Control of transcription factors by signal transduction pathways: the beginning of the end.

Signal transduction pathways regulate gene expression by modulating the activity of nuclear transcription factors. The mechanisms that control the activity of two groups of sequence-specific transcription factors, the AP-1 and CREB/ATF proteins, are described. These factors serve as a paradigm explaining the transfer of regulatory information from the cell surface to the nucleus.

Mechanisms of cellular senescence.

Aging is a near universal process, yet the molecular mechanisms that underlie cellular senescence have remained elusive. Recent progress in determining the roles of various genetic influences in controlling the rate of cellular aging has made this an exciting time in aging research. Genetic screens designed to isolate long-lived mutants in Saccharomyces cerevisiae and Caenorhabditis elegans have implicated factors involved in transcriptional silencing and the dauer pathway in the control of aging. The gene responsible for Werner's syndrome, a disease with symptoms of premature aging, was isolated and found to be a member of the RecQ subfamily of DNA helicases. The regulation of telomere length and its role in senescence and cellular immortalization has been found to be more complex than expected. In C. elegans, mutations have been isolated in maternal-effect genes that presumably control its biological clocks and can dramatically extend its lifespan. Indeed, aging research within the past year has implicated a variety of mechanisms ranging from the control of gene expression, stress resistance, and DNA metabolism to the overall 'rate of living'.

Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73.

In resting cells, c-Jun is phosphorylated on five sites. Three of these sites reside next to its DNA binding domain and negatively regulate DNA binding. In response to expression of oncogenic Ha-Ras, phosphorylation of these sites decreases, while phosphorylation of two other sites within c-Jun's activation domain is greatly enhanced. Phosphorylation of these residues, serines 63 and 73, stimulates the transactivation function of c-Jun and is required for oncogenic cooperation with Ha-Ras. We now show that the same changes in c-Jun phosphorylation are elicited by a variety of transforming oncoproteins with distinct biochemical activities. These oncoproteins, v-Sis, v-Src, Ha-Ras, and Raf-1, participate in a signal transduction pathway that leads to increased phosphorylation of serines 63 and 73 on c-Jun. While oncogenic Ha-Ras is a constitutive stimulator of c-Jun activity and phosphorylation, the normal c-Ha-Ras protein is a serum-dependent modulator of c-Jun's activity. c-Jun is therefore a downstream target for a phosphorylation cascade involved in cell proliferation and transformation.

c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases.

c-Jun transcriptional activity is stimulated by phosphorylation at two N-terminal sites: Ser-63 and -73. Phosphorylation of these sites is enhanced in response to a variety of extracellular stimuli, including growth factors, cytokines, and UV irradiation. New members of the mitogen-activated protein (MAP) kinase group of signal-transducing enzymes, termed JNKs, bind to the activation domain of c-Jun and specifically phosphorylate these sites. However, the N-terminal sites of c-Jun were also suggested to be phosphorylated by two other MAP kinases, ERK1 and ERK2. Despite these reports, we find that unlike the JNKs, ERK1 and ERK2 do not phosphorylate the N-terminal sites of c-Jun in vitro; instead they phosphorylate an inhibitory C-terminal site. Furthermore, the phosphorylation of c-Jun in vivo at the N-terminal sites correlates with activation of the JNKs but not the ERKs. The ERKs are probably involved in the induction of c-fos expression and thereby contribute to the stimulation of AP-1 activity. Our study suggests that two different branches of the MAP kinase group are involved in the stimulation of AP-1 activity through two different mechanisms.

The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALK(F1174L)/MYCN Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

Malignant wound management: what dressings do nurses use?

This paper reports on the findings from the first part of a three-phase project that aimed to identify nursing strategies used in the management of malignant wounds. The difficulties relating to the management of these wounds and the significant physical and psychological impact on patients are described. A quantitative postal survey aiming to identify the types of dressing used in the care of malignant wounds was sent to specialist nurses working in oncology and palliative care in New South Wales, Australia. Additional qualitative data showed that the major issues were coping with odour and meeting the financial costs of the dressing products. The long list of products compiled for this research demonstrates the complexities nurses face when selecting dressings for the management of malignant wounds. Furthermore, there are no clear recommendations to guide nursing practice. This study provides a framework for subsequent phases of the project and will hopefully lead to the development of guidelines for best practice in malignant wound management.

