Ralf Janknecht

Activation of ternary complex factor Elk-1 by MAP kinases.

Ternary complex factors (TCFs), one of which is Elk‐1, have been implicated in mediation of c‐fos induction. They have been shown to be phosphorylated by mitogen‐activated protein kinases (MAPKs) in vitro. We demonstrate that recombinant Elk‐1 is hyperphosphorylated in vivo upon joint overexpression of MAPKs and constitutively activated Raf‐1 kinase, the latter serving as an indirect in vivo activator of MAPKs. This phosphorylation is accompanied by a conformational change and results in an elevated transactivation potential of Elk‐1. Mutation of mapped in vivo phosphorylation sites, which are potential targets for MAPKs, reduced Elk‐1‐mediated transcription. Thus, MAPKs are very probably controlling Elk‐1 activity by direct phosphorylation in vivo. Furthermore, Elk‐1 was shown to stimulate transcription from both the c‐fos serum response element and also from an Ets binding site. While binding of TCFs to the c‐fos promoter is dependent on the serum response factor, TCFs can autonomously interact with Ets binding sites. This indicates that TCFs may participate in the transcriptional regulation of two different sets of genes.

THE KIT RECEPTOR PROMOTES CELL SURVIVAL VIA ACTIVATION OF PI 3-KINASE AND SUBSEQUENT AKT-MEDIATED PHOSPHORYLATION OF BAD ON SER136

The c-kit-encoded receptor protein tyrosine kinase for stem cell factor (Kit/SCF-R) is essential for the development of cells within the hematopoietic, melanogenic and gametogenic lineages. SCF stimulation induces activation of phosphatidylinositol (PI) 3-kinase, which is required for SCF-induced mitogenesis and cell survival, and for activation of the serine/threonine, we found that, in response to SCF Akt became activated and mediated phosphorylation of Bad, a pro-apoptotic molecule, in a PI-3-kinase-dependent manner. Phosphorylation of Bad was restricted to Ser112 and Ser136 in vivo, but only the Akt phosphorylation sit Ser136 was essential for SCF-promoted cell survival. Furthermore, Bad and Akt interacted and colocalized in intact cells. A Kit/SCF-R gain-of-function mutant that has increased mitogenic and PI 3-kinase activation potential, due to the absence of the two protein kinase C negative feedback phosphorylation site, enhanced both Akt activation and Bad phosphorylation and also resulted in increased cell survival. Such a mechanism may account for how deregulated PI 3-kinase activity and naturally occurring gain-of-function point mutants of Kit/SCF-R lead to cellular transformation and fatal malignancies in man.

Convergence of MAP kinase pathways on the ternary complex factor Sap-1a.

The serum response element (SRE), which is pivotal for transcriptional up‐regulation of the c‐fos proto‐oncogene, is constitutively occupied by a protein complex comprising the serum response factor and a ternary complex factor (TCF). Phosphorylation of the TCFs Elk‐1 and Sap‐1a by the ERK and JNK subclasses of MAP kinases triggers c‐fos transcription. We demonstrate here that Elk‐1 is barely activated by a third subclass of MAP kinases (p38), most likely because the critical residues Ser383 and Ser389 are poorly phosphorylated by p38 MAP kinase. In contrast, the TCF Sap‐1a is efficiently phosphorylated by p38 MAP kinase in vitro and in vivo on the homologous residues Ser381 and Ser387. Mutation of these sites to alanine severely reduces c‐fos SRE‐dependent transcription mediated by Sap‐1a and p38 MAP kinase. Thus, Sap‐1a may be an important target for mitogens, stress and apoptotic signals to elicit a nuclear response. However, signaling from p38 MAP kinase to Sap‐1a or from Sap‐1a to the basal transcription machinery does not occur in all cell types nor at promoters other than the c‐fos SRE, which may ensure the specificity of signaling.

Transcriptional control: Versatile molecular glue

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.

