Transcriptional activation of the cyclin D1 by oncogenic Ras appears to be mediated by several pathways leading to the activation of multiple transcription factors which interact with distinct elements of the cyclin D1 promoter. The present investigations revealed that cyclin D1 induction by transforming Ha-Ras is MEKand Rac-dependent and requires the PKC isotypes ⑀, , and , but not cPKC-␣. This conclusion is based on observations indicating that cyclin D1 induction by transforming Ha-Ras was depressed in a dose-dependent manner by PD98059, a selective inhibitor of the mitogenactivated kinase kinase MEK-1, demonstrating that HaRas employs extracellular signal-regulated kinases (ERKs) for signal transmission to the cyclin D1 promoter. Evidence is presented that PKC isotypes ⑀ and , but not are required for the Ras-mediated activation of ERKs. Expression of kinase-defective, dominant negative (DN) mutants of nPKC-⑀ or aPKC-inhibit ERK activation by constitutively active Raf-1. Phosphorylation within the TEY motif and subsequent activation of ERKs by constitutively active MEK-1 was significantly inhibited by DN aPKC-, indicating that aPKC-functions downstream of MEK-1 in the pathway leading to cyclin D1 induction. In contrast, TEY phosphorylation induced by constitutively active MEK-1 was not effected by nPKC-⑀, suggesting another position for this kinase within the cascade investigated. Transformation by oncogenic Ras requires activation of several Ras effector pathways which may be PKC-dependent and converge on the cyclin D1 promoter. Therefore, we investigated a role for PKC isotypes in the Ras-Rac-mediated transcriptional regulation of cyclin D1. We have been able to reveal that cyclin D1 induction by oncogenic Ha-Ras is Rac-dependent and requires the PKC isotypes ⑀, , and , but not cPKC-␣. Evidence is presented that aPKC-acts upstream of Rac, between Ras and Rac, whereas the PKC isotypes ⑀ and act downstream of Rac and are required for the activation of ERKs.Transformation by oncogenic Ras requires activation of several Ras effectors which, in turn, stimulate different signaling pathways. These Ras effectors include Raf, Ral-GDS, PI 3-kinase, and Rac-1 (1). Activation of Rac-1 by oncogenic Ras is mediated by PI 3-kinase 1 -dependent (2, 3) and PI 3-kinaseindependent mechanisms (2). These Ras effector pathways may converge on the cyclin D1 promoter (1, 4). Evidence for a positive regulation of the cyclin D1 promoter by Ras has been presented (4 -7).The Ras-Raf-ERK cascade has been described as the dominant pathway by which Ras transmits signals to the cyclin D1 promoter (4, 8 -10), although in nontransformed cells the situation may be different (1). One effect of the stimulation of ERK1 by Ras seems to be the activation of Ets-2, since expression of plasmids encoding DN Ets (Ets-LacZ) antagonized ERKdependent activation of the cyclin D1 promoter (4) and mutation of an Ets-binding site (termed EtsB (6)) strongly reduced basal and also Ras-induced activation of cyclin D1.Another Ras effector, Rac-1, has also been...

The action of the glucocorticoid receptor (GR) on ␤-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the ␤-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent ␤-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for ␤-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.Modulation of gene expression by the glucocorticoid receptor (GR) involves a combination of several mechanisms such as modulation of chromatin structure (5, 27); binding to specific DNA response elements (24); interaction with sequence-specific transcription factors, coactivators, and corepressors; and ligand-dependent alterations in the balance of corepressors and coactivators bound to the receptor (20). The actual type of mechanism employed by the receptor strongly depends on the genes that are regulated and on the cellular context. There is a differential requirement for domains in the GR, depending on the prevalent mechanism utilized by the receptor. For instance, a specific subset of GR-regulated genes is affected in transgenic mice in which the wild-type GR is replaced by a mutant defective in dimerization (26). Since this mutant is strongly impaired in binding to palindromic canonical glucocorticoid response elements (GREs) (7), it can no longer regulate genes that contain functional GREs. Consistent with the observation that in most cases the binding of the GR to DNA is a prerequisite for transactivation but not for transrepression, transgenic mice expressing the dimerization mutant predominantly exhibit a defect in the expression of genes induced by glucocorticoids.One of the exception...

