Neither the homeodomain nor the activation domain of Bicoid is specifically required for its down-regulation by the Torso receptor tyrosine kinase cascade

Development ◽  
1996 ◽  
Vol 122 (11) ◽  
pp. 3499-3508 ◽  
Author(s):  
Y. Bellaiche ◽  
R. Bandyopadhyay ◽  
C. Desplan ◽  
N. Dostatni
Keyword(s):  
Dna Binding ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Chimeric Protein ◽  
Drosophila Embryo ◽  
Activation Domain ◽  
Down Regulation ◽  
Dna Binding Sites ◽  
Early Embryos ◽  
Kinase Cascade

Bicoid (Bcd) is a maternal morphogen responsible for patterning the head and thorax of the Drosophila embryo. Correct specification of head structure, however, requires the activity of the Torso receptor tyrosine kinase cascade, which also represses expression of Bcd targets at the most anterior tip of the embryo. Here, we investigate the role of both the homeodomain (HD) and the activation domain of Bcd in the anterior repression of its targets. When a Bcd mutant protein whose HD has been replaced by the Gal4 DNA-binding domain is expressed in early embryos, a reporter gene driven by Gal4 DNA-binding sites is first activated in an anterior domain and then repressed from the anterior pole. The down-regulation of Bcd-Gal4 activity requires torso function but does not depend on endogenous bcd activity, indicating that the Bcd protein alone and none of its targets is required to mediate the effect of torso. Functional analysis of a chimeric protein, whose activation domain has been replaced by a generic activation domain, indicates that the activation domain of Bcd is also not specifically required for its down-regulation by Torso. We propose that Torso does not affect the ability of Bcd to bind DNA, but instead directs modification of Bcd or of a potential Bcd co-factor, which renders the Bcd protein unable to activate transcription.

Torso signalling regulates terminal patterning in Drosophila by antagonising Groucho-mediated repression

Development ◽  
1997 ◽  
Vol 124 (19) ◽  
pp. 3827-3834 ◽  
Author(s):  
Z. Paroush ◽  
S.M. Wainwright ◽  
D. Ish-Horowicz
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Drosophila Embryo ◽  
Gap Gene ◽  
Gap Genes ◽  
Developmental Contexts

Patterning of the non-segmental termini of the Drosophila embryo depends on signalling via the Torso receptor tyrosine kinase (RTK). Activation of Torso at the poles of the embryo triggers restricted expression of the zygotic gap genes tailless (tll) and huckebein (hkb). In this paper, we show that the Groucho (Gro) corepressor acts in this process to confine terminal gap gene expression to the embryonic termini. Embryos lacking maternal gro activity display ectopic tll and hkb transcription; the former leads, in turn, to lack of abdominal expression of the Kruppel and knirps gap genes. We show that torso signalling permits terminal gap gene expression by antagonising Gro-mediated repression. Thus, the corepressor Gro is employed in diverse developmental contexts and, probably, by a variety of DNA-binding repressors.

The Receptor Tyrosine Kinase RON Is Constitutively Activated in Acute Myeloid Leukemia and May Represent a New Therapeutic Target.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 994-994
Author(s):  
Camille Fialin ◽  
Veronique De Mas ◽  
Stephane Manenti ◽  
Bernard Payrastre ◽  
Serge Roche ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Protein Level ◽  
Leukemic Cell ◽  
Down Regulation ◽  
Hematopoietic Stem ◽  
Inhibition Of Proliferation ◽  
Si Rna

Abstract Abstract 994 Poster Board I-16 Recepteur d'Origine Nantais (RON) is a receptor tyrosine kinase (RTK) expressed mostly on epithelial cells but also in normal hematopoietic stem cells and macrophages. RON is closely related to c-Met in terms of homology and function. Its oncogenic properties have been documented in solid tumors leading to the clinical development of small molecules inhibitors. However, the role of RON in haematological malignancies and specifically in AML, has received little attention. Interestingly, a truncated form of RON lacking most of the RON receptor extracellular domain but retaining the whole transmembrane and intracellular domains has been described in the leukemic cell line KG1 (Bardella C et al, Cancer Res 64, 5154–5161, 2004). Since RTK and TK play a crucial role in leukemogenesis, we have assessed the expression and the role of RON in both AML cell lines and patient samples. The expression of both full length (fl) and short form (sf) of RON was assessed in 71 AML samples by RT-PCR. fl-RON and sf-RON were found in 48/71 pts (68%) and 26/71 (37%) respectively, whereas both forms were undetectable in 17 samples (24%). RON expression was further confirmed at the protein level by western blot analysis in 20 samples. Conversely, c-Met was not expressed at the protein level in 10 cases tested. In leukemic cell lines, fl-RON and sf-RON were expressed both at the mRNA and protein levels in KG1 and KG1a but not in HL60 and U937. Immunoprecipitation analysis showed that fl-RON and sf-RON were constitutively phosphorylated on tyrosine in KG1/KG1a cells. This phosphorylation was fully inhibited by the dual c-Met/RON inhibitor, SU-11274. Cell signaling induced by RON has also been explored. Specific down regulation by si-RNA to RON induced a significant decrease of Lyn phosphorylation. Conversely, AKT phosphorylation was not influenced by RON down regulation. We then assessed the activity of SU-11274 on the proliferation and survival of KG1 (RON+) and HL60 (RON-). The proliferation of KG1 (IC50=3.5μM) but not HL60 (IC50 not reached at 10μM) was strongly inhibited in a time and dose dependant manner. This inhibition of proliferation was mostly due to apoptosis induction. Accordingly using clonogenic and cytotoxic assays, we show that only RON positive samples from AML patients responded to SU-11274 (n=8). Moreover, specific down regulation of RON by si-RNA inhibited the clonogenic properties of KG1 cells. Altogether, these data demonstrate that the tyrosine kinase RON is aberrantly deregulated in AML cells, control cell proliferation and could represent a new target for the treatment of AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Insulin receptor desensitization correlates with attenuation of tyrosine kinase activity, but not of receptor endocytosis

