Reconstitution of protein kinase A regulation of the rat prolactin promoter in HeLa nonpituitary cells: identification of both GHF-1/Pit-1-dependent and -independent mechanisms.

Pit-1, a pituitary-specific POU homeodomain transcription factor, specifies three anterior pituitary lineages; governs growth hormone, prolactin, and thyrotropin gene expression; and mediates basal and Rasstimulated prolactin promoter activity in GH 4 pituitary cells. Alternate splicing of the Pit-1 message produces the Pit-1␤ isoform, which contains a 26-amino acid insertion, the ␤-domain, within the amino-terminal transactivation domain. The ␤-domain functions as a molecular switch, such that Pit-1␤ blocks both basal and Rasstimulated prolactin promoter activity in GH 4 pituitary cells yet preferentially enhances protein kinase A-stimulated prolactin promoter activity in a HeLa reconstitution system. To determine whether the amino acid sequence of the ␤-domain dictates function, we replaced it with five different 26-amino acid sequences. These mutants fail to block basal or Ras-stimulated rat prolactin promoter activity and fail to optimally enhance the protein kinase A response of prolactin promoter. These data demonstrate that the amino acid sequence of the ␤-domain specifies its role as a molecular switch. Additionally, the presence of both Pit-1 and Pit-1␤ in pituitary cells allows diverse incoming signals to utilize structurally different forms of the same gene product, which can interact with distinct co-factors, integrating multiple signaling pathways at the level of the nucleus.

Section: ␤-Domain-specific Sequences Interfere With the Functional Inmentioning

confidence: 62%

A 26-Amino Acid Insertion Domain Defines a Functional Transcription Switch Motif in Pit-1β

Diamond¹,

Gutierrez‐Hartmann

1996

“…1) are required for activation of the rPRL promoter in response to several hormones and growth factors (35,43). FPI and FPIII are necessary for activation of the rPRL promoter by thyrotropin-releasing hormone (44), and FPI is also critical to mediate cAMP induction of PRL transcription (36,(45)(46)(47). Interestingly, these DNA cis-elements have also been implicated in the response of the rPRL promoter to both EGF (44,48) and insulin (37,49), both of which are known to transduce their signals via the Ras pathway in other cell types (50,51).…”

Section: Mapping Of the Ras-responsive Element Of The Rat Prl Pro-mentioning

confidence: 99%

GHF-1/Pit-1 Functions as a Cell-specific Integrator of Ras Signaling by Targeting the Ras Pathway to a Composite Ets-1/GHF-1 Response Element

Bradford

Conrad

Tran

et al. 1996

Activation of the rat prolactin (rPRL) promoter by Ras is a prototypical example of tissue-specific transcriptional regulation in a highly differentiated cell type. Using a series of site-specific mutations and deletions of the proximal rPRL promoter we have mapped the major Ras/Raf response element (RRE) to a composite Ets-1/ GHF-1 binding site located between positions ؊217 and ؊190. Mutation of either the Ets-1 or GHF-1 binding sites inhibits Ras and Raf activation of the rPRL promoter, and insertion of this RRE into the rat growth hormone promoter confers Ras responsiveness. We show that Ets-1 is expressed in GH 4 cells and, consistent with their functional synergistic interaction, both Ets-1 and GHF-1 are able to bind specifically to this bipartite RRE. We confirm that Ets-1 or a related Ets factor is the nuclear target of the Ras pathway leading to activation of the rPRL promoter and demonstrate that Elk-1 and Net do not mediate the Ras response. Thus, the pituitary-specific POU homeodomain transcription factor, GHF-1, serves as a cell-specific signal integrator by functionally interacting with an Ets-1-like factor, at uniquely juxtaposed binding sites, thereby targeting an otherwise ubiquitous Ras signaling pathway to a select subset of cell-specific GHF-1-dependent genes.The p21 Ras proto-oncogene is a critical component of a network of signaling pathways that mediate the control of cell growth, metabolism, and differentiation (1) Signals initiated at transmembrane receptors are transduced via Ras and propagated, by a phosphorylation cascade, to the nucleus, resulting in changes in the activity of specific transcription factors (2, 3). Distinct signaling components of the Ras pathway may be present in different cell types, allowing the signal to be interpreted in a cell-specific manner (4, 5) Indeed, cell-specific, phenotypic sequelae of Ras activation are exemplified by the differential effects of oncogenic Ras in PC12 pheochromocytoma, TT medullary carcinoma, and FRTL5 thyroid cells (6 -8), whereby V-12 Ras induces terminal differentiation of the first two cell lines but causes transformation of the last. Thus, the characterization of cell-specific endogenous nuclear factors that may act as effectors of the Ras signaling pathway and the identification of specific Ras-responsive cis-acting DNA elements are important unanswered questions. Several Ras/Raf response elements (RREs) 1 or oncogene response units have been identified to date, implicating members of the Ets, AP-1, and ATF/CREB families of transcription factors as nuclear components of the Ras signaling pathway (2, 3).Tandem c-Ets-2 binding sites have been shown to confer Ras responsiveness in NIH-3T3 cells (9), and dominant-negative Ets constructs inhibit both Ras-induced mitogenesis (10) and transformation (11). The serum response is governed by MAP kinase phosphorylation of Elk-1, a member of the Ets family of transcription factors (12, 13), thus facilitating its interaction with serum response factor (SRF). Similarly, the Drosophila Ets fact...

