c-Jun Transactivation Research Articles

Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun.

The stress-activated protein kinases (SAPKs), which are distantly related to the MAP kinases, are the dominant c-Jun amino-terminal protein kinases activated in response to a variety of cellular stresses, including treatment with tumour-necrosis factor-alpha and interleukin-beta (refs 1, 2). SAPK phosphorylation of c-Jun probably activates the c-Jun transactivation function. SAPKs are part of a signal transduction cascade related to, but distinct from, the MAPK pathway. We have now identified a novel protein kinase, called SAPK/ERK kinase-1 (SEK1), which is structurally related to the MAP kinase kinases (MEKs). SEK1 is a potent activator of the SAPKs in vitro and in vivo. An inactive SEK1 mutant blocks SAPK activation by extracellular stimuli without interfering with the MAPK pathway. Although alternative mechanisms of SAPK activation may exist, as an immediate upstream activator of the SAPKs, SEK1 further defines a signalling cascade that couples cellular stress agonists to the c-Jun transcription factor.

Polyomavirus middle-sized tumor antigen modulates c-Jun phosphorylation and transcriptional activity.

Polyomavirus middle-sized tumor antigen (MT) increases the expression of c-jun through a phorbol 12-O-tetradecanoate 13-acetate response element in the c-jun promoter. To investigate the cellular signaling pathways affected by MT, we studied the role of the c-Ras and Raf-1 proteins in MT-induced transactivation of c-jun and cell transformation. There was an increase in GTP complexed to Ras in MT-expressing cells, indicating an increase in Ras activity. Coexpression of dominant inhibitory mutants of Ha-ras and raf-1 with MT inhibited MT-mediated transactivation and focus formation. Studies of the phosphorylation of c-Jun showed that MT expression increased the phosphorylation of Ser-63 and Ser-73 in the transactivation domain and decreased the phosphorylation of a peptide containing Ser-243, Ser-249, and Thr-231 in the DNA binding domain. MT increased the transcriptional activating ability of c-Jun but failed to increase the transcriptional activating ability of c-Jun mutants with Ser-63 and Ser-73 changed to nonphosphorylatable Ala, indicating that MT modulates c-Jun activity through phosphorylation. The dominant inhibitory mutants of Ha-ras and raf-1 interfered with the ability of MT to activate c-Jun. The results indicate that MT induces a phosphorylation cascade through the activation of c-Ras and Raf-1 and that c-Jun is one of the downstream targets that may cause changes in gene expression leading to cell transformation.

The stress-activated protein kinase subfamily of c-Jun kinases.

The mitogen-activated protein (MAP) kinases Erk-1 and Erk-2 are proline-directed kinases that are themselves activated through concomitant phosphorylation of tyrosine and threonine residues. The kinase p54 (M(r) 54,000), which was first isolated from cycloheximide-treated rats, is proline-directed like Erks-1/2, and requires both Tyr and Ser/Thr phosphorylation for activity. p54 is, however, distinct from Erks-1/2 in its substrate specificity, being unable to phosphorylate pp90rsk but more active in phosphorylating the c-Jun transactivation domain. Molecular cloning of p54 reveals a unique subfamily of extracellularly regulated kinases. Although they are 40-45% identical in sequence to Erks-1/2, unlike Erks-1/2 the p54s are only poorly activated in most cells by mitogens or phorbol esters. However, p54s are the principal c-Jun N-terminal kinases activated by cellular stress and tumour necrosis factor (TNF)-alpha, hence they are designated stress-activated protein kinases, or SAPKs. SAPKs are also activated by sphingomyelinase, which elicits a subset of cellular responses to TNF-alpha (ref. 9). SAPKs therefore define a new TNF-alpha and stress-activated signalling pathway, possibly initiated by sphingomyelin-based second messengers, which regulates the activity of c-Jun.

JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain

The ultravlolet (UV) response of mammalian cells is characterized by a rapid and selective increase in gene expression mediated by AP-1 and NF-kB. The effect on AP-1 transcriptional activity results, in part, from enhanced phosphorylation of the c-Jun NH2-terminal activation domain. Here, we describe the molecular cloning and characterization of JNK1, a distant relative of the MAP kinase group that is activated by dual phosphorylation at Thr and Tyr during the UV response. Significantly, Ha-Ras partially activates JNK1 and potentiates the activation caused by UV. JNK1 binds to the c-Jun transactivation domain and phosphorylates it on Ser-63 and Ser-73. Thus, JNK1 is a component of a novel signal transduction pathway that is activated by oncoproteins and UV irradiation. These properties indicate that JNK1 activation may play an important role in tumor promotion.

Molecular events in the pathogenesis of hepadnavirus-associated hepatocellular carcinoma.