ER71 Acts Downstream of BMP, Notch, and Wnt Signaling in Blood and Vessel Progenitor Specification

FLK1-expressing (FLK1(+)) mesoderm generates blood and vessels. Here, we show that combined BMP, Notch, and Wnt signaling is necessary for efficient FLK1(+) mesoderm formation from embryonic stem cells (ESCs). Inhibition of BMP, Notch, and Wnt signaling pathways greatly decreased the generation of FLK1(+) mesoderm and expression of the Ets transcription factor Er71. Enforced expression of ER71 in ESCs resulted in a robust induction of FLK1(+) mesoderm; rescued the generation of FLK1(+) mesoderm when blocked by BMP, Notch, and Wnt inhibition; and enhanced hematopoietic and endothelial cell generation. Er71-deficient mice had greatly reduced FLK1 expression, died early in gestation, and displayed severe blood and vessel defects that are highly reminiscent of the Flk1 null mouse phenotype. Collectively, we provide compelling evidence that ER71 functions downstream of BMP, Notch, and Wnt signals and regulates FLK1(+) mesoderm, blood, and vessel development.

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.

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.

p38-2, a Novel Mitogen-activated Protein Kinase with Distinct Properties

Mitogen-activated protein (MAP) kinases are involved in many cellular processes. Here we describe the cloning and characterization of a new MAP kinase, p38-2. p38-2 belongs to the p38 subfamily of MAP kinases and shares with it the TGY phosphorylation motif. The complete p38-2 cDNA was isolated by polymerase chain reaction. It encodes a 364-amino acid protein with 73% identity to p38. Two shorter isoforms missing the phosphorylation motif were identified. Analysis of various tissues demonstrated that p38-2 is differently expressed from p38. Highest expression levels were found in heart and skeletal muscle. Like p38, p38-2 is activated by stress-inducing signals and proinflammatory cytokines. The preferred upstream kinase is MEK6. Although p38-2 and p38 phosphorylate the same substrates, the site specificity of phosphorylation can differ as shown by two-dimensional phosphopeptide analysis of Sap-1a. Additionally, kinetic studies showed that p38-2 appears to be about 180 times more active than p38 on certain substrates such as ATF2. Both kinases are inhibited by a class of pyridinyl imidazoles. p38-2 phosphorylation of ATF2 and Sap-1a but not Elk1 results in increased transcriptional activity of these factors. A sequential kinetic mechanism of p38-2 is suggested by steady state kinetic analysis. In conclusion, p38-2 may be an important component of the stress response required for the homeostasis of a cell.

KDM4/JMJD2 Histone Demethylases: Epigenetic Regulators in Cancer Cells

Lysine methylation is one of the most prominent histone posttranslational modifications that regulate chromatin structure. Changes in histone lysine methylation status have been observed during cancer formation, which is thought to be a consequence of the dysregulation of histone lysine methyltransferases or the opposing demethylases. KDM4/JMJD2 proteins are demethylases that target histone H3 on lysines 9 and 36 and histone H1.4 on lysine 26. This protein family consists of three ~130-kDa proteins (KDM4A-C) and KDM4D/JMJD2D, which is half the size, lacks the double PHD and Tudor domains that are epigenome readers and present in the other KDM4 proteins, and has a different substrate specificity. Various studies have shown that KDM4A/JMJD2A, KDM4B/JMJD2B, and/or KDM4C/JMJD2C are overexpressed in breast, colorectal, lung, prostate, and other tumors and are required for efficient cancer cell growth. In part, this may be due to their ability to modulate transcription factors such as the androgen and estrogen receptor. Thus, KDM4 proteins present themselves as novel potential drug targets. Accordingly, multiple attempts are under way to develop KDM4 inhibitors, which could complement the existing arsenal of epigenetic drugs that are currently limited to DNA methyltransferases and histone deacetylases.

Independence of Repressive Histone Marks and Chromatin Compaction during Senescent Heterochromatic Layer Formation

The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into nonoverlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells, heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of presenescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks, nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events.