“…2 The site-specific mutants in FP I (pA 3 ⌬1luc), FP II (pA 3 ⌬2luc), and FP II together with a loop-out deletion of the Ϫ112 to Ϫ80 basal transcription element (BTE) region (pA 3 ⌬2,Dluc) were constructed in the Ϫ425 to ϩ73 rPRL promoter as described previously (39,43). The FP I/FP III double mutant (pA 3 ⌬1,3luc) was constructed in the Ϫ425 to ϩ73 rPRL promoter as described previously (45). Plasmids pRSVc-jun, pRSVv-jun, and pRS-VGHF-1 were generously provided by M. Karin (University of California, San Diego, CA) (3,46,47).…”

Section: Methodsmentioning

confidence: 99%

“…The plasmid pCMVluc was kindly provided by Dr. Mike Smith (University of Colorado Health Sciences Center, Denver, CO). Plasmids pRSVluc400 and pA 3 SV40luc have been previously described (45,49). Plasmids pGem4 and pGem7 were both obtained from Promega Corp. (Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

“…To address these points, we used a nonpituitary gene transfer reconstitution system, whereby the ability of co-transfected effector expression vectors (e.g. GHF-1) to reconstitute the activity of the rPRL promoter in HeLa cells, which otherwise fail to express endogenous PRL or GHF-1, can be determined (45). As shown in Fig.…”

Section: The Absence Of Pituitary-specific Factor(s) Switches C-jun Fmentioning

confidence: 99%

See 1 more Smart Citation

The c-Jun δ-Domain Inhibits Neuroendocrine Promoter Activity in a DNA Sequence- and Pituitary-specific Manner

Farrow

Manning

Schaufele

et al. 1996

The transcription and transformation activity of cJun is governed by a 27-amino acid regulatory motif, labeled the ␦-domain, which is deleted in v-Jun. We have previously shown that c-Jun is a potent inhibitor of the rat prolactin (rPRL) promoter activity induced by either oncogenic Ras or phorbol esters. Here, we have characterized the structural and cell-specific requirements for this c-Jun inhibitory response, and we show that this c-Jun inhibitory response mapped to the rPRL footprint II repressor site, was pituitary-specific and required the c-Jun ␦-domain. Moreover, alteration of any one of these features (e.g., cis-element, trans-factor, or cell-specific background) switched c-Jun to a transcriptional activator of the rPRL promoter. In HeLa nonpituitary cells, c-Jun alone activated the rPRL promoter via the most proximal GHF-1/Pit-1 binding site, footprint I, and synergized with GHF-1. Finally, recombinant GHF-1 interacted directly with c-Jun but not c-Fos proteins. These data provide important fundamental insights into the molecular mechanisms by which the c-Jun ␦-domain functions as a modulatory switch and further imply that the functional role of c-Jun is dictated by cell-specific influences and the ␦-domain motif.