indicated that Plk interacts with alpha- and beta-tubulins directly. In addition, mitotic Plk was able to phosphorylate associated tubulins in vitro. Finally, we show that the kinase domain of the Plk molecule is both required and sufficient for its binding to tubulins in vivo. The specific interaction between Plk and tubulins might provide a molecular basis for the physiological functions of Plk in regulating the cell cycle, particularly in establishing the normal bipolar spindle.

Elucidating molecular links between cell-fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell-fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem-cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS-box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem-cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS-domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4-dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning.

The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.

Protein phosphorylation is a key molecular switch used to transmit information in biological signalling networks. The output of these signalling circuits is governed by the counteracting activities of protein kinases and phosphatases that determine the direction of the switch. Whereas many kinases have been functionally characterized, it has been difficult to ascribe precise cellular roles to plant phosphatases, which are encoded by enlarged gene families that may provide a high degree of genetic redundancy. In this work we have analysed the role in planta of catalytic subunits of protein phosphatase 2A (PP2A), a family encoded by five genes in Arabidopsis. Our results indicate that the two members of subfamily II, PP2A-C3 and PP2A-C4, have redundant functions in controlling embryo patterning and root development, processes that depend on auxin fluxes. Moreover, polarity of the auxin efflux carrier PIN1 and auxin distribution, determined with the DR5(pro) :GFP proxy, are affected by mutations in PP2A-C3 and PP2A-C4. Previous characterization of mutants in putative PP2A regulatory subunits had established a link between this class of phosphatases and PIN dephosphorylation and subcellular distribution. Building on those findings, the results presented here suggest that PP2A-C3 and PP2A-C4 catalyse this reaction and contribute critically to the establishment of auxin gradients for proper plant development.

OBJECTIVE

To investigate clinical significance of Pin1 and beta-catenin expression in colorectal cancers and to demonstrate the relationship of their expression.

METHODS

The role of Pin1 and beta-catenin protein in colorectal tumorigenesis and their clinicopathologic significance were analyzed by immunohistochemistry, and the correlation between Pin1 and beta-catenin protein expressions was also studied in 124 patients with colorectal cancer who were surgically treated.

RESULTS

Normal colonic epithelium either failed to express or showed focal and weak expression of Pin1 and beta-catenin. Overexpression of Pin1 and beta-catenin protein was found in 23 (18.54%) and 50 (40.3%) of 124 colorectal cancers, respectively. Overexpression of both proteins was not related to the lymph node metastasis, tumor stage and survival period after excision. Survival analysis results indicated that tumor stage was a valuable predictor of survival. Interestingly, a significant correlation was found between Pin1 and beta-catenin protein expression.

CONCLUSIONS

Overexpression of Pin1 and beta-catenin may be closely related with the development and/or progression of colorectal carcinoma and further supports that Pin1 overexpression might contribute to the upregulation of beta-catenin.

Backbone cyclization is often used in attempts to enhance protein stability, but is not always successful as it is possible to remove stabilizing or introduce destabilizing interactions in the process. Cyclization of the PIN1 WW domain, a 34-residue three-stranded beta-sheet structure, removes a favorable electrostatic interaction between its termini. Nevertheless, optimization of the linker connecting the N- and C-termini using information based on the previously determined ensemble of NMR structures leads to beta-sheets that are more stable than those derived from the linear sequence. Linkers that are too short or too long introduce strain, likely disrupting native interactions, leading to cyclic folds that are less stable than that of the linear sequence.

