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R Janknecht

Publications and source records attributed to R Janknecht.

At least 37 records · Page 2Linked to original sources
PubMed1996

The ETS-related transcription factor ERM is a nuclear target of signaling cascades involving MAPK and PKA.

Recent studies support a model for signal transduction from activated receptor tyrosine kinases to Ras which, in turn, activates the pathway of the mitogen-activated protein kinase (MAPK). Although some members of the Ets transcription factor family have been shown to be activated by this signaling pathway, no data are available on the activation of the PEA3 group of Ets proteins. This group is composed of three members -- PEA3, ER81 and ERM -- which are very similar in the DNA-binding domain, the ETS domain, in the 32 residue amino-terminal acidic domain and in the 61 residue carboxy-terminal domain. First of all we demonstrated that ERM-transfected cells contain a positive labeling in the nucleus, and we concluded that a nuclear localization signal might be situated in the ETS domain. We then showed that of four putative reporter plasmids, ERM activated the artificial 3 x TORU plasmid which contains an Ets binding site contiguous to an AP1 one. This transactivation enhancement requires the presence of the ERM amino-terminal domain. In contrast, although the lack of the carboxy-terminal domain induced a decrease in transactivation, this latter domain is not crucial. By using the E74-reporter plasmid system which is not basically activated by ERM, we showed that the activation of the Ras/Raf-1/MAPK pathway significantly enhanced ERM-mediated transactivation. The deletion of the amino-terminal transactivation domain abolished the capacity of stimulated MAPK to activate ERM. We also demonstrated that ERM can also be activated through the protein kinase A (PKA), another signaling pathway. Nevertheless, the MAPK and PKA activation of ERM are not synergistic. Finally, we showed that this Ets transcription factor is in vitro phosphorylated by both activated ERK-2 and activated PKA. ERM has thus been identified as a transcription factor which is a target for two different signaling pathways and might therefore be involved in the mitogenic response of cells.

Animals↗
PubMed1996

Versatile molecular glue. Transcriptional control.

CBP and p300 are versatile coactivators that physically connect many DNA-binding factors to the basal transcription machinery. Phosphorylation by cyclin-dependent or signal-induced protein kinases may regulate their function.

Acetyltransferases↗
PubMed1996

Interaction of the co-activator CBP with Myb proteins: effects on Myb-specific transactivation and on the cooperativity with NF-M.

The oncoprotein v-Myb is a potent inducer of myeloid leukemias, and its cellular homolog c-Myb plays a crucial role in the regulation of hematopoiesis. Both proteins function as transcriptional regulators. We demonstrate that this function is mediated at least in part by the nuclear co-activator CREB binding protein (CBP). This protein interacts directly with both c-Myb and v-Myb and potentiates Myb-specific transcription as measured on the mim-1 promoter. In contrast, dominant negative mutants of CBP lead to repression, as does E1A, an antagonist of CBP function. Phosphorylation of c-Myb does not appear to be required for interaction with CBP, thus indicating that the binding may be constitutive. Furthermore, the C/EBP family member NF-M, which cooperates with c-Myb in transactivating the mim-1 promoter through an adjacent DNA binding site, is co-activated by CBP in a Ras-dependent manner. Not only the individual activities of c-Myb and NF-M are stimulated by CBP, but also their synergistic transcriptional function, while it is negatively regulated by dominant negative forms of CBP. These data suggest that CBP is recruited by both Myb proteins and NF-M and potentiates their transcriptional activity. We suggest that CBP can bridge between c-Myb and NF-M, thus providing an explanation for the strong synergism between these two proteins.

Animals↗
PubMed1996

Regulation of the c-fos promoter by the ternary complex factor Sap-1a and its coactivator CBP.

The c-fos proto-oncogene is activated by a plethora of signals via the transcription factors Sap-1a and CREB. Recently, the coactivator CBP has been demonstrated to act in concert with CREB when CREB is phosphorylated by protein kinase A. We show that CBP also binds directly to Sap-1a. While phosphorylation of Sap-1a by mitogen-activated protein kinases is not necessary for CBP/Sap-1a interaction, functional cooperation between these two proteins requires Sap-1a to become phosphorylated. CBP-antagonists impair Sap-1a-mediated transactivation. Similarly, the CBP antagonist E1A suppresses c-fos upregulation by phosphorylated CREB, indicating that CBP is a central component of c-fos regulation. Furthermore, CBP is phosphorylated by protein kinase A in vitro and the transactivation potential of the carboxy-terminal region of CBP is enhanced in the presence of active protein kinase A in vivo. Thus, CBP, in addition to CREB, is a target for cAMP-dependent signaling. However, combined phosphorylation of CBP by protein kinase A and mitogen-activated protein kinases appears to be non-cooperative, suggesting that CBP serves the function of a dampening integrator of two different signaling pathways.

