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1.
Mol Cell ; 58(3): 453-67, 2015 May 07.
Article in English | MEDLINE | ID: mdl-25866244

ABSTRACT

Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers.


Subject(s)
Cell Nucleus/metabolism , Homeodomain Proteins/metabolism , Phosphatidylinositols/metabolism , Transcription, Genetic , Amino Acid Sequence , Animals , Cell Differentiation/genetics , Cell Line , Cell Nucleus/genetics , Electrophoresis, Polyacrylamide Gel , Gene Expression Profiling , Histones/metabolism , Homeodomain Proteins/genetics , Lysine/metabolism , Methylation , Mice , Minor Histocompatibility Antigens , Molecular Sequence Data , Mutation , Myoblasts/cytology , Myoblasts/metabolism , Oligonucleotide Array Sequence Analysis , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Binding , RNA Interference , Sequence Homology, Amino Acid , TATA-Binding Protein Associated Factors , Transcription Factor TFIID/genetics , Transcription Factor TFIID/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism
2.
Int J Biochem Cell Biol ; 94: 71-78, 2018 01.
Article in English | MEDLINE | ID: mdl-29203233

ABSTRACT

Notch signaling plays an essential role in the proliferation, differentiation and cell fate determination of various tissues, including the developing pancreas. One regulator of the Notch pathway is GDE2 (or GDPD5), a transmembrane ecto-phosphodiesterase that cleaves GPI-anchored proteins at the plasma membrane, including a Notch ligand regulator. Here we report that Gdpd5-knockdown in zebrafish embryos leads to developmental defects, particularly, impaired motility and reduced pancreas differentiation, as shown by decreased expression of insulin and other pancreatic markers. Exogenous expression of human GDE2, but not catalytically dead GDE2, similarly leads to developmental defects. Human GDE2 restores insulin expression in Gdpd5a-depleted zebrafish embryos. Importantly, zebrafish Gdpd5 orthologues localize to the plasma membrane where they show catalytic activity against GPI-anchored GPC6. Thus, our data reveal functional conservation between zebrafish Gdpd5 and human GDE2, and suggest that strict regulation of GDE2 expression and catalytic activity is critical for correct embryonic patterning. In particular, our data uncover a role for GDE2 in regulating pancreas differentiation.


Subject(s)
Gene Expression Regulation, Developmental , Organogenesis , Pancreas/metabolism , Phosphoric Diester Hydrolases/metabolism , Zebrafish Proteins/metabolism , Animals , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/diagnostic imaging , Embryo, Nonmammalian/metabolism , Gene Knockdown Techniques , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Morpholinos/metabolism , Pancreas/diagnostic imaging , Pancreas/embryology , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Phylogeny , Protein Domains , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Zebrafish , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/chemistry , Zebrafish Proteins/genetics
3.
Sci Rep ; 7(1): 7327, 2017 08 04.
Article in English | MEDLINE | ID: mdl-28779098

ABSTRACT

Autophagy is an evolutionarily conserved process that degrades cellular components to restore energy homeostasis under limited nutrient conditions. How this starvation-induced autophagy is regulated at the whole-body level is not fully understood. Here, we show that the tumor suppressor Lkb1, which activates the key energy sensor AMPK, also regulates starvation-induced autophagy at the organismal level. Lkb1-deficient zebrafish larvae fail to activate autophagy in response to nutrient restriction upon yolk termination, shown by reduced levels of the autophagy-activating proteins Atg5, Lc3-II and Becn1, and aberrant accumulation of the cargo receptor and autophagy substrate p62. We demonstrate that the autophagy defect in lkb1 mutants can be partially rescued by inhibiting mTOR signaling but not by inhibiting the PI3K pathway. Interestingly, mTOR-independent activation of autophagy restores degradation of the aberrantly accumulated p62 in lkb1 mutants and prolongs their survival. Our data uncover a novel critical role for Lkb1 in regulating starvation-induced autophagy at the organismal level, providing mechanistic insight into metabolic adaptation during development.


Subject(s)
Autophagy , Protein Serine-Threonine Kinases/metabolism , Starvation , Stress, Physiological , Tumor Suppressor Proteins/metabolism , Animals , Autophagy/genetics , Biomarkers , Fluorescent Antibody Technique , Immunohistochemistry , Larva , Mutation , Protein Serine-Threonine Kinases/genetics , Stress, Physiological/genetics , TOR Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/genetics , Zebrafish
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