Estrogen receptor-alpha (ERα) is an important biomarker used to classify and direct therapy decisions in breast cancer (BC). Both ERα protein and its transcript, ESR1, are used to predict response to tamoxifen therapy, yet certain tumors have discordant levels of ERα protein and ESR1, which is currently unexplained. Cellular ERα protein levels can be controlled post-translationally by the ubiquitin-proteasome pathway through a mechanism that depends on phosphorylation at residue S118. Phospho-S118 (pS118-ERα) is a substrate for the peptidyl prolyl isomerase, Pin1, which mediates cis-trans isomerization of the pS118-P119 bond to enhance ERα transcriptional function. Here, we demonstrate that Pin1 can increase ERα protein without affecting ESR1 transcript levels by inhibiting proteasome-dependent receptor degradation. Pin1 disrupts ERα ubiquitination by interfering with receptor interactions with the E3 ligase, E6AP, which also is shown to bind pS118-ERα. Quantitative in situ assessments of ERα protein, ESR1, and Pin1 in human tumors from a retrospective cohort show that Pin1 levels correlate with ERα protein but not to ESR1 levels. These data show that ERα protein is post-translationally regulated by Pin1 in a proportion of breast carcinomas. As Pin1 impacts both ERα protein levels and transactivation function, these data implicate Pin1 as a potential surrogate marker for predicting outcome of ERα-positive BC.

Peptidylprolyl isomerase Pin1 regulates the function and/or stability of phosphoproteins by altering the conformation of specific pSer/pThr-Pro peptide bonds. In this work, a cyclic peptide library was synthesized and screened against the catalytic domain of human Pin1. The selected inhibitors contained a consensus motif of D-pThr-Pip-Nal (where Pip is L-piperidine-2-carboxylic acid and Nal is L-2-naphthylalanine). Representative compounds were tested for binding to Pin1 by isothermal titration calorimetry and inhibition of Pin1 activity, and the most potent inhibitors had K(D) (and K(I)) values in the low nanomolar range. Treatment of breast cancer cells with the inhibitors, which were rendered membrane permeable by attachment of an octaarginine sequence, inhibited cell proliferation and increased the protein levels of two previously established Pin1 substrates, PML and SMRT. Finally, a second generation of cell permeable Pin1 inhibitors was designed by replacing the noncritical residues within the cyclic peptide ring with arginine residues and shown to have antiproliferative activity against the cancer cells.

One mechanism by which plants defend themselves against insect herbivores is the production of plant proteinase inhibitors, which can inhibit digestion in the midgut, thus affecting growth and survival. In this work, the effect of Colorado potato beetle (CPB) [Leptinotarsa decemlineata (Say)] regurgitant on Solanum lycopersicum defenses was investigated. When regurgitant from fourth-instar CPB was applied to wounded S. lycopersicum leaves, the wound-induced transcripts for the proteinase inhibitors pin1 and pin2 were reduced. Boiling the regurgitant abolished its ability to reduce the pin transcripts. Ultrafiltration of the regurgitant demonstrated that it contained a component between 10 and 30 kDa molecular weight that inhibited wound-induced pin1 and pin2 expression, suggesting that it may be a protein. This may represent a mechanism that the CPB has evolved to elude the plant's induced response to infestation.

