STK25 is a candidate gene for pseudopseudohypoparathyroidism.

We determined the chromosomal location of the mouse gene Stk25, encoding a member of the Ste20/PAK family of serine/threonine kinases, by interspecific backcross analysis. We mapped Stk25 to the central region of mouse chromosome 1 linked to Chrng (formerly Acrg) and En1. This central region of mouse chromosome 1 shares a region of homology with the long arm of human chromosome 2, suggesting that the human homologue of Stk25 would also map to 2q. We proved this prediction of syntenic homology correct by mapping human STK25 to 2q37. Deletion of the 2q37 region has been implicated in the expression of pseudopseudohypoparathyroidism (PPHP), a disease which shares features of the Albright hereditary osteodystrophy (AHO) phenotype. To investigate a pathogenetic relationship between STK25 and PPHP, we carried out fluorescence in situ hybridization (FISH) using an STK25 gene probe and chromosomes from PPHP patients characterized as having small deletions near the distal end of 2q. PPHP patient DNA showed no hybridization to STK25 genomic DNA, indicating that STK25 is contained within the deleted chromosomal region. This finding, in conjunction with previous studies demonstrating the role of Ste20/PAK kinases in heterotrimeric G protein signaling, suggests that STK25 is a positional candidate gene for PPHP.

Identification of the NIMA family kinases NEK6/7 as regulators of the p70 ribosomal S6 kinase.

BACKGROUND: The p70 S6 kinase, like several other AGC family kinases, requires for activation the concurrent phosphorylation of a site on its activation loop and a site carboxyterminal to the catalytic domain, situated in a hydrophobic motif site FXXFS/TF/Y, e.g.,Thr412 in p70 S6 kinase (alpha 1). Phosphorylation of the former site is catalyzed by PDK1, whereas the kinase responsible for the phosphorylation of the latter site is not known. RESULTS: The major protein kinase that is active on the p70 S6 kinase hydrophobic regulatory site, Thr412, was purified from rat liver and identified as the NIMA-related kinases NEK6 and NEK7. Recombinant NEK6 phosphorylates p70 S6 kinase at Thr412 and other sites and activates the p70 S6 kinase in vitro and in vivo, in a manner synergistic with PDK1. Kinase-inactive NEK6 interferes with insulin activation of p70 S6 kinase. The activity of recombinant NEK6 is dependent on its phosphorylation, but NEK6 activity is not regulated by PDK1 and is only modestly responsive to insulin and PI-3 kinase inhibitors. CONCLUSION: NEK6 and probably NEK7 are novel candidate physiologic regulators of the p70 S6 kinase.

BAD Ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival.

The BH3 domain of BAD mediates its death-promoting activities via heterodimerization to the Bcl-XL family of death regulators. Growth and survival factors inhibit the death-promoting activity of BAD by stimulating phosphorylation at multiple sites including Ser-112 and Ser-136. Phosphorylation at these sites promotes binding of BAD to 14-3-3 proteins, sequestering BAD away from the mitochondrial membrane where it dimerizes with Bcl-XL to exert its killing effects. We report here that the phosphorylation of BAD at Ser-155 within the BH3 domain is a second phosphorylation-dependent mechanism that inhibits the death-promoting activity of BAD. Protein kinase A, RSK1, and survival factor signaling stimulate phosphorylation of BAD at Ser-155, blocking the binding of BAD to Bcl-XL. RSK1 phosphorylates BAD at both Ser-112 and Ser-155 and rescues BAD-mediated cell death in a manner dependent upon phosphorylation at both sites.

FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions.

The FKBP12-rapamycin associated protein (FRAP, also RAFT, mTOR) belongs to a family of phosphatidylinositol kinase-related kinases. These kinases mediate cellular responses to stresses such as DNA damage and nutrient deprivation in a variety of eukaryotes from yeast to humans. FRAP regulates G(1) cell cycle progression and translation initiation in part by controlling the phosphorylation states of a number of translational and cell cycle regulators. Although FRAP is known to be phosphorylated in vivo and to phosphorylate several proteins (including itself) in vitro, FRAP's phosphorylation sites and substrate specificity are unknown. We report here the identification of a FRAP autophosphorylation site. This site, Ser-2481, is located in a hydrophobic region near the conserved carboxyl-terminal FRAP tail. We demonstrate that the COOH-terminal tail is required for FRAP kinase activity and for signaling to the translational regulator p70(s6k) (ribosomal subunit S6 kinase). Phosphorylation of wild-type but not kinase-inactive FRAP occurs at Ser-2481 in vivo, suggesting that Ser-2481 phosphorylation is a marker of FRAP autokinase activity in cells. FRAP autophosphorylation is blocked completely by wortmannin treatment but not by rapamycin treatment, amino acid deprivation, or serum withdrawal, treatments that lead to acute dephosphorylation of eIF4E-binding protein (4E-BP1) and p70(s6k). Ser-2481 phosphorylation increases slightly upon c-Akt/PKB activation and dramatically upon calyculin A treatment of T-cells. These results suggest that FRAP-responsive dephosphorylation of 4E-BP1 and p70(s6k) occurs through a mechanism other than inhibition of intrinsic FRAP kinase activity.

p90(RSK) blocks bad-mediated cell death via a protein kinase C-dependent pathway.

Although activation of protein kinase C (PKC) is known to promote cell survival and protect against cell death, the PKC targets and pathways that serve this function have remained elusive. Here we demonstrate that two potent activators of PKC, 12-O-tetradecanoylphorbol-13-acetate and bryostatin, both stimulate phosphorylation of Bad at Ser(112), a site known to regulate apoptotic cell death by interleukin-3. PKC inhibitors but not PI 3-kinase/Akt inhibitors block 12-O-tetradecanoylphorbol-13-acetate-stimulated Bad phosphorylation. PKC isoforms tested in vitro were unable to phosphorylate Bad at Ser(112), suggesting that PKC acts indirectly to activate a downstream Bad kinase. p90(RSK) and family members RSK-2 and RSK-3 are activated by phorbol ester and phosphorylate Bad at Ser(112) both in vitro and in vivo. p90(RSK) stimulates binding of Bad to 14-3-3 and blocks Bad-mediated cell death in a Ser(112)-dependent manner. These findings suggest that p90(RSK) can function in a PKC-dependent pathway to promote cell survival via phosphorylation and inactivation of Bad-mediated cell death.

mu-Opioid receptor activates signaling pathways implicated in cell survival and translational control.

The mu-opioid receptor mediates the analgesic and addictive properties of morphine. Despite the clinical importance of this G-protein-coupled receptor and many years of pharmacological research, few intracellular signaling mechanisms triggered by morphine and other mu-opioid agonists have been described. We report that mu-opioid agonists stimulate three different effectors of a phosphoinositide 3-kinase (PI3K)-dependent signaling cascade. By using a cell line stably transfected with the mu-opioid receptor cDNA, we show that the specific agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) stimulates the activity of Akt, a serine/threonine protein kinase implicated in protecting neurons from apoptosis. Activation of Akt by DAMGO correlates with its phosphorylation at serine 473. The selective PI3K inhibitors wortmannin and LY294002 blocked phosphorylation of this site, previously shown to be necessary for Akt enzymatic activity. DAMGO also stimulates the phosphorylation of two other downstream effectors of PI3K, the p70 S6 kinase and the repressors of mRNA translation, 4E-BP1 and 4E-BP2. Upon mu-opioid receptor stimulation, p70 S6 kinase is activated and phosphorylated at threonine 389 and at threonine 421/serine 424. Phosphorylation of p70 S6 kinase and 4E-BP1 is also repressed by PI3K inhibitors as well as by rapamycin, the selective inhibitor of FRAP/mTOR. Consistent with these findings, DAMGO-stimulated phosphorylation of 4E-BP1 impairs its ability to bind the translation initiation factor eIF-4E. These results demonstrate that the mu-opioid receptor activates signaling pathways associated with neuronal survival and translational control, two processes implicated in neuronal development and synaptic plasticity.

Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies.

The p70 S6 kinase is activated by diverse stimuli through a multisite phosphorylation directed at three separate domains as follows: a cluster of (Ser/Thr) Pro sites in an autoinhibitory segment in the noncatalytic carboxyl-terminal tail; Thr-252 in the activation loop of the catalytic domain; and Ser-394 and Thr-412 in a segment immediately carboxyl-terminal to the catalytic domain. Phosphorylation of Thr-252 in vitro by the enzyme phosphatidylinositol 3-phosphate-dependent kinase-1 or mutation of Thr-412 --> Glu has each been shown previously to engender some activation of the p70 S6 kinase, whereas both modifications together produce 20-30-fold more activity than either alone. We employed phospho-specific anti-peptide antibodies to examine the relative phosphorylation at several of these sites in wild type and various p70 mutants, in serum-deprived cells, and in response to activators and inhibitors of p70 S6 kinase activity. Substantial phosphorylation of p70 Thr-252 and Ser-434 was present in serum-deprived cells, whereas Thr-412 and Thr-444/Ser-447 were essentially devoid of phospho-specific immunoreactivity. Activation of p70 by insulin was accompanied by a coordinate increase in phosphorylation at all sites examined, together with a slowing in mobility on SDS-PAGE of a portion of p70 polypeptides. Upon addition of rapamycin or wortmannin to insulin-treated cells, the decrease in activity of p70 was closely correlated with the disappearance of anti-Thr-412(P) immunoreactivity and the most slowly migrating p70 polypeptides, whereas considerable phosphorylation at Ser-434 and Thr-252 persisted after the disappearance of 40 S kinase activity. The central role of Thr-412 phosphorylation in the regulation of kinase activity was further demonstrated by the close correlation of the effects of various deletions and point mutations on p70 activity and Thr-412 phosphorylation. In conclusion, although p70 activity depends on a disinhibition from the carboxyl-terminal tail and the simultaneous phosphorylation at both Thr-252 and Thr-412, p70 activity in vivo is most closely related to the state of phosphorylation at Thr-412.

A mitogen-activated protein kinase pathway is required for mu-opioid receptor desensitization.

The mu-opioid receptor mediates not only the beneficial painkilling effects of opiates like morphine but also the detrimental effects of chronic exposure such as tolerance and dependence. Different studies have linked tolerance to opioid receptor desensitization. Agonist activation of the mu-opioid receptor stimulates a mitogen-activated protein kinase (MAPK) activity, but the functional significance of this pathway remains unclear. We have focused on the MAPK signaling cascade to study mu-opioid receptor desensitization. We report that inhibition of the MAPK pathway blocks desensitization of mu-opioid receptor signaling as well as the loss of receptor density due to internalization. Our results suggest that a feedback signal emanating from the MAPK cascade is required for mu-opioid receptor desensitization.

Human T-cell leukemia virus type 1 Tax releases cell cycle arrest induced by p16INK4a.

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein causes cellular transformation by deregulating important cellular processes such as DNA repair, transcription, signal transduction, proliferation, and growth. Although it is clear that normal cell cycle control is deregulated during HTLV-1-induced cellular transformation, the effects of Tax on cell cycle control are not well understood. Flow cytometric analyses of human T cells indicate that cell cycle arrest in late G1, at or before the G1/S restriction point, by p16INK4a is relieved by Tax. Furthermore, Tax-dependent stimulation of 5-bromo-2'-deoxyuridine incorporation and transcriptional activation is inhibited by p16INK4a. This result suggests that p16INK4a is able to block Tax-dependent stimulation of DNA synthesis and cell cycle progression into S phase. In vitro binding assays with recombinant glutathione S-transferase fusion proteins and [35S]methionine-labeled proteins indicate that Tax binds specifically with p16INK4a but not with either p21cip1 or p27kip1. Furthermore, sequential immunoprecipitation assays with specific antisera and [35S]methionine-labeled cell lysates subsequent to coexpression with Tax and p16INK4a indicate that the two proteins form complexes in vivo. Immunocomplex kinase assays with cyclin-dependent kinase 4 antiserum indicate that Tax blocks the inhibition of cdk4 kinase activity by p16INK4a. This study identifies p16INK4a as a novel cellular target for Tax and suggests that the inactivation of p16INK4a function is a mechanism of cell cycle deregulation by Tax.

FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2.

Fibroblast growth factor (FGF) activates a protein kinase cascade in SK-N-MC cells that regulates gene expression at a cyclic-AMP response element (CRE) by stimulating the transcriptional activity of CREB. The activation of CREB is prevented by a dominant negative mutant of Ras and triggered via the same site (Ser133) that becomes phosphorylated in response to cyclic AMP and Ca2+. However, the effect of FGF is not mediated by cyclic AMP-dependent protein kinase, TPA-sensitive isoforms of protein kinase-C, p70S6K or p90rsk (all of which phosphorylate CREB at Ser133 in vitro). Instead, we identify the FGF-stimulated CREB kinase as MAP kinase-activated protein (MAPKAP) kinase-2, an enzyme that lies immediately downstream of p38 MAP kinase, in a pathway that is also stimulated by cellular stresses. We show that MAPKAP kinase-2 phosphorylates CREB at Ser133 in vitro, that the FGF- or stress-induced activation of MAPKAP kinase-2 and phosphorylation of CREB and ATF-1 are prevented by similar concentrations of the specific p38 MAP kinase inhibitor SB 203580, and that MAPKAP kinase-2 is the only detectable SB 203580-sensitive CREB kinase in SK-N-MC cell extracts. We also show that transfection of RK/p38 MAP kinase in SK-N-MC cells, but not transfection of p44 MAP kinase, activates Gal4-CREB-dependent transcription via Ser133. These findings identify a new growth factor and stress-activated signaling pathway that regulates gene expression at the CRE.

Fibroblast growth factor and cyclic AMP (cAMP) synergistically activate gene expression at a cAMP response element.

Growth factors and cyclic AMP (cAMP) are known to activate distinct intracellular signaling pathways. Fibroblast growth factor (FGF) activates ras-dependent kinase cascades, resulting in the activation of MAP kinases, whereas cAMP activates protein kinase A. In this study, we report that growth factors and cAMP act synergistically to stimulate proenkephalin gene expression. Positive synergy between growth factor- and cAMP-activated signaling pathways on gene expression has not been previously reported, and we suggest that these synergistic interactions represent a useful model for analyzing interactions between these pathways. Transfection and mutational studies indicate that both FGF-dependent gene activation and cAMP-dependent gene activation require cAMP response element 2 (CRE-2), a previously characterized cAMP-dependent regulatory element. Furthermore, multiple copies of this element are sufficient to confer FGF regulation upon a minimal promoter, indicating that FGF and cAMP signaling converge upon transcription factors acting at CRE-2. Among many different ATF/AP-1 factors tested, two factors, ATF-3 and c-Jun, stimulate proenkephalin transcription in an FGF- or Ras-dependent fashion. Finally, we show that ATF-3 and c-Jun form heterodimeric complexes in SK-N-MC cells and that the levels of both proteins are increased in response to FGF but not cAMP. Together, these results indicate that growth factor- and cAMP-dependent signaling pathways converge at CRE-2 to synergistically stimulate gene expression and that ATF-3 and c-Jun regulate proenkephalin transcription in response to both growth factor- and cAMP-dependent intracellular signaling pathways.

Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element.

Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes. This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate. Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation. Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription. Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation. In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A. Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax. Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3. Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax. Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax.

Activating transcription factor-3 stimulates 3',5'-cyclic adenosine monophosphate-dependent gene expression.

Activating transcription factor-3 (ATF-3) is one member of a large family of leucine zipper transcription factors which bind to promoters responsive to cAMP and phorbol ester at the related cAMP (CRE) and phorbol ester response elements. We report here that ATF-3 is coexpressed with the neuropeptide precursor proenkephalin in human neuroblastoma SK-N-MC cells. Cotransfection experiments indicate that activation of proenkephalin gene expression by ATF-3 is dependent upon both the catalytic subunit of the cAMP-dependent protein kinase and the CRE-2 element. The CRE-2 element is essential for second messenger-inducible expression and is known to bind AP-1-like transcription factors. ATF-3 expressed in bacteria or from rabbit reticulocyte lysates binds to the proenkephalin CRE-2 element as a homodimer and as a heterodimer with Jun-D, another activator of proenkephalin transcription. ATF-3 stimulates binding of Jun-D to the proenkephalin CRE-2 element and acts synergistically with Jun-D to induce proenkephalin gene expression. Sequential immunoprecipitations of ATF-3 from SK-N-MC cells expressing proenkephalin indicate that ATF-3 is complexed with Jun-D in vivo and that both proteins are highly phosphorylated. Together, our results suggest that ATF-3 may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways.

Everything activates c-fos--how can it matter?

It is probably safe to say that the regulation of c-fos expression by drug treatment and other stimulus paradigms has biological specificity and mechanistic significance. However, as this brief essay makes clear, the immunohistochemical detection of c-fos is only the tip of the biological iceberg. IEGs deserve to be studied as critical components of the intracellular signaling machinery that may transduce intercellular signals, including those produced by drugs of abuse, into longer term changes in cellular function. However, the study of IEG expression must be related to biologically significant target genes and must be complemented by the study of phosphorylation of constitutively expressed transcription factors. Moreover, as illustrated by the proenkephalin gene, the regulation of genes that play important roles in differentiated cell function may differ depending on which cell types and stimulus conditions are investigated. Finally, attention must be paid to the complexities of gene regulation under conditions of repetitive and chronic stimulation if phenomena such as the neural substrates of drug dependence are to be understood.

Expression and regulation of a proenkephalin beta-galactosidase fusion gene in the reproductive system of transgenic mice.

A fusion gene containing 3 kilobases of human proenkephalin 5'-flanking sequences and 1 kilobase of human proenkephalin 3'-flanking sequence and the easily visualized histochemical marker, Escherichia coli beta-galactosidase, was used to study the function of cis-regulatory elements within the human proenkephalin gene in transgenic mice. Here data are presented on expression and regulation of this fusion gene in the reproductive system of three independent lines of transgenic mice. Within the male reproductive system, the fusion gene is expressed in the proximal epididymis and in developing germinal cells but not in mature or elongating spermatids. In the female reproductive system, the transgene was expressed at low basal levels, but expression was dramatically stimulated in the ovary and oviduct by hormonal stimulation and pregnancy; additionally, expression was induced at the uteroplacental junction in pregnant mice. Taken together these observations suggest that critical sequences for expression and regulation of the proenkephalin gene within the reproductive system are contained within sequences of the construct.

Regulation of proenkephalin expression in cultured skin mesenchymal cells.

Proenkephalin, a classically defined opioid encoding gene, is transiently expressed in nondifferentiated mesodermal cells during organogenesis. We examined the hypothesis that this expression is associated with mesenchymal cell proliferation. For this purpose, we established a cell culture derived from fetal skin mesenchyme that specifically expresses proenkephalin mRNA in correlation with hypodermis development. These mesenchymal cells also produce and secrete significant amounts of proenkephalin-derived peptides. Using this model system, we observed a marked increase in proenkephalin mRNA expression in response to serum. This effect is time dependent and reaches peak levels during the G1/S transition. Similarly, 12-O-tetradecanoyl-phorbol-13-ester, whose biological actions have been shown to be mediated by the activity of protein kinase C (PKC), up-regulates proenkephalin expression. Desensitization of PKC by prolonged exposure of cells to 12-O-tetradecanoyl-phorbol-13-ester attenuates the serum induction of proenkephalin. The results presented in this report demonstrate that proenkephalin expression in mesenchymal cells is regulated by serum factors via mechanisms that involve PKC activity. A possible association between proenkephalin expression and cell proliferation is suggested.

Regulation of opioid gene expression: a model to understand neural plasticity.

The recent finding that neurotransmitters and drugs that affect neurotransmission have important influences on gene expression suggests that drug-induced alterations in gene expression may underlie many long-term effects of addictive drugs, for example, dependence and drug-seeking behaviors. These long-term adaptive responses to opiate drugs have been particularly difficult to understand at a mechanistic level. Data presented here indicate that the gene encoding the opioid precursor proenkephalin is highly regulated by neural activity, second-messenger pathways, and PKA. These observations raise the possibility that drugs of abuse (e.g., opiates acting through opiate receptors) may act at the genetic level to modulate the expression of endogenous opiates and that these effects may underlie one component of the brain's long-term adaptive response to exogenous opiates. The transgenic animals described above can be used to investigate opiate drug-induced changes in proenkephalin gene expression, allowing rapid analysis of changes in proenkephalin gene expression in highly restricted populations of neurons in a fashion previously impossible. In addition, by analyzing the effects of specific enhancer mutations on tissue-specific and transsynaptic regulation of proenkephalin expression, transgenic models will permit mechanistic investigations within the intact nervous system that cannot otherwise be undertaken. Investigation of mechanisms underlying this process requires the analysis of intracellular signaling pathways, responsive DNA regulatory elements, and the transcription factors transducing synaptic signals into gene regulation. In the studies described herein, we demonstrate that AP-1 complexes consisting of different Jun proteins differentially regulate proenkephalin transcription at the CRE-2 element. c-Jun constitutively activates proenkephalin transcription, whereas JunD activates in a fashion completely dependent on the activation of second-messenger pathways and the cAMP-dependent PKA. JunB alone has no effect on proenkephalin gene expression, yet this molecule effectively blocks activation mediated by JunD and, hence, may act as a repressor. These data are consistent with a model (figure 4) in which preexisting JunD mediates the rapid cAMP-dependent activation of the proenkephalin enhancer, whereas IEGs such as JunB or c-Fos mediate the protein synthesis-dependent inactivation. Because c-Jun activates proenkephalin transcription constitutively, induction of c-Jun may lead to a further and prolonged activation of proenkephalin gene expression. Hence, the ratio of c-Jun to JunB induction may determine whether proenkephalin is repressed or further activated.

cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins.

We demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester-, and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c-Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways.

NF-I proteins from brain interact with the proenkephalin cAMP inducible enhancer.

A short region of the human proenkephalin promoter has been shown previously to mediate transcriptional regulation in response to activation of the cAMP, TPA, and Ca+ + dependent intracellular signalling pathways. Two adjacent DNA elements, CRE-1 and CRE-2, are essential for this regulation although neither element alone is sufficient for inducible expression. The CRE-2 element consists of overlapping binding sites for the transcription factors AP-1 and AP-4. The CRE-1 element has been shown to interact with a DNA binding factor called ENKTF-1. Here we characterize proteins from bovine brain which bind the CRE-1 element of the human proenkephalin gene. Interactions between proteins binding the CRE-1 and CRE-2 elements are characterized in vitro using affinity purified DNA binding proteins. We demonstrate that CRE-1 binding proteins from bovine brain consist of three different polypeptides each belonging to the NF-I family of transcription factors. Point mutation analysis of the contacts of these proteins with the CRE-1 element indicate that NF-I proteins contact the inducible enhancer at the sequence CTGGCxxxxxxCCT which overlaps the CRE-1 element (underlined) defined by in vivo point mutation analysis. