Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast.

Tor proteins, homologous to DNA-dependent protein kinases, participate in a signal transduction pathway in yeast that regulates protein synthesis and cell wall expansion in response to nutrient availability. The anti-inflammatory drug rapamycin inhibits yeast cell growth by inhibiting Tor protein signaling. This leads to diminished association of a protein, Tap42, with two different protein phosphatase catalytic subunits; one encoded redundantly by PPH21 and PPH22, and one encoded by SIT4. We show that inactivation of either Cdc55 or Tpd3, which regulate Pph21/22 activity, results in rapamycin resistance and that this resistance correlates with an increased association of Tap42 with Pph21/22. Furthermore, we show Tor-dependent phosphorylation of Tap42 both in vivo and in vitro and that this phosphorylation is rapamycin sensitive. Inactivation of Cdc55 or Tpd3 enhances in vivo phosphorylation of Tap42. We conclude that Tor phosphorylates Tap42 and that phosphorylated Tap42 effectively competes with Cdc55/Tpd3 for binding to the phosphatase 2A catalytic subunit. Furthermore, Cdc55 and Tpd3 promote dephosphorylation of Tap42. Thus, Tor stimulates growth-promoting association of Tap42 with Pph21/22 and Sit4, while Cdc55 and Tpd3 inhibit this association both by direct competition and by dephosphorylation of Tap42. These results establish Tap42 as a target of Tor and add further refinement to the Tor signaling pathway.     

CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration

Cylindromatosis (CYLD), a deubiquitinase involved in inflammation and tumorigenesis via the modulation of cell signaling, has recently been identified as a critical regulator of microtubule dynamics. CYLD has also been shown to stimulate cell migration and thereby contribute to normal physiological processes. However, it remains elusive how the regulation of microtubule dynamic properties by CYLD is connected to its role in mediating cell migration. In this study, we performed yeast 2-hybrid screening with CYLD as bait and identified 7 CYLD-interacting proteins, including end-binding protein 1 (EB1). The CYLD–EB1 interaction was confirmed both in cells and in vitro, and these 2 proteins colocalized at the plus ends of microtubules. Interestingly, the association of CYLD with EB1 was significantly increased upon the stimulation of cell migration. CYLD coordinated with EB1 to orchestrate tail retraction, centrosome reorientation, and leading-edge microtubule stabilization in migratory cells. In addition, CYLD acted in concert with EB1 to regulate microtubule assembly in vitro, microtubule nucleation at the centrosome, and microtubule growth at the cell periphery. These data provide mechanistic insights into the actions of CYLD in the regulation of microtubule dynamics and cell migration. These findings also support the notion that coordinated actions of microtubule-binding proteins are critical for microtubule-mediated cellular events.     

LncRNAs PSMG3-AS1 and MEG3 negatively regulate each other to participate in endometrial carcinoma cell proliferation

Mammalian Genome - Endometrial carcinoma (EC), also known as corpus cancer or corpus uterine cancer, is the most frequently diagnosed genital cancer among women in developed countries. Our...     

Diego and Prickle regulate Frizzled planar cell polarity signalling by competing for Dishevelled binding

Epithelial planar cell polarity (PCP) is evident in the cellular organization of many tissues in vertebrates and invertebrates. In mammals, PCP signalling governs convergent extension during gastrulation and the organization of a wide variety of structures, including the orientation of body hair and sensory hair cells of the inner ear. In Drosophila melanogaster, PCP is manifest in adult tissues, including ommatidial arrangement in the compound eye and hair orientation in wing cells. PCP establishment requires the conserved Frizzled/Dishevelled PCP pathway. Mutations in PCP-pathway-associated genes cause aberrant orientation of body hair or inner-ear sensory cells in mice, or misorientation of ommatidia and wing hair in D. melanogaster. Here we provide mechanistic insight into Frizzled/Dishevelled signalling regulation. We show that the ankyrin-repeat protein Diego binds directly to Dishevelled and promotes Frizzled signalling. Dishevelled can also be bound by the Frizzled PCP antagonist Prickle. Strikingly, Diego and Prickle compete with one another for Dishevelled binding, thereby modulating Frizzled/Dishevelled activity and ensuring tight control over Frizzled PCP signalling.     

S-nitrosylation and S-palmitoylation reciprocally regulate synaptic targeting of PSD-95

PSD-95, a principal scaffolding component of the postsynaptic density, is targeted to synapses by palmitoylation, where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA-mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction.     