Dendritic cells (DC) play a critical role in modulating antigen-specific immune responses elicited by T cells via engagement of the prototypic T cell costimulatory receptor CD28 by the cognate ligands CD80/CD86, expressed on DC. Although CD28 signaling in T cell activation has been well characterized, it has only recently been shown that CD80/CD86, which have no demonstrated binding domains for signaling proteins in their cytoplasmic tails, nonetheless also transduce signals to the DC. Functionally, CD80/CD86 engagement results in DC production of the pro-inflammatory cytokine IL-6, which is necessary for full T cell activation. However, ligation of CD80/CD86 by CTLA4 also induces DC production of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO), which depletes local pools of the essential amino acid tryptophan, resulting in blockade of T cell activation. Despite the significant role of CD80/CD86 in immunological processes and the seemingly opposing roles they play by producing IL-6 and IDO upon their activation, how CD80/CD86 signal remains poorly understood. We have now found that cross-linking CD80/CD86 in human DC activates the PI3K/AKT pathway. This results in phosphorylation/inactivation of its downstream target, FOXO3A, and alleviates FOXO3A-mediated suppression of IL-6 expression. A second event downstream of AKT phosphorylation is activation of the canonical NF-κB pathway, which induces IL-6 expression. In addition to these downstream pathways, we unexpectedly found that CD80/CD86-induced PI3K signaling is regulated by previously unrecognized cross-talk with NOTCH1 signaling. This cross-talk is facilitated by NOTCH-mediated up-regulation of the expression of prolyl isomerase PIN1, which in turn increases enzyme activity of casein kinase II. Subsequently, phosphatase and tensin homolog (which suppresses PI3K activity) is inactivated via phosphorylation by casein kinase II. This results in full activation of PI3K signaling upon cross-linking CD80/CD86. Similar to IL-6, we have found that CD80/CD86-induced IDO production by DC at late time points is also dependent upon the PI3K → AKT → NF-κB pathway and requires cross-talk with NOTCH signaling. These data further suggest that the same signaling pathways downstream of DC CD80/CD86 cross-linking induce early IL-6 production to enhance T cell activation, followed by later IDO production to self-limit this activation. In addition to characterizing the pathways downstream of CD80/CD86 in IL-6 and IDO production, identification of a novel cross-talk between NOTCH1 and PI3K signaling may provide new insights in other biological processes where PI3K signaling plays a major role.

Protein kinase CK2 represents a small family of highly conserved protein kinases involved in a complex series of cellular events. Furthermore, CK2 has been localised to many discrete cellular sites and has an extensive and diverse array of substrates and interaction partners in cells. Despite considerable investigation, the precise mechanism(s) of regulation of CK2 in cells remains poorly understood. In consideration of the prospect that cells contain many distinct sub-populations of CK2 that are distinguished on the basis of localisation and/or interactions with other cellular components, one possibility is that there may be differential regulation of specific sub-populations of CK2. With this in mind, some of the individual sub-populations of CK2 may be regulated through particular protein-protein interactions that may play a role in recruiting CK2 into the vicinity of its substrates and/or modulating its ability to phosphorylate specific cellular targets. In this respect, here we examine two CK2-interacting proteins, namely Pin1 and CKIP-1 that have been shown to participate in the modulation of CK2 specificity or the subcellular localisation of CK2, respectively. One aspect of this work has been focused on the prospect that Pin1 interacts with CK2 in response to UV stimulation in a manner analogous to the phosphorylation-dependent interactions of CK2 that occur following the mitotic phosphorylation of CK2. A second aspect of this work involves an examination of the structural basis for interactions between CK2 and CKIP-1 with emphasis on a putative HIKE domain in CK2.

The stability and activity of tumor suppressor p53 are tightly regulated and partially depend on the p53 proline-rich domain (PRD). We recently analyzed mice expressing p53 with a deletion of the PRD (p53(DeltaP)). p53(DeltaP), a weak transactivator hypersensitive to Mdm2-mediated degradation, is unable to suppress oncogene-induced tumors. This phenotype could result from the loss of two motifs: Pin1 sites proposed to influence p53 stabilization and PXXP motifs proposed to mediate protein interactions. We investigated the importance of these motifs by generating mice encoding point mutations in the PRD. p53(TTAA) contains mutations suppressing all putative Pin1 sites in the PRD, while p53(AXXA) lacks PXXP motifs but retains one intact Pin1 site. Both mutant proteins accumulated in response to DNA damage, although the accumulation of p53(TTAA) was partially impaired. Importantly, p53(TTAA) and p53(AXXA) are efficient transactivators and potent suppressors of oncogene-induced tumors. Thus, Pin1 sites in the PRD may modulate p53 stability but do not significantly affect function. In addition, PXXP motifs are not essential, but structure dictated by the presence of prolines, PXXXXP motifs that may mediate protein interactions, and/or the length of this region appears to be functionally significant. These results may explain why the sequence of the p53 PRD is so variable in evolution.