1987 ◽  
Vol 245 (2) ◽  
pp. 357-364 ◽  
Author(s):  
A D Blake ◽  
N S Hayes ◽  
E E Slater ◽  
C D Strader
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Glycogen Synthesis ◽  
Insulin Receptors ◽  
Beta Subunit ◽  
Tyrosine Kinase Activity ◽  
Down Regulation ◽  
Receptor Endocytosis

A model of insulin-receptor down-regulation and desensitization has been developed and described. In this model, both insulin-receptor down-regulation and functional desensitization are induced in the human HepG2 cell line by a 16 h exposure of the cells to 0.1 microM-insulin. Insulin-receptor affinity is unchanged, but receptor number is decreased by 50%, as determined both by 125I-insulin binding and by protein immunoblotting with an antibody to the beta-subunit of the receptor. This down-regulation is accompanied by a disproportionate loss of insulin-stimulated glycogen synthesis, yielding a population of cell-surface insulin receptors which bind insulin normally but which are unable to mediate insulin-stimulated glycogen synthesis within the cell. Upon binding of insulin, the desensitized receptors are internalized rapidly, with characteristics indistinguishable from those of control cells. In contrast, this desensitization is accompanied by a loss of the insulin-sensitive tyrosine kinase activity of insulin receptors isolated from these cells. Receptors isolated from control cells show a 5-25-fold enhancement of autophosphorylation of the beta-subunit by insulin; this insulin-responsive autophosphorylation is severely attenuated after desensitization to a maximum of 0-2-fold stimulation by insulin. Likewise, the receptor-mediated phosphorylation of exogenous angiotensin II, which is stimulated 2-10-fold by insulin in receptors from control cells, is completely unresponsive to insulin in desensitized cells. These data provide evidence that the insulin-receptor tyrosine kinase activity correlates with insulin stimulation of an intracellular metabolic event. The data suggest that receptor endocytosis is not sufficient to mediate insulin's effects, and thereby argue for a role of the receptor tyrosine kinase activity in the mediation of insulin action.

Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization

1991 ◽  
Vol 11 (2) ◽  
pp. 954-962
Author(s):  
C V Dang ◽  
J Barrett ◽  
M Villa-Garcia ◽  
L M Resar ◽  
G J Kato ◽  
...  
Keyword(s):  
Dna Binding ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Chimeric Protein ◽  
Dna Binding Domain ◽  
Cytoplasmic Protein ◽  
Activation Domain ◽  
In Cells

The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein. In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction. These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.

scholarly journals Functional Analysis of H-Ryk, an Atypical Member of the Receptor Tyrosine Kinase Family

1999 ◽  
Vol 19 (9) ◽  
pp. 6427-6440 ◽  
Author(s):  
R. M. Katso ◽  
R. B. Russell ◽  
T. S. Ganesan
Keyword(s):  
Catalytic Activity ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Catalytic Domain ◽  
Homology Modelling ◽  
Mitogen Activated Protein Kinase ◽  
In Vitro Mutagenesis ◽  
Chimeric Receptor ◽  
Activation Domain

ABSTRACT H-Ryk is an atypical receptor tyrosine kinase which differs from other members of this family at a number of conserved residues in the activation and nucleotide binding domains. Using a chimeric receptor approach, we demonstrate that H-Ryk has impaired catalytic activity. Despite the receptor’s inability to undergo autophosphorylation or phosphorylate substrates, we demonstrate that ligand stimulation of the chimeric receptor results in activation of the mitogen-activated protein kinase pathway. The ability to transduce signals is abolished by mutation of the invariant lysine (K334A) in subdomain II of H-Ryk. Further, by in vitro mutagenesis, we show that the amino acid substitutions in the activation domain of H-Ryk account for the loss of catalytic activity. In addition to the essential aspartate residue, either phenylalanine or glycine is required in the activation domain to maintain proper conformation of the catalytic domain and thus ensure receptor autophosphorylation. Homology modelling of the catalytic domain of H-Ryk provides a rationale for these findings. Thus, the signalling properties of H-Ryk are divergent from those of other classical receptor tyrosine kinases.

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