E6AP and calmodulin reciprocally regulate estrogen receptor stability

Estrogen promotes the proliferation of human breast epithelial cells by interacting with the estrogen receptor (ER). Physiological responses of cells to estrogen are regulated in part by degradation of the ER. Previous studies revealed that calmodulin binds directly to the ER, thereby enhancing its stability. Consistent with these findings, cell-permeable calmodulin antagonists dramatically reduced the number of ER in MCF-7 human breast epithelial cells. Here we investigated the molecular mechanism by which calmodulin attenuates ER degradation. MG132 and lactacystin, inhibitors of the ubiquitin-proteasome pathway, prevented the calmodulin antagonist CGS9343B from reducing the amount of ER in MCF-7 cells. In contrast, protease inhibitors afforded no protection. Moreover, CGS9343B enhanced ER ubiquitination. A point mutant ER construct that is unable to bind calmodulin, termed ERDeltaCaM, is ubiquitinated to a greater extent than wild type ER. The ubiquitin-protein isopeptide ligase E6-associated protein (E6AP) associated with and promoted the degradation of ER. The possible convergence of calmodulin and E6AP on ER degradation was examined. ERDeltaCaM bound E6AP with higher affinity than that of wild type ER. Moreover, calmodulin attenuated the in vitro interaction between ER and E6AP in a Ca(2+)-dependent manner. Collectively, our data reveal that E6AP is a component of ER degradation via the ubiquitin-proteasome pathway and that Ca(2+)/calmodulin modulates this degradation mechanism. These results have potential implications for the development of selectively targeted therapeutic agents for breast cancer.     

Band 4.1 proteins regulate integrin-dependent cell spreading

Integrins link the extracellular matrix (ECM) to the cytoskeleton to control cell behaviors including adhesion, spreading and migration. Band 4.1 proteins contain 4.1, ezrin, radixin, moesin (FERM) domains that likely mediate signaling events and cytoskeletal reorganization via integrins. However, the mechanisms by which Band 4.1 proteins and integrins are functionally interconnected remain enigmatic. Here we have investigated roles for Band 4.1 proteins in integrin-mediated cell spreading using primary astrocytes as a model system. We demonstrate that Proteins 4.1B and 4.1G show dynamic patterns of sub-cellular localization in astrocytes spreading on fibronectin. During early stages of cell spreading Proteins 4.1B and 4.1G are enriched in ECM adhesion sites but become more diffusely localized at later stages of spreading. Combinatorial inactivation of Protein 4.1B and 4.1G expression leads to impaired astrocyte spreading. Furthermore, in exogenous expression systems we show that the isolated Protein 4.1 FERM domain significantly enhances integrin-mediated cell spreading. Protein 4.1B is dispensable for reactive astrogliosis in experimental models of cortical injury, likely due to functional compensation by related Protein 4.1 family members. Collectively, these findings reveal that Band 4.1 proteins are important intracellular components for integrin-mediated cell spreading.     

ErbB-2 and met reciprocally regulate cellular signaling via plexin-B1

Sema4D-induced activation of plexin-B1 has been reported to evoke different and sometimes opposing cellular responses. The mechanisms underlying the versatility of plexin-B1-mediated effects are not clear. Plexin-B1 can associate with the receptor tyrosine kinases ErbB-2 and Met. Here we show that Sema4D-induced activation and inactivation of RhoA require ErbB-2 and Met, respectively. In breast carcinoma cells, Sema4D can have pro- and anti-migratory effects depending on the presence of ErbB-2 and Met, and the exchange of the two receptor tyrosine kinases is sufficient to convert the cellular response to Sema4D from pro- to anti-migratory and vice versa. This work identifies a novel mechanism by which plexin-mediated signaling can be regulated and explains how Sema4D can exert different biological activities through the differential association of its receptor with ErbB-2 and Met.     

Dishevelled phosphorylation, subcellular localization and multimerization regulate its role in early embryogenesis

Dishevelled (Dsh) induces a secondary axis and can translocate to the membrane when activated by Frizzleds; however, dominant-negative approaches have not supported a role for Dsh in primary axis formation. We demonstrate that the Dsh protein is post-translationally modified at the dorsal side of the embryo: timing and position of this regulation suggests a role of Dsh in dorsal-ventral patterning in Xenopus. To create functional links between these properties of Dsh we analyzed the influence of endogenous Frizzleds and the Dsh domain dependency for these characteristics. Xenopus Frizzleds phosphorylate and translocate Xdsh to the membrane irrespective of their differential ectopic axes inducing abilities, showing that translocation is insufficient for axis induction. Dsh deletion analysis revealed that axis inducing abilities did not segregate with Xdsh membrane association. The DIX region and a short stretch at the N-terminus of the DEP domain are necessary for axis induction while the DEP region is required for Dsh membrane association and its phosphorylation. In addition, Dsh forms homomeric complexes in embryos suggesting that multimerization is important for its proper function.     

Nuclear retinoic-acid-binding proteins and receptors in retinoic-acid-responsive cell lines

Nuclear retinoic-acid-binding activity and the expression of retinoic acid receptor mRNA (RAR-alpha and RAR-beta) were assayed in the F9 embryonal carcinoma, HeLa, HL-60 promyelocytic leukaemia and S91 melanoma cell lines. A 4-svedberg nuclear retinoic-acid-binding activity was detected in all 4 cell lines, but the levels in the HeLa and HL-60 cells were lower than in the F9 and S91 lines. RAR-alpha mRNA was expressed in all 4 cell lines, although at a very low level in S91 cells. Conversely, RAR-beta mRNA was expressed in S91 cells and, at a lower level, in F9 cells but was undetectable in HeLa and HL-60 cells. RAR-beta, transcribed and translated in vitro from the cloned cDNA coding region, sedimented at 4 S and this suggests that the 4-svedberg nuclear retinoic-acid-binding activity may represent the retinoic acid receptors.     

Deoxyribinucleic acid-binding proteins in virus-transformed cell lines.

The synthesis of proteins with affinity for DNA has been studied in clones of a Syrian hamster cell line (NIL) and subclones of this line transformed by polyoma virus (NIL-Py) or hamster sarcoma virus (NIL-HSV). The results show that the synthesis of DNA-binding proteins in NIL and in its virus-transformed derivatives NIL-Py and NIL-HSV is very similar in exponentially growing cells, but in dense culture there is a very significant difference in the level of a protein (P8), which is much higher in the transformed lines than in untransformed NIL. The high levels of P8 in dense transformed cells have been observed in all the clones of transformed cells examined, indicating that this behavior of P8 is related to transformation and not simply due to a fortuitous clonal selection from the NIL. Experiments with synchronized cells indicate that the time of maximal P8 synthesis relative to cellular DNA synthesis in NIL-HSV precedes that observed in NIL cells. P8 has a molecular weight of 30,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and is present in large amounts in the transformed cells in dense culture, where it makes up 0.5 to 1% of the total soluble protein.     

APC/C and SCF: controlling each other and the cell cycle

Regulated protein degradation has emerged as a key recurring theme in multiple aspects of cell-cycle regulation. Importantly, the irreversible nature of proteolysis makes it an invaluable complement to the intrinsically reversible regulation through phosphorylation and other post-translational modifications. Consequently, ubiquitin-protein ligases, the protagonists of regulated protein destruction, have gained prominence that compares to that of the cyclin-dependent kinases (Cdks) in driving the eukaryotic cell-cycle clock. This review will focus on the two main players, the related ubiquitin-protein ligases APC/C and SCF, and how they control cell-cycle progression. I will also try to delineate the regulation and interplay of these destruction mechanisms, which are intricately connected to the kinase network as well as to extrinsic signals. Moreover, cell-cycle ubiquitin-protein ligases are themselves subject to proteolytic control in cis as well as in trans. Finally, a careful comparison of the functions and regulation of APC/C and SCF shows that, in certain aspects, their logic of action is fundamentally different.     

The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density

Macrophages regulate lymphatic vasculature development; however, the molecular mechanisms regulating their recruitment to developing, and adult, lymphatic vascular sites are not known. Here, we report that resting mice deficient for the inflammatory chemokine‐scavenging receptor, ACKR2, display increased lymphatic vessel density in a range of tissues under resting and regenerating conditions. This appears not to alter dendritic cell migration to draining lymph nodes but is associated with enhanced fluid drainage from peripheral tissues and thus with a hypotensive phenotype. Examination of embryonic skin revealed that this lymphatic vessel density phenotype is developmentally established. Further studies indicated that macrophages and the inflammatory CC‐chemokine CCL2, which is scavenged by ACKR2, are associated with this phenotype. Accordingly, mice deficient for the CCL2 signalling receptor, CCR2, displayed a reciprocal phenotype of reduced lymphatic vessel density. Further examination revealed that proximity of pro‐lymphangiogenic macrophages to developing lymphatic vessel surfaces is increased in ACKR2‐deficient mice and reduced in CCR2‐deficient mice. Therefore, these receptors regulate vessel density by reciprocally modulating pro‐lymphangiogenic macrophage recruitment, and proximity, to developing, resting and regenerating lymphatic vessels.