Chronic hepadnavirus infection is associated with hepatocellular carcinoma (HCC) in natural hosts such as humans, woodchucks, and Beechey ground squirrels. Several possible oncogenic mechanisms have been identified, including a potential role of the hepadnavirus x (hbx) gene, which transactivates transcription regulated by certain cis-acting sequences, e.g. regulatory sequences of the hepatitis B virus (HBV) and heterologous regulatory sequences of other viruses and cellular genes. The oncogenic potential of hbx is suggested by the observation of HCCs in hbx transgenic mice, the oncogenic transformation of cells expressing hbx in culture, and the transactivation of oncogenes c-myc and c-jun by hbx. Cis-activation of cellular oncogenes N-myc and c-myc by viral promoter insertion has been a common finding in woodchuck hepatitis virus (WHV)-associated HCCs of woodchucks. No such cis-activation of any cellular gene has been shown in virus-associated HCCs of ground squirrels or humans. Amplification and overexpression of the c-myc gene has been a common finding in HCCs of ground squirrels, and is rare in woodchuck or human HCCs. Point mutations in the p53 gene and allelic deletion of p53 have been common findings in human HCCs, but have not been found in HCCs in woodchucks and have been found rarely in ground squirrels. How each of these genetic changes in the different hosts contributes to HCC remains to be determined, but apparently different changes in different HCCs of hepadnavirus-infected hosts suggest that several separate genetic events may contribute to the development of HCC. These events may differ in each host, and some may not result from a direct virus-specific mechanism. Chronic hepadnavirus infection is often associated with chronic necroinflammatory liver disease and cirrhosis, a pathologic process common to several other risk factors for HCC. This suggests that this pathologic process (necroinflammatory disease) may be hepatocarcinogenic regardless of the inciting agent. Thus hepadnavirus infection may play an important role in the development of HCC by causing chronic hepatitis and HCC with the same mechanisms by which other risk factors for HCC cause chronic necroinflammatory liver disease and HCC.

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.

The PML-retinoic acid receptor alpha translocation converts the receptor from an inhibitor to a retinoic acid-dependent activator of transcription factor AP-1.

We report here that the fusion of PML, a nuclear protein defined by the t(15;17) chromosomal translocation in acute promyelocytic leukemia, with retinoic acid receptor alpha (RAR alpha) changes the RAR alpha from a retinoic acid (RA)-dependent inhibitor to a RA-dependent activator of AP-1 transcriptional activity. The PML-RAR alpha chimera cooperates with c-Jun and, strikingly, with c-Fos to stimulate the transcription of both synthetic and natural reporter genes containing an AP-1 site. Stimulation is dependent on the concentration of RA and its dose-response curve is comparable to that for activation by RAR alpha of transcription on RA-responsive genes. Further, in the absence of RA, a circumstance in which RAR alpha has no effect on AP-1 activity, PML-RAR alpha is an inhibitor. Deletion of the dimerization, transactivation, or DNA-binding domains of c-Jun and removal of the PML dimerization domain in the PML-RAR alpha hybrid abrogates their transcriptional cooperatively. In view of the association between AP-1 activity and hemopoietic differentiation, we suggest that these properties of PML-RAR alpha could contribute to the leukemic phenotype and its response to RA.

Phorbolester stimulates the activity of human corticotropin-releasing hormone gene promoter via 3',5'-cyclic adenosine monophosphate response element in transiently transfected chicken macrophages

We previously characterized the transcriptional activity of the human CRH (hCRH) gene promoter in the mouse anterior pituitary cell line AtT20. Here, we show that phorbolester stimulated through the cAMP response element (CRE), located between -227 and -220 relative to the putative cap site, about 2.6-fold the expression of a chimeric hCRH-chloramphenicol-acetyl-transferase gene which was transiently transfected into chicken macrophages (HD11 cell line). The induction was dependent on the cell types, and was not observed when AtT20 were used as target cells. Elevated mouse c-fos protein expressed from a cotransfecting plasmid pRSVfos or human c-jun protein expressed from RSVjun led to 2.1-fold and 3.1-fold stimulation of the activity of the hCRH promoter. Tetradecanoyl-phorbol-13-acetate stimulated the c-fos and c-jun transactivation by a factor of 10 and 2.6, indicating a modification of c-fos and c-jun is required for the transactivation induced by protein kinase C activation. Mutation within the cAMP response element abolished this transcriptional activation. This result suggests an involvement of the protein kinase C pathway in the regulation of the CRH gene expression.

Phorbolester stimulates the activity of human corticotropin-releasing hormone gene promoter via 3',5'-cyclic adenosine monophosphate response element in transiently transfected chicken macrophages.

We previously characterized the transcriptional activity of the human CRH (hCRH) gene promoter in the mouse anterior pituitary cell line AtT20. Here, we show that phorbolester stimulated through the cAMP response element (CRE), located between -227 and -220 relative to the putative cap site, about 2.6-fold the expression of a chimeric hCRH-chloramphenicol-acetyl-transferase gene which was transiently transfected into chicken macrophages (HD11 cell line). The induction was dependent on the cell types, and was not observed when AtT20 were used as target cells. Elevated mouse c-fos protein expressed from a cotransfecting plasmid pRSVfos or human c-jun protein expressed from RSVjun led to 2.1-fold and 3.1-fold stimulation of the activity of the hCRH promoter. Tetradecanoyl-phorbol-13-acetate stimulated the c-fos and c-jun transactivation by a factor of 10 and 2.6, indicating a modification of c-fos and c-jun is required for the transactivation induced by protein kinase C activation. Mutation within the cAMP response element abolished this transcriptional activation. This result suggests an involvement of the protein kinase C pathway in the regulation of the CRH gene expression.