Altering the specificity of signal transduction cascades: positive regulation of c-Jun transcriptional activity by protein kinase A.

Protein phosphorylation is commonly used to modulate transcription factor activity. However, all existing genetic evidence for stimulation of transcription factor activity by phosphorylation rests on loss-of-function mutations. To demonstrate conclusively that phosphorylation of a transcription factor potentiates its transactivation potential in vivo, we constructed a c-Jun mutant that is phosphorylated by the cAMP-sensitive protein kinase A (PKA) instead of the UV- and Ras-responsive protein kinase JNK. The transcriptional activity of this mutant is enhanced by PKA, but not by JNK activation. These results provide a positive and conclusive proof that phosphorylation of c-Jun on a critical site (Ser73) located in its activation domain is directly responsible for enhancing its transactivation function.

Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain.

Ha-Ras augments c-Jun-mediated transactivation by potentiating the activity of the c-Jun activation domain. Ha-Ras also causes a corresponding increase in phosphorylation of specific sites in that part of the c-Jun protein. A Ha-Ras-induced protein kinase cascade resulting in hyperphosphorylation of the c-Jun activation domain could explain how these oncoproteins cooperate to transform rat embryo fibroblasts.

The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1.

Binding of the human transcription factor Jun/AP-1 to a conserved 8 bp nucleotide sequence (TRE) is responsible for increased transcription of different cellular genes in response to tumor promoters, such as TPA, and serum factors. Enhanced Jun/AP-1 activity in TPA-stimulated cells is regulated by two different mechanisms: a posttranslational event acting on pre-existing Jun/AP-1 molecules, and transcriptional activation of jun gene expression leading to an increase in the total amount of Jun/AP-1. Induction of jun transcription in response to TPA is mediated by binding of Jun/AP-1 to a high-affinity AP-1 binding site in the jun promoter region. Site-specific mutagenesis of this binding site prevents TPA induction and trans-activation by Jun/AP-1. These results clearly demonstrate that jun transcription is directly stimulated by its own gene product. This positive regulatory loop is likely to be responsible for prolonging the transient signals generated by activation of protein kinase C.

Jun and v-jun contain multiple regions that participate in transcriptional activation in an interdependent manner.

Transcription factor AP1 is a heteromeric complex composed of the Jun and Fos proteins. It has been shown that by associating with Jun, Fos increases Jun's ability to bind DNA and activate transcription. To determine the roles of the two proteins, we undertook the functional analysis described here. We show that both the cellular Jun and its viral counterpart, v-Jun, are efficient transcriptional activators even in the absence of Fos. The Jun proteins contain at least three separate regions responsible for transcriptional activation in F9 cells, which act in an additive manner. All of these regions contain several acidic amino acid residues that appear to be functionally important and interact with a titratable target. Although trans-activation by Fos was previously shown to be dependent on the presence of Jun, by fusing Fos to a heterologous DNA-binding domain we show that once given the ability to bind DNA on its own, Fos is also an independent trans-activator. Both Jun and Fos contribute to trans-activation by the AP1 complex, and the augmentation of Jun activity by Fos is probably due to the increased DNA-binding activity of the Jun:Fos heterodimer.

Different requirements for formation of Jun: Jun and Jun: Fos complexes.

The cFos proto-oncoprotein associates with cJun to form a heterodimer with increased DNA binding and transcriptional activities. It has been suggested that dimerization of these proteins is mediated by the interdigitation of an orderly repeat of leucine residues forming a leucine zipper. In agreement with this model, we find that binding to the AP-1 site requires dimerization of these proteins. Although cFos, itself, does not seem to dimerize and bind to the AP-1 site, Jun: Fos heterodimers have higher stability than Jun homodimers, which accounts for their increased DNA binding activity. Mutational analysis indicates that at least three of the repeated leucines of cJun are important for homodimer formation. However, these residues can be mutated without affecting formation of Jun: Fos heterodimers. In addition, several other residues present between the leucines are also important for both homo- and heterodimerization. These findings provide support for the recent proposal that these proteins dimerize via formation of a coiled coil and suggest that residues other than leucines provide specificity for this interaction. Assuming that dimerization is required for proper alignment of the DNA recognition sites, we generated a cJun mutant containing a small insertion between the dimerization and the DNA recognition domains. This mutant fails to bind DNA, but it acts as a trans-dominant inhibitor of cJun and cFos because it still dimerizes with the wild-type proteins.

The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases.

Exposure of mammalian cells to DNA-damaging agents induces the ultraviolet (UV) response, involving transcription factor AP-1, composed of Jun and Fos proteins. We investigated the mechanism by which UV irradiation induces the c-jun gene. The earliest detectable step was activation of Src tyrosine kinases, followed by activation of Ha-Ras and Raf-1. The response to UV was blocked by tyrosine kinase inhibitors and dominant negative mutants of v-src, Ha-ras, and raf-1. This signaling cascade leads to increased phosphorylation of c-Jun on two serine residues that potentiate its activity. These results strongly suggest that the UV response is initiated at or near the plasma membrane rather than the nucleus. The response may be elicited by oxidative stress, because it is inhibited by elevation of intracellular glutathione. Using tyrosine kinase inhibitors, we demonstrate that the UV response has a protective function.

Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain.

The activity of c-Jun is regulated by phosphorylation. Various stimuli including transforming oncogenes and UV light, induce phosphorylation of serines 63 and 73 in the amino-terminal activation domain of c-Jun and thereby potentiate its trans-activation function. We identified a serine/threonine kinase whose activity is stimulated by the same signals that stimulate the amino-terminal phosphorylation of c-Jun. This novel c-Jun amino-terminal kinase (JNK), whose major form is 46 kD, binds to a specific region within the c-Jun trans-activation domain and phosphorylates serines 63 and 73. Phosphorylation results in dissociation of the c-Jun-JNK complex. Mutations that disrupt the kinase-binding site attenuate the response of c-Jun to Ha-Ras and UV. Therefore the binding of JNK to c-Jun is of regulatory importance and suggests a mechanism through which protein kinase cascades can specifically modulate the activity of distinct nuclear targets.

Villin sequence and peptide map identify six homologous domains.

Site-specific proteases and antisera to the amino terminus of villin have been used to show that villin is organized into seven protease-resistant domains. Six are contained in the amino-terminal Mr 87,000 villin core, a Ca2+-regulated actin-severing fragment, whereas the carboxyl-terminal domain includes the villin "headpiece," a fragment involved in bundling of actin filaments. Ca2+ inhibits proteolytic cleavage between domains in the amino-terminal half of villin. The protein sequence of villin deduced from a single cDNA clone contains a conserved sequence that is repeated six times and is found in each domain of the villin core. The conserved repeats are found in other actin-severing proteins but not in the villin headpiece. Our results suggest that actin-severing proteins are organized around a common Mr 14,000-17,000 domain.

The c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive genes.

Cell lines stably transfected with metal inducible, MT-fos chimeric genes were used to study the ability of the c-fos gene product, Fos, to act as a transcriptional trans-activator. In 3T3MTfos cells, induction of Fos expression led to specific trans-activation of an AP-1 responsive reporter gene. Induction of Fos expression in F9MTfos cells, however, did not lead to trans-activation. Since, unlike NIH3T3 cells, F9 cells do not contain detectable levels of AP-1, we examined whether a c-Jun/AP-1 expression vector can restore the trans-activating effect of Fos in F9MTfos cells. Transfection with a functional c-Jun/AP-1 vector restored the specific trans-activating effect of Fos on AP-1 responsive constructs. When incubated with nondenatured cell extracts, anti-cFos antisera precipitated a protein complex composed of Fos and several Fos associated proteins (FAP). One of these, FAP p39, is structurally identical to c-Jun/AP-1. These results suggest that Fos is a trans-acting factor that is capable of stimulating gene expression not by direct binding to DNA but by interaction with the sequence-specific transcription factor AP-1. Therefore recognition of specific cis-elements by AP-1 is a prerequisite for Fos-mediated stimulation of gene expression.

Loss of transcriptional silencing causes sterility in old mother cells of S. cerevisiae.

We show that sterility is an aging-specific phenotype in S. cerevisiae and, by genetic and physical means, demonstrate that this phenotype results from a loss of silencing in most old cells by the SIR complex at the HM loci. This loss of silencing is specific because transcription of genes, such as ME14 and DCM1, normally induced by sporulation, is not observed, while transcription of HMRa is observed. These findings pinpoint the molecular cause of an aging-specific phenotype in yeast. Further, they provide direct evidence for a breakdown of silencing in old cells, as predicted from earlier findings that SIR4 is a determinant of life span in this organism.

Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73.

Recent advances indicate a link between tumour promoters, transformation, and AP-1 activity. Protein kinase C activation increases AP-1 DNA-binding activity independently of new protein synthesis. AP-1 is also stimulated by transforming oncoproteins and growth factors. These proteins are thought to participate in a signalling cascade affecting the nuclear AP-1 complex composed of the Jun and Fos proteins. Because c-Jun is the most potent transactivator in the AP-1 complex and is elevated in Ha-ras-transformed cells, in which c-Fos is downregulated, we focused on it as a potential target. c-Jun could convert input from an oncogenic signalling cascade into changes in gene expression. Indeed, transformation of rat embryo fibroblasts by c-Jun requires an intact transcriptional activation domain and cooperation with oncogenic Ha-ras. Expression of oncogenic Ha-ras augments transactivation by c-Jun and stimulates its phosphorylation. Here we describe the mapping of the Ha-ras-responsive phosphorylation sites to serines 63 and 73 of c-Jun. Site-directed mutagenesis indicates that phosphorylation of these serines is essential for stimulation of c-Jun activity and for cooperation with Ha-ras in ocogenic transformation.

DNA-binding activity of Jun is increased through its interaction with Fos.

Transcription factor AP-1 mediates induction of a set of genes in response to the phorbol ester tumor promoter TPA. Recently, AP-1 preparations from HeLa cells were shown to contain a product of the c-JUN protooncogene (Jun/AP-1) which forms a tight complex with the Fos protein. In this paper, we examine the role of the Fos protein in the DNA-binding activity of the AP-1 complex. We show that the DNA-binding activity of bacterially expressed trpE-Jun fusion proteins is increased many-fold upon their interaction with Fos (or a Fos-related antigen) expressed from a baculovirus vector. The site of Fos interaction is within the DNA-binding domain of Jun/AP-1, and anti-Fos antibodies interfere with the binding of affinity purified AP-1 to DNA. These results suggest that, by associating with Jun/AP-1, Fos is responsible for the formation of a multimeric protein complex that has greater affinity for the target sequence than does Jun/AP-1 alone.

Activation of cAMP and mitogen responsive genes relies on a common nuclear factor.

A number of signalling pathways stimulate transcription of target genes through nuclear factors whose activities are primarily regulated by phosphorylation. Cyclic AMP regulates the expression of numerous genes, for example, through the protein kinase-A (PKA)-mediated phosphorylation of transcription factor CREB at Ser 133. Although phosphorylation may stimulate transcriptional activators by modulating their nuclear transport or DNA-binding affinity, CREB belongs to a class of proteins whose phosphorylation appears specifically to enhance their trans-activation potential. Recent work describing a phospho-CREB binding protein (CBP) which interacts specifically with the CREB trans-activation domain prompted us to examine whether CBP is necessary for cAMP regulated transcription. We report here that microinjection of an anti-CBP antiserum into fibroblasts can inhibit transcription from a cAMP responsive promoter. Surprisingly, CBP also cooperates with upstream activators such as c-Jun, which are involved in mitogen responsive transcription. We propose that CBP is recruited to the promoter through interaction with certain phosphorylated factors, and that CBP may thus play a critical role in the transmission of inductive signals from cell surface receptor to the transcriptional apparatus.

Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity.

In resting human epithelial and fibroblastic cells, c-Jun is phosphorylated on serine and threonine at five sites, three of which are phosphorylated in vitro by glycogen synthase kinase 3 (GSK-3). These three sites are nested within a single tryptic peptide located just upstream of the basic region of the c-Jun DNA-binding domain (residues 227-252). Activation of protein kinase C results in rapid, site-specific dephosphorylation of c-Jun at one or more of these three sites and is coincident with increased AP-1-binding activity. Phosphorylation of recombinant human c-Jun proteins in vitro by GSK-3 decreases their DNA-binding activity. Mutation of serine 243 to phenylalanine blocks phosphorylation of all three sites in vivo and increases the inherent trans-activation ability of c-Jun at least 10-fold. We propose that c-Jun is present in resting cells in a latent, phosphorylated form that can be activated by site-specific dephosphorylation in response to protein kinase C activation.