Prolactin has been reported to induce distinct sets of signal transducers and activators of transcription (STAT) in a cell-type-specific fashion. In the mammary epithelium, although STAT1, STAT3, STAT5A, STAT5B and STAT6 are present in a latent form, only STAT5A and STAT5B are activated. This selective activation of STAT5 by prolactin was also observed in COS-7 cells cotransfected with the long form of the mouse prolactin receptor (PRL-R) and expression vectors for STAT1, STAT3, STAT5 and STAT6. Mutated PRL-Rs and chimeric erythropoietin/gp130 (EPO/gp130) receptors with a tyrosine-containing motif attached at the carboxy terminus were employed to determine the sites in the PRL-R required for the specific activation of STAT5. The experiments revealed the importance of two motifs containing Y477 and Y578 in the PRL-R. When linked to the EPO/gp130 receptor, these sequences were sufficient to specifically induce DNA binding of STAT5 and to activate transcription from the β-casein gene promoter. By contrast, only weakly they induced DNA binding of STAT6 and STAT3 and did not induce STAT1. A synthetic nonapeptide with phosphorylated Y477 was able to disrupt STAT5 DNA binding in vitro.Our results define structural domains within the carboxy terminus of the PRL-R which recruit STAT5 for signalling and which are capable of distinguishing STAT5 from other STAT proteins. The activity of STAT5 forms with deletions of the carboxy terminus was induced more strongly than that of their fulllength counterparts 2 min after activation of the PRL-R. This effect was not dependent on the presence of Y477 and Y578 in the PRL-R, indicating that facilitated activation of short STAT5 isoforms relies on mechanisms other than increased coupling to specific regions of the PRL-R. Fax: ϩ3 512 5072872. E-mail: Wolfgang.Doppler@uibk.ac.at Abbreviations. EMSA, electromobility shift assay; Eg, erythropoietin/gp130 chimeric receptor; EPO, erythropoietin; EPO-R, erythropoietin receptor; IFN, interferon ; IL, interleukin ; sIL-6R, soluble interleukin-6 receptor A; JAK, janus kinase; LHRR, lactogenic hormone response region; PRL, prolactin; PRL-R, prolactin receptor; SH-2, src homology domain ; STAT, signal transducer and activator of transcription ; SIE, sis-inducible element.be relevant for activating STAT5 fit to the derived consensus. Docking of STAT5 to specific tyrosine-phosphorylated domains of JAK2 has been described [2] and has served to explain the relatively broad activation pattern of this STAT protein by various cytokines, hormones and growth factors.In the present study, we have investigated the selectivity of the coupling of STAT1, STAT3, STAT5A, STAT5B and STAT6 to the mouse PRL-R in co-transfection experiments using COS-7 cells. PRL-R induction of DNA binding by STAT1, STAT3 and STAT6 was at least one order of magnitude weaker than by STAT5. We show that two domains within the carboxy terminus, which are conserved in mammals, birds and fish, mediate the selective activation of STAT5A and STAT5B. Each of the two domains alone was suff...

The activation of phospholipase D (PLD) by transforming Ras is well documented. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned from mammalian cells, it has remained unclear whether both isoenzymes are activated by Ras and, if this is the case, whether they are stimulated by a common mechanism. In the present study we show that expression of transforming Ras in HC11 mouse mammary epithelial cells enhanced the activity of endogenous PLD. Co-expression of Ras with either PLD1b or PLD2 resulted in elevated activities of both PLD isoenzymes in HC11 cells, indicating that transforming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF109203X, which preferentially affects conventional- and novel-type PKCs, but sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. Co-transfection of atypical PKC-iota with either PLD1b or PLD2 led to a selective activation of PLD2 by PKC-iota, whereas PLD1b was not affected. PLD1b, however, was found to be a potent activator of PKC-iota, whereas PLD2 was less effective in this respect. The data suggest that PKC-iota acts upstream of PLD2 and that PLD1b is implicated in the activation of PKC-iota. The data are discussed as indicating a putative signalling cascade comprising Ras-->PLD1b-->PKC-iota-->PLD2. Evidence for the implication of this pathway in the transcriptional regulation of cyclin D1 is also presented.

The activation of phospholipase D (PLD) by transforming Ras is well documented. Although two distinct PLD isoforms, PLD1 and PLD2, have been cloned from mammalian cells, it has remained unclear whether both isoenzymes are activated by Ras and, if this is the case, whether they are stimulated by a common mechanism. In the present study we show that expression of transforming Ras in HC11 mouse mammary epithelial cells enhanced the activity of endogenous PLD. Co-expression of Ras with either PLD1b or PLD2 resulted in elevated activities of both PLD isoenzymes in HC11 cells, indicating that transforming Ras was capable of activating both PLD isoforms in vivo. Ras-induced activation of PLD was resistant to the protein kinase C (PKC) inhibitor GF109203X, which preferentially affects conventional- and novel-type PKCs, but sensitive to Ro-31-8220, which inhibits atypical PKCs more effectively. Co-transfection of atypical PKC-ι with either PLD1b or PLD2 led to a selective activation of PLD2 by PKC-ι, whereas PLD1b was not affected. PLD1b, however, was found to be a potent activator of PKC-ι, whereas PLD2 was less effective in this respect. The data suggest that PKC-ι acts upstream of PLD2 and that PLD1b is implicated in the activation of PKC-ι. The data are discussed as indicating a putative signalling cascade comprising Ras → PLD1b →PKC-ι → PLD2. Evidence for the implication of this pathway in the transcriptional regulation of cyclin D1 is also presented.