Marc-Étienne Huot

BRK phosphorylates PSF promoting its cytoplasmic localization and cell cycle arrest

BReast tumor Kinase (BRK) also known as protein kinase 6 (PTK6) is a non-receptor tyrosine kinase overexpressed in the majority of human breast carcinoma. The expression of BRK is a known prognostic marker of breast carcinoma. BRK has been shown to lie downstream of epidermal growth factor (EGF) signaling and mediate effects on cell proliferation and migration. To identify BRK substrates and interacting proteins, we undertook a proteomic approach. BRK immune complexes were purified from the BT-20 breast cancer cell line and analyzed by mass spectrometry. Herein, we report the identification of PSF, the polypyrimidine tract-binding (PTB) protein-associated splicing factor, as a BRK-interacting protein and substrate. BRK and PSF co-eluted in a large protein complex that was regulated by EGF stimulation. Furthermore, BRK and PSF co-immunoprecipitated in BT-20 cells and we defined the interaction as being an SH3 domain-polyproline interaction. The C-terminal tyrosines of PSF were the site of phosphorylation by BRK. Moreover, tyrosine phosphorylation of PSF was also observed upon EGF stimulation, consistent with a role of PSF and BRK downstream of the EGF receptor. Interestingly, the tyrosine phosphorylation promoted the cytoplasmic relocalization of PSF, impaired its binding to polypyrimidine RNA, and led to cell cycle arrest. Our findings show that BRK targets the PSF RNA-binding protein during EGF stimulation.

Muscle specific fragile X related protein 1 isoforms are sequestered in the nucleus of undifferentiated myoblast.

BackgroundThe family of Fragile X Mental Retardation Proteins is composed of three members: Fragile Mental Retardation 1, Fragile X Related 1 and X Related 2 proteins. These proteins are associated with mRNPs within translating ribosomes and have the capacity to shuttle between the nucleus and the cytoplasm. Great attention has been given to FMRP due to its implication in human hereditary mental retardation while FXR1P and FXR2P have only recently been studied.ResultsUsing antibodies directed against several epitopes of FXR1P, we have detected protein isoforms generated by small peptides pocket inserts. Four isoforms of MW 70, 74, 78, 80 kDa are widely distributed in mouse organs, while in striated muscles these isoforms are replaced by proteins of 82 and 84 kDa containing an extra pocket of 27 aa. Expression of these muscle isoforms is an early event during in vitro differentiation of myoblasts into myotubes and correlates with the activation of muscle-specific genes. However, while FXR1P82,84 are associated with cytoplasmic mRNPs in myotubes, they are sequestered in the nuclei of undifferentiated myoblasts. These observations suggest that, in addition to a cytoplasmic function yet to be defined, FXR1P82,84 may play a nuclear role in pre-mRNA metabolism.ConclusionsThe pattern of subcellular partitioning of FXR1P isoforms during myogenesis is unique among the family of the FXR proteins. The model system described here should be considered as a powerful tool for ongoing attempts to unravel structure-function relationships of the different FMR family members since the potential role(s) of FXR1P as a compensatory factor in Fragile X syndrome is still elusive.

The oncometabolite 2-hydroxyglutarate activates the mTOR signalling pathway.

The identification of cancer-associated mutations in the tricarboxylic acid (TCA) cycle enzymes isocitrate dehydrogenases 1 and 2 (IDH1/2) highlights the prevailing notion that aberrant metabolic function can contribute to carcinogenesis. IDH1/2 normally catalyse the oxidative decarboxylation of isocitrate into α-ketoglutarate (αKG). In gliomas and acute myeloid leukaemias, IDH1/2 mutations confer gain-of-function leading to production of the oncometabolite R-2-hydroxyglutarate (2HG) from αKG. Here we show that generation of 2HG by mutated IDH1/2 leads to the activation of mTOR by inhibiting KDM4A, an αKG-dependent enzyme of the Jumonji family of lysine demethylases. Furthermore, KDM4A associates with the DEP domain-containing mTOR-interacting protein (DEPTOR), a negative regulator of mTORC1/2. Depletion of KDM4A decreases DEPTOR protein stability. Our results provide an additional molecular mechanism for the oncogenic activity of mutant IDH1/2 by revealing an unprecedented link between TCA cycle defects and positive modulation of mTOR function downstream of the canonical PI3K/AKT/TSC1-2 pathway.

Identification of a Sam68 Ribonucleoprotein Complex Regulated by Epidermal Growth Factor

Sam68, Src associated in mitosis of 68 kDa, is a known RNA-binding protein and a signaling adaptor protein for tyrosine kinases. However, the proteins associated with Sam68 and the existence of a Sam68 complex, its mass, and regulation are, however, unknown. Herein we identify a large Sam68 complex with a mass >1 MDa in HeLa cells that is composed of ∼40 proteins using an immunoprecipitation followed by a mass spectrometry approach. Many of the proteins identified are RNA-binding proteins and are known components of a previously identified structure termed the spreading initiation center. The large Sam68 complex is a ribonucleoprotein complex, as treatment with RNases caused a shift in the molecular mass of the complex to 200–450 kDa. Moreover, treatment of HeLa cells with phorbol 12-myristate 13-acetate or epidermal growth factor induced the disassociation of Sam68 from the large complex and the appearance of Sam68 within the smaller complex. Actually, in certain cell lines such as breast cancer cell lines MCF-7 and BT-20, Sam68 exists in equilibrium between a large and a small complex. The appearance of the small Sam68 complex in cells correlates with the ability of Sam68 to promote the alternative splicing of CD44 and cell migration. Our findings show that Sam68 exists in equilibrium in transformed cells between two complexes and that extracellular signals, such as epidermal growth factor stimulation, promote alternative splicing by modulating the composition of the Sam68 complex.

Lost Once, the Fragile X Mental Retardation Protein is Now Back onto Brain Polyribosomes

The Fragile X Mental Retardation protein (FMRP) is an RNA-binding protein and its absence leads to the Fragile X syndrome, the most common form of inherited mental retardation. Because it has been acknowledged for a long time that FMRP is associated with polyribosomal mRNPs in all non-neuronal cellular systems studied so far, it is thought that it regulates translation in neurons also; however, its exact function remains elusive. Recently, it has been reported that, contrary to non-neuronal cells, brain FMRP is not associated with the translation machinery, but is part of repressed small RNP complexes excluded from polyribosomes.27 To elucidate this puzzling result, Stefani et al.17 and Khandjian et al.32 have optimized methods to analyze brain polyribosomes and now provide definitive evidence for the association of FMRP with brain polyribosomes. In addition, the data presented in these two reports clearly indicate that FMRP’s function resides at the translation control level.

Characterization of Fragile X Mental Retardation Protein Recruitment and Dynamics in Drosophila Stress Granules

The RNA-binding protein Fragile X Mental Retardation (FMRP) is an evolutionarily conserved protein that is particularly abundant in the brain due to its high expression in neurons. FMRP deficiency causes fragile X mental retardation syndrome. In neurons, FMRP controls the translation of target mRNAs in part by promoting dynamic transport in and out neuronal RNA granules. We and others have previously shown that upon stress, mammalian FMRP dissociates from translating polysomes to localize into neuronal-like granules termed stress granules (SG). This localization of FMRP in SG is conserved in Drosophila. Whether FMRP plays a key role in SG formation, how FMRP is recruited into SG, and whether its association with SG is dynamic are currently unknown. In contrast with mammalian FMRP, which has two paralog proteins, Drosophila FMR1 (dFMRP) is encoded by a single gene that has no paralog. Using this genetically simple model, we assessed the role of dFMRP in SG formation and defined the determinants required for its recruitment in SG as well as its dynamics in SG. We show that dFMRP is dispensable for SG formation in vitro and ex vivo. FRAP experiments showed that dFMRP shuttles in and out SG. The shuttling activity of dFMRP is mediated by a protein-protein interaction domain located at the N-terminus of the protein. This domain is, however, dispensable for the localization of dFMRP in SG. This localization of dFMRP in SG requires the KH and RGG motifs which are known to mediate RNA binding, as well as the C-terminal glutamine/asparagine rich domain. Our studies thus suggest that the mechanisms controlling the recruitment of FMRP into SG and those that promote its shuttling between granules and the cytosol are uncoupled. To our knowledge, this is the first demonstration of the regulated shuttling activity of a SG component between RNA granules and the cytosol.

Oncogenic Activities of IDH1/2 Mutations: From Epigenetics to Cellular Signaling

Gliomas and leukemias remain highly refractory to treatment, thus highlighting the need for new and improved therapeutic strategies. Mutations in genes encoding enzymes involved in the tricarboxylic acid (TCA) cycle, such as the isocitrate dehydrogenases 1 and 2 (IDH1/2), are frequently encountered in astrocytomas and secondary glioblastomas, as well as in acute myeloid leukemias; however, the precise molecular mechanisms by which these mutations promote tumorigenesis remain to be fully characterized. Gain-of-function mutations in IDH1/2 have been shown to stimulate production of the oncogenic metabolite R-2-hydroxyglutarate (R-2HG), which inhibits α-ketoglutarate (αKG)-dependent enzymes. We review recent advances on the elucidation of oncogenic functions of IDH1/2 mutations, and of the associated oncometabolite R-2HG, which link altered metabolism of cancer cells to epigenetics, RNA methylation, cellular signaling, hypoxic response, and DNA repair.

DDX3 regulates endoplasmic reticulum stress-induced ATF4 expression

Accumulation of unfolded and potentially toxic proteins in the endoplasmic reticulum (ER) activates a cell stress adaptive response, which involves a reprogramming of general gene expression. ATF4 is a master stress-induced transcription factor that orchestrates gene expression in cells treated with various ER stress inducers including those used to treat cancers. ER stress-induced ATF4 expression occurs mainly at the translational level involving the activity of the phosphorylated (P) translation initiation factor (eIF) eIF2α. While it is well established that under ER stress PeIF2α drives ATF4 expression through a specialised mode of translation re-initiation, factors (e.g. RNA-binding proteins and specific eIFs) involved in PeIF2α-mediated ATF4 translation remain unknown. Here we identified the RNA-binding protein named DDX3 as a promotor of ATF4 expression in cancer cells treated with sorafenib, an ER stress inducer used as a chemotherapeutic. Depletion experiments showed that DDX3 is required for PeIF2α-mediated ATF4 expression. Luciferase and polyribosomes assays showed that DDX3 drives ER stress-induced ATF4 mRNA expression at the translational level. Protein-interaction assays showed that DDX3 binds the eIF4F complex, which we found to be required for ER stress-induced ATF4 expression. This study thus showed that PeIF2α-mediated ATF4 mRNA translation requires DDX3 as a part of the eIF4F complex.

Manipulating the Fragile X Mental Retardation Proteins in the Frog

The frog is a model of choice to study gene function during early development, since a large number of eggs are easily obtained and rapidly develop external to the mother. This makes it a highly flexible model system in which direct tests of gene function can be investigated by microinjecting RNA antisense reagents. Two members of the Fragile X Related (FXR) gene family, namely xFmr1 and xFxr1 have been identified in Xenopus. While the tissue distribution of their products was found to be identical to that in mammals, the pattern of isoform expression is less complex. Translational silencing of the xFmr1 and xFxr1 mRNAs by microinjection of antisense morpholino oligonucleotides (MO) induced dramatic morphological alterations, revealing tissue-specific requirements for each protein during development and in maintaining the steady state levels of a range of transcripts in these tissues. The power and versatility of the frog model is that the MO-induced phenotypes can be rescued by microinjection of the corresponding MO-insensitive mRNAs. Most importantly, this animal model allows one rapidly to determine whether any member of the FXR family can compensate for the absence of another, an approach that cannot be performed in other animal models.

The phosphoinositide 3-kinase (PI3K) pathway and glycogen synthase kinase-3 (GSK-3) positively regulate the activity of metal-responsive transcription factor-1 (MTF-1) in response to zinc ions.

Metal-responsive transcription factor-1 (MTF-1) is a metal-regulatory transcription factor essential for induction of the genes encoding metallothioneins (MTs) in response to transition metal ions. Activation of MTF-1 is dependent on the interaction of zinc with the zinc fingers of the protein. In addition, phosphorylation is essential for MTF-1 transactivation. We previously showed that inhibition of phosphoinositide 3-kinase (PI3K) abrogated Mt expression and metal-induced MTF-1 activation in human hepatocellular carcinoma (HCC) HepG2 and mouse L cells, thus showing that the PI3K signaling pathway positively regulates MTF-1 activity and Mt gene expression. However, it has also been reported that inhibition of PI3K has no significant effects on Mt expression in immortalized epithelial cells and increases Mt expression in HCC cells. To further characterize the role of the PI3K pathway on the activity of MTF-1, transfection experiments were performed in HEK293 and HepG2 cells in presence of glycogen synthase kinase-3 (GSK-3), mTOR-C1, and mTOR-C2 inhibitors, as well as of siRNAs targeting Phosphatase and TENsin homolog (PTEN). We showed that inhibition of the mTOR-C2 complex inhibits the activity of MTF-1 in HepG2 and HEK293 cells, while inhibition of the mTOR-C1 complex or of PTEN stimulates MTF-1 activity in HEK293 cells. These results confirm that the PI3K pathway positively regulates MTF-1 activity. Finally, we showed that GSK-3 is required for MTF-1 activation in response to zinc ions.