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

The monoclonal antibody MPM-2 recognizes a subset of M phase phosphoproteins in a phosphorylation-dependent manner. It is believed that phosphorylation at MPM-2 antigenic sites could regulate mitotic events since most of the MPM-2 antigens identified to date have M phase functions. In addition, many of these proteins are substrates of the mitotic regulator Pin1, a peptidyl-prolyl isomerase which is present throughout the cell cycle and which is thought to alter its mitotic targets by changing their conformation. In interphase cells, most MPM-2 reactivity is confined to nuclear speckles. We report here that a hyperphosphorylated form of the RNA polymerase II largest subunit is the major MPM-2 interphase antigen. These findings were made possible by the availability of another monoclonal antibody, CC-3, that was previously used to identify a 255 kDa nuclear matrix protein associated with spliceosomal components as a hyperphosphorylated form of the RNA polymerase II largest subunit. MPM-2 recognizes a phosphoepitope of the large subunit that becomes hyperphosphorylated upon heat shock in contrast to the phosphoepitope defined by CC-3, whose reactivity is diminished by the heat treatment. Therefore, these two antibodies may discriminate between distinct functional forms of RNA polymerase II. We also show that RNA polymerase II large subunit interacts with Pin1 in HeLa cells. Pin1 may thus regulate transcriptional and post-transcriptional events by catalyzing phosphorylation-dependent conformational changes of the large RNA polymerase II subunit.

Throughout the history, olive (Olea europea L.) leaves have been heavily exploited for the prevention or the treatment of hypertension, carcinogenesis, diabetes, atherosclerosis and so many other traditional therapeutic uses. These activities are thought to be the output of olive micronutrients especially polyphenols. Hydroxytyrosol and oleuropein are considered as major polyphenolic compounds in olive leaf. In this work, a hydroxytyrosol rich olive leaves extract was investigated for potential anti-tumoral activities. In vitro cytotoxic effects against MCF-7 breast cancer cells were examined using MTT and neutral red tests. The anti-tumor activities were further investigated by flow cytometry and western blotting. Cytotoxicity assays resulted in a dose dependent growth inhibition of MCF-7 cells. This inhibition was due to the cell cycle arrest in the G0/G1 phase. The understanding of the molecular mechanism by which olive leaves extract arrested cell growth showed a down-expression of the peptidyl-prolyl cis-trans isomerase Pin1 which in turn decreased the level of a G1 key protein; Cyclin D1. Additionally, olive leaves extract treatment up-regulated the AP1 transcription factor member, c-jun. Therefore, olive leaves extract will necessitate further deep investigation for a probable use as a cancer preventive food additive.

OBJECTIVE

The Pin1 prolyl isomerase acts in concert with proline-directed protein kinases to regulate function of protein substrates through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline. We sought to determine whether Pin1 interacts with endothelial nitric oxide synthase (eNOS) in endothelial cells in a manner that depends on proline-directed phosphorylation of the eNOS enzyme and whether this interaction influences basal or agonist-stimulated eNOS activity.

RESULTS

Inhibitors of the extracellular-regulated kinase (ERK) 1/2 MAP kinases inhibit proline-directed phosphorylation of eNOS at serine 116 (Ser116) in bovine aortic endothelial cells (BAECs). Moreover, eNOS and Pin1 can be coimmunoprecipitated from BAECs only when Ser116 is phosphorylated. In addition, phosphomimetic Ser116Asp eNOS, but not wild-type eNOS, can be coimmunoprecipitated with Pin1 coexpressed in COS-7 cells. Inhibition of Pin1 in BAECs by juglone or by dominant negative Pin1 increases basal and agonist-stimulated NO release from the cells, whereas overexpression of wild-type Pin1 in BAECs suppresses basal and agonist-stimulated NO production. Overexpression of wild-type Pin1 in intact aortae also reduces agonist-induced relaxation of aortic rings.

CONCLUSIONS

Our results demonstrate a novel form of eNOS regulation in endothelial cells and blood vessels through Ser116 phosphorylation-dependent interaction of eNOS with Pin1.

Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1-HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis.