The Hedgehog (Hh) signaling pathway plays crucial roles in embryonic development and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Aberrant activation of Hh signaling is ...associated with various developmental abnormalities and several types of cancer. Genetic and biochemical studies ascertain serine/threonine kinase Fused (Fu) as a protein involved in Hh signaling in
Drosophila. However, the role of Fu is not fully conserved in mammals suggesting involvement of other kinases in the mammalian Hh signaling pathway. In search of potential homologues to
Drosophila and human Fu, we have cloned human serine/threonine kinase ULK3 and assessed its ability to regulate GLI transcription factors, mediators of SHH signaling. We demonstrate that ULK3 enhances endogenous and over-expressed GLI1 and GLI2 transcriptional activity in cultured cells, as assessed by GLI-luciferase reporter assay. Besides that, ULK3 alters subcellular localization of GLI1, as assessed by immunofluorescent staining and immunoblotting assays. We show that ULK3 is an autophosphorylated kinase and phosphorylates GLI proteins
in vitro. We also demonstrate that ULK3 catalytical activity is crucial for its function in SHH pathway. We show that
ULK3 is widely expressed and its expression is higher in a number of tissues where Shh signaling is known to be active. Our data suggest that serine/threonine kinase ULK3 is involved in the SHH pathway as a positive regulator of GLI proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. ...During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12-37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Hedgehog signaling pathway is involved in both development and cancer induction in a wide range of organisms. The end
point of the Hedgehog signal-transduction cascade is the Gli/Ci, zinc-finger ...transcription factors. Proteins such as Fused,
Suppressor of fused (SUFU), Costal-2, and protein kinase A are essential for regulation of Gli/Ci processing, activity, and
localization. Coimmunoprecipitation and Far Western assays, coupled with truncation analysis and mutagenesis have been used
to define the region of interaction between Gli proteins and SUFU. We identify a novel motif SYGH in Gli/Ci family proteins,
which is required for the interaction with SUFU. Mutational studies revealed that Gly 122 and His 123 are crucial for binding to SUFU, suggesting the importance of hydrophobicity for the correct binding conformation. Functional
analysis revealed that the activity of GLI transcription factors with mutations in this motif is no longer suppressed by co-expression
of SUFU. Moreover, we have found that a C-terminal 19-amino acid deletion in SUFU (Î465) is sufficient to abrogate interaction
with GLI1. Interestingly, this SUFU mutant localizes in the nucleus, most probably because it is not efficiently sequestered
in the cytoplasm. Taken together, we identified a novel motif in the Gli/Ci family of proteins that is essential both for
protein-protein interaction with SUFU and for functional repression of GLI1 by SUFU.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Sonic hedgehog, Patched and Gli are components of a mammalian signalling pathway that has been conserved during evolution and which has a central role in the control of pattern formation and cellular ...proliferation during development. Here we identify the human Suppressor-of-Fused (SUFUH) complementary DNA and show that the gene product interacts physically with the transcriptional effector GLI-1, can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signalling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signalling that may affect nuclear-cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The PTCH1 tumor suppressor gene encodes a receptor for secreted hedgehog (HH) ligands and is important for proper proliferation, differentiation and patterning in almost every tissue and organ during ...embryogenesis. The PTCH1 protein works as a negative regulator of the HH-signaling pathway by repressing downstream signaling by the coreceptor smoothened (SMOH). Mutations in
PTCH1 lead to constitutive expression of HH target genes and a relationship between mutated
PTCH1 and the most common tumor form in the Western world, Basal Cell Carcinoma (BCC) has been clearly established. We here show that
PTCH1 is transcriptionally regulated by three independent promoters generating transcripts with alternative first exons. We demonstrate that only one of two putative Gli-binding sites that were identified in the promoter region of
PTCH1 is functional, and that the transactivating Gli proteins, GLI1, Gli2 and GLI3, bind and enhance transcription through this site. Moreover, a strong repression of both basal and induced PTCH1 transcription was observed following expression of a truncated version of GLI3. Most interestingly, the upstream components in the HH-signaling cascade, Sonic HH (SHH) and SMOH, solely operate through the functional Gli-binding site because mutation of the Gli-binding site resulted in the disappearance of the enhanced transcription induced by the Gli proteins, as well as by SHH or SMOH. This finding suggests that transcriptional activation of the
PTCH1 gene mediated via the HH-signaling pathway is dependent on the single functional Gli-binding site.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Sonic hedgehog signaling pathway is important in developmental processes like dorsoventral neural tube patterning, neural stem cell proliferation and neuronal and glial cell survival. Shh is also ...implicated in the regulation of the adult hippocampal neurogenesis. Recently, nonpeptidyl Smoothened activators of the Shh pathway have been identified. The aim of this study was to investigate the effects of chlorobenzothiophene-containing molecule, Smo agonist (SAG), which has been shown to activate Shh signaling pathway, in neurogenesis and neuronal survival in
in vitro and
in vivo models. Our
in vitro experiments showed that SAG induces increased expression of
Gli1 mRNA, transcriptional target and mediator of Shh signal.
In vitro experiments also demonstrated that SAG in low-nanomolar concentrations induces proliferation of neuronal and glial precursors without affecting the differentiation pattern of newly produced cells. In contrast to Shh, SAG did not induce neurotoxicity in neuronal cultures. The SAG and Shh treatment also promoted the survival of newly generated neural cells in the dentate gyrus after their intracerebroventricular administration to adult rats. We propose that SAG and similar compounds represent attractive molecules to be developed for treatment of disorders where stimulation of the generation and survival of new neural cells would be beneficial.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
To investigate the cellular role of dual specificity Yak1-related kinase (Dyrk) 1, a nuclear localized dual specificity protein
kinase, we examined its effect on transcriptional regulation using ...reporter gene assays. We found that Dyrk1 can substantially
enhance Gli1-dependent, but not LEF-1-, c-Jun-, or Elk-dependent, gene transcription. In part, Dyrk1 does this through retaining
Gli1 in the nucleus. However, we also demonstrate that Dyrk1 can enhance the transcriptional activity of Gli1-AHA, a nuclear
export mutant, suggesting that Dyrk1 may be more directly involved in regulating the transcriptional activity of Gli1. In
addition, Dyrk1 acted synergistically with Sonic hedgehog (Shh) to induce gene transcription and differentiation in mouse
C3H10T1/2 cells. The failure of Shh to stimulate Dyrk1 kinase activity suggests that Dyrk1 may not be directly regulated by
the Shh signaling pathway but functionally interacts with it. Thus, Gli1 transcriptional activity may be subjected to further
regulation in the cell nucleus by a pathway distinct from Shh signaling, one mediated by Dyrk1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed ...the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull-down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The transcription factor Gli3 is acting mainly as a transcriptional repressor in the Sonic hedgehog signal transduction pathway. Gli3 contains a repressor domain in its N-terminus from residue G106 ...to E236. In this study we have characterized the intracellular structure of the Gli3 repressor domain using a combined bioinformatics and experimental approach. According to our findings the Gli3 repressor domain while being intrinsically disordered contains predicted anchor sites for partner interactions. The obvious interaction partners to test were Ski and DNA; however, with both of these the structure of Gli3 repressor domain remained disordered. To locate residues important for the repressor function we mutated several residues within the Gli3 repressor domain. Two of these, H141A and H157N, targeting predicted helical regions, significantly decreased transcriptional repression and thus identify important functional parts of the domain.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The PTCH1 gene is a human tumour suppressor gene frequently mutated in basal cell carcinoma (BCC) and several other tumour types. It encodes a receptor for soluble factors of the hedgehog family. ...Binding of hedgehog to the receptor relieves its inhibitory action on the transmembrane co-receptor Smoh. In this study we describe alternative first exons of the PTCH1 tumour suppressor gene and show that they are differentially regulated in normal tissues, exon 1B being expressed at very low levels and the major mRNA species containing exon 1 or 1A. Exon 1B transcripts were found to be specifically upregulated in nodular BCCs. The different PTCH1 transcripts all encode proteins that interact with Smoh in doubly transfected cells. Furthermore, functional assays demonstrated that whereas all PTCH1 isoforms can inhibit the activity of SHH, only the PTCH1B isoform is capable of fully inhibiting Smoh activity. The results indicate that in tumour cells the PTCH1B promoter is specifically activated and importantly, that the N-terminal part of PTCH1 including exon 1B is required for full inhibition of Smoh signaling but not for physical interaction with Smoh.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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