Animals↗
PubMed1996

Signalling pathways: jack of all cascades.

The transcription factors that bind the c-fos promoter element SRE are targeted by multiple, independent signalling cascades; the identities of these signalling pathways and their modes of activation are being elucidated.

Animals↗
PubMed1996

Quantitative structure-activity analysis correlating Ras/Raf interaction in vitro to Raf activation in vivo.

Binding of Ras to c-Raf-1 is a pivotal step of many mitogenic signalling pathways. Based on the recent crystal structure of the complex of Rap1A with the Ras-binding domain of Raf, mutations were introduced in c-Raf-1 and their effects on Ras/Raf binding affinity in vitro and Ras/Raf regulated gene expression in vivo were analysed. Our data reveal an empirical semilogarithmic correlation between dissociation constants and Raf-induced gene activity. The functional epitope that primarily determines binding affinity consists of residues Gln 66, Lys 84 and Arg 89 in Raf. This quantitative structure-activity investigation may provide a general approach to correlate structure-guided biochemical analysis with biological function of protein-protein interactions.

Amino Acid Sequence↗
PubMed1996

Ras/Rap effector specificity determined by charge reversal.

Members of the Ras subfamily of small GTP-binding proteins have been shown to be promiscuous towards a variety of putative effector molecules such as the protein kinase c-Raf and the Ral-specific guanine nucleotide exchange factor (Ral-GEF). To address the question of specificity of interactions we have introduced the mutations E30D and K31E into Rap and show biochemically, by X-ray structure analysis and by transfection in vivo that the identical core effector region of Ras and Rap (residues 32-40) is responsible for molecular recognition, but that residues outside this region are responsible for the specificity of the interaction. The major determinant for the switch in specificity is the opposite charge of residue 31--Lys in Rap, Glu in Ras--which creates a favourable complementary interface for the Ras-Raf interaction.

Amino Acid Sequence↗
PubMed1996

Analysis of the ERK-stimulated ETS transcription factor ER81.

A plethora of extracellular signals leads to the stimulation of Ras, which triggers intracellular protein kinase cascades, resulting in activation of transcription factors and thus in enhanced gene activity. In this report, it is demonstrated that the ETS transcription factor ER81, which appears to be localized within the cell nucleus by virtue of its DNA binding domain, is transcriptionally activated by oncogenic Ras. Since this activation was dependent on the presence of Raf-1 and ERK-1, ER81 is a target of the Ras/Raf/MEK/ERK signaling cascade. Consistently, activated ERK-1 is capable to phosphorylate ER81. However, the carboxy-terminal region of ER81, which contains no potential ERK phosphorylation sites, is also transcriptionally activated by ERK-1, suggesting that an ERK-stimulated protein kinase phosphorylates and thus stimulates ER81 activity. Two acidic stretches of amino acids, which are conserved in the related PEA3 and ERM proteins, are localized within the amino-and carboxy-terminal transactivation domains of ER81. In addition, an inhibitory domain may dampen the activation function of these two domains. In conclusion, ER81 is a target of Ras-dependent signaling cascades and may thus contribute to the nuclear response upon stimulation of cells and also to cellular transformation due to oncogenic Ras.

Animals↗
PubMed1995

High affinity DNA binding of native full length c-Myb and differential proteolytic sensitivity of its N- and C-terminal domains.

c-Myb is the prototype of a family of transcription factors characterised by a unique DNA binding domain. Previous analyses have concentrated on truncated versions of c-Myb as it has been very difficult to produce full length c-Myb. To overcome these difficulties we expressed full length c-Myb in HeLa cells using a recombinant vaccinia virus. Partially purified native full length c-Myb bound efficiently and specifically to DNA with a dissociation constant similar to that obtained with bacterially expressed DNA binding domains. No evidence was found for a negative effect of the leucine zipper on DNA binding. Furthermore the DNA binding domain was protease resistant in contrast to the transactivation and negative regulatory domains. Phosphorylation had no apparent effect on this differential protease sensitivity. The increased sensitivity of the C-terminal domain suggests a more open conformation, which may be relevant in the integration of signals and/or in protein-protein interactions.

Amino Acid Sequence↗
PubMed1995

SAP1a is a nuclear target of signaling cascades involving ERKs.

The Ets protein SAP1a has been shown to interact with the c-fos serum response element upon recruitment by the serum response factor. We demonstrate that SAP1a is a nuclear protein stimulating transcription via the c-fos serum response element, and additionally via an Ets binding site independently of the serum response factor. However, transactivation has only been observed under conditions leading to the activation of extracellular signal-regulated protein kinases (ERKs). The transcriptional activation domain of SAP1a resides within the C-terminal region, the function of which may be impeded by the N-terminus. Several potential ERK consensus sites within the C-terminal region of SAP1a can modulate its transactivation efficacy, implicating that SAP1a is a direct target of ERKs. Since ERKs are activated by a broad range of signals, SAP1a may play an important role in the transformation of extracellular stimuli into a nuclear response.

Amino Acid Sequence↗
PubMed1995

Transcriptional repression mediated by the serum response factor.

The serum response element (SRE) contributes to transcriptional repression of the c-fos proto-oncogene. We show that the transcription factor SRF is able to repress SRE-dependent transcription, apparently by sequestering a co-activator. Only the DNA-binding core region is required for this SRE-dependent repression. Furthermore the phosphorylation status at potential casein kinase II sites within an N-terminal repression domain affects SRE-independent transcription. SRF may thus pleiotropically influence cellular transcription, representing a novel aspect of SRF function.

3T3 Cells↗
PubMed1994

Co-occurrence of CArG boxes and TCF sites within viral genomes.

The transcription factor SRF is involved in the transduction of extracellular signals into nuclear responses, often in conjunction with ternary complex factors (TCFs). Here we report the identification of CArG box SRF binding-sites, and neighboring TCF binding-sites, in viral genomes. SRF binds and recruits TCFs to CMV, RSV and HTLV-1 viral genomes. At least one of two specific CArG boxes occurred in cytomegaloviruses in the 5' proximal region of the major immediate early gene, one always accompanied by a TCF site. This conservation was striking since neither the flanking sequences nor the spacing to the CAP site were conserved. Thus the ubiquitous SRF and TCF molecules may control events in the life cycle of viruses.

3T3 Cells↗
PubMed1994

Heterogeneity of ternary complex factors in HeLa cell nuclear extracts.

Ternary complex factors (TCFs) interact with the serum response factor and DNA containing the c-fos serum response element to form a ternary complex that mediates induction of c-fos. TCF activities were partially purified from HeLa cell nuclear extracts by DNA-cellulose and anion-exchange chromatography followed by two-dimensional gel electrophoresis. Four different protein spots (p60TCF, p62TCF, p62.5TCF, and p64TCF) show renaturable TCF activity. One, p62TCF, was indistinguishable from the Ets protein Elk-1 in gel shift analyses, while none of the HeLa TCFs resembled the other cloned TCFs, SAP-1a and SAP-1b. In two-dimensional gel analysis, Elk-1, SAP-1a, and SAP-1b displayed different pI and M(r) values and in vitro synthesized Elk-1 comigrated with the p62TCF spot. Both reacted with Elk-1 specific antisera, as did the major proportion of TCF activity present in HeLa crude extracts. We conclude that p62TCF is composed of Elk-1, whereas the identities of the other identified TCFs (p60TCF, p62.5TCF, and p64TCF) are still unknown.

Base Sequence↗
PubMed1994

Regulatory squelching.

An important function of transcription factors may be to sequester coactivators or corepressors of transcription. In this manner transcription factors could regulate in trans the activity of promoters to which they do not bind. This may be of widespread significance as a mechanism to control cell cycle-dependent and differentiation-specific transcriptional activity within eukaryotic cells. Therefore squelching in vivo may be important than hitherto appreciated.

Animals↗
PubMed1994

Functional dissection of the transcription factor Elk-1.

The ternary complex factor Elk-1 belongs to the Ets oncoprotein family. We demonstrate that this transcription factor is localized predominantly in the nucleus, for which at least two regions of Elk-1 are required. One of these regions is part of the N-terminal ETS-domain, while the other encompasses amino acids 137-157. In conjunction with the ETS-domain, which mediates autonomous binding of Elk-1 to some DNA target sequences, the conserved B-region is both necessary and sufficient for ternary complex formation with the c-fos serum response element and the serum response factor. However, the B-region must be linked to the ETS-domain by a spacer. Furthermore, the B-region impedes autonomous DNA-binding, possibly by masking the ETS-domain. A point mutation within the ETS-domain, homologous to the ts1.1 point mutation of v-Ets in the E26 virus, affects DNA-binding of Elk-1 in a temperature-dependent manner, which by analogy might be causative for the altered phenotype of ts1.1 E26. Finally we show that amino acids 83-428 contribute to Elk-1 mediated transactivation. In particular, the region 376-404 is indispensable for transactivation, while flanking amino acids on both sides are only required for enhancement of transcriptional efficacy.

Amino Acid Sequence↗