Kumiko Tamura

Physical interaction between a novel domain of the receptor Notch and the transcription factor RBP-Jκ/Su(H)

BACKGROUND The mammalian transcription factor RBP-J kappa binds to the DNA sequence motif CGTGGGAA and is involved in the regulation of gene expression; for example, it plays a part in the transactivation of viral and cellular genes by Epstein-Barr virus nuclear antigen-2. The Drosophila homologue of RBP-J kappa is the product of the Suppressor of Hairless (Su(H)) gene. Su(H) is a neurogenic gene that acts downstream of Notch, which encodes a cell-surface receptor. Furthermore, in the mouse, the phenotypes of homozygous mutant Notch1 embryos are very similar to those of homozygous mutant RBP-J kappa embryos. Recent studies, using the yeast two-hybrid system, have led to the suggestion that the CDC10/ankyrin-like repeats of the Drosophila Notch protein interact with the Su(H) protein. RESULTS We searched for proteins that interact with mouse RBP-J kappa using the yeast two-hybrid system, and in this way identified a short intracellular region (mRAM23) of the mouse Notch1 protein that lacks any known sequence motif. In vitro interaction studies, using proteins fused to glutathione-S-transferase, showed that RBP-J kappa and Su(H) bind directly to the RAM23 regions of mouse Notch1 and Drosophila Notch, respectively. Immunoprecipitation analysis showed that RBP-J kappa and the mRAM23 region of mouse Notch1 also interact in vivo. Further studies, including site-directed mutagenesis experiments, narrowed down the region of mouse Notch1 that interacts with RBP-J kappa. The results indicate that this region is less than 50 amino-acid residues in length, and lies immediately downstream of the transmembrane region. CONCLUSIONS We show that the transcription factor RBP-J kappa/Su(H) interacts directly with a novel intracellular domain of the cell-surface receptor Notch. RBP-J kappa/Su(H) does not appear to interact with Notch via the CDC10/ankyrin repeats implicated in previous studies.

p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps.

ABSTRACT The extracellular signals which regulate the myogenic program are transduced to the nucleus by mitogen-activated protein kinases (MAPKs). We have investigated the role of two MAPKs, p38 and extracellular signal-regulated kinase (ERK), whose activities undergo significant changes during muscle differentiation. p38 is rapidly activated in myocytes induced to differentiate. This activation differs from those triggered by stress and cytokines, because it is not linked to Jun–N-terminal kinase stimulation and is maintained during the whole process of myotube formation. Moreover, p38 activation is independent of a parallel promyogenic pathway stimulated by insulin-like growth factor 1. Inhibition of p38 prevents the differentiation program in myogenic cell lines and human primary myocytes. Conversely, deliberate activation of endogenous p38 stimulates muscle differentiation even in the presence of antimyogenic cues. Much evidence indicates that p38 is an activator of MyoD: (i) p38 kinase activity is required for the expression of MyoD-responsive genes, (ii) enforced induction of p38 stimulates the transcriptional activity of a Gal4-MyoD fusion protein and allows efficient activation of chromatin-integrated reporters by MyoD, and (iii) MyoD-dependent myogenic conversion is reduced in mouse embryonic fibroblasts derived from p38α−/− embryos. Activation of p38 also enhances the transcriptional activities of myocyte enhancer binding factor 2A (MEF2A) and MEF2C by direct phosphorylation. With MEF2C, selective phosphorylation of one residue (Thr293) is a tissue-specific activating signal in differentiating myocytes. Finally, ERK shows a biphasic activation profile, with peaks of activity in undifferentiated myoblasts and postmitotic myotubes. Importantly, activation of ERK is inhibitory toward myogenic transcription in myoblasts but contributes to the activation of myogenic transcription and regulates postmitotic responses (i.e., hypertrophic growth) in myotubes.

Requirement for p38α in Erythropoietin Expression

Activity of the p38alpha MAP kinase is stimulated by various stresses and hematopoietic growth factors. A role for p38alpha in mouse development and physiology was investigated by targeted disruption of the p38alpha locus. Whereas some p38alpha(-/-) embryos die between embryonic days 11.5 and 12.5, those that develop past this stage have normal morphology but are anemic owing to failed definitive erythropoiesis, caused by diminished erythropoietin (Epo) gene expression. As p38alpha-deficient hematopoietic stem cells reconstitute lethally irradiated hosts, p38alpha function is not required downstream of Epo receptor. Inhibition of p38 activity also interferes with stabilization of Epo mRNA in human hepatoma cells undergoing hypoxic stress. The p38alpha MAP kinase plays a critical role linking developmental and stress-induced erythropoiesis through regulation of Epo expression.

Delta-induced Notch Signaling Mediated by RBP-J Inhibits MyoD Expression and Myogenesis

Signaling induced by interaction between the receptor Notch and its ligand Delta plays an important role in cell fate determination in vertebrates as well as invertebrates. Vertebrate Notch signaling has been investigated using its constitutively active form, i.e. the truncated intracellular region which is believed to mimic Notch-Delta signaling by interaction with a DNA-binding protein RBP-J. However, the molecular mechanism for Notch signaling triggered by ligand binding, which leads to inhibition of differentiation, is not clear. We have established a myeloma cell line expressing mouse Delta1 on its cell surface which can block muscle differentiation by co-culture with C2C12 muscle progenitor cells. We showed that Delta-induced Notch signaling stimulated transcriptional activation of RBP-J binding motif, containing promoters including the HES1 promoter. Furthermore, ligand-induced Notch signaling up-regulated HES1 mRNA expression within 1 h and subsequently reduced expression of MyoD mRNA. Since cycloheximide treatment did not inhibit induction of HES1 mRNA, the HES1 promoter appears to be a primary target of activated Notch. In addition, a transcriptionally active form of RBP-J, i.e. VP16-RBP-J, inhibited muscle differentiation of C2C12 cells by blocking the expression of MyoD protein. These results suggest that HES1 induction by the Delta1/Notch signaling is mediated by RBP-J and blocks myogenic differentiation of C2C12 cells by subsequent inhibition of MyoD expression.

The stress-induced MAP kinase p38 regulates endocytic trafficking via the GDI : Rab5 complex

Early endocytic membrane traffic is regulated by the small GTPase Rab5, which cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle depends on GDI, which functions as a Rab vehicle in the aqueous environment of the cytosol. Here, we report that formation of the GDI:Rab5 complex is stimulated by a cytosolic factor that we purified and then identified as p38 MAPK. We find that p38 regulates GDI in the cytosolic cycle of Rab5 and modulates endocytosis in vivo. Our observations reveal the existence of a cross-talk between endocytosis and the p38-dependent stress response, thus providing molecular evidence that endocytosis can be regulated by the environment.

Functional conservation of mouse Notch receptor family members

All the known members of the mouse Notch receptor family were examined for their biochemical function by interaction with a DNA binding protein RBP‐Jκ. mNotch2, mNotch3 and int3 (= mNotch4) were shown to interact with RBP‐Jκ by the GST‐fusion pull down assay and dominant negative competition with Epstein Barr virus nuclear antigen 2. Furthermore the intracellular region of int3 was shown to transactivate the Epstein Barr virus TP1 promoter. These results indicate that all mouse Notch family members have biochemical functions similar to mNotch1, which transduces proliferative signal by direct interaction with the DNA binding protein RBP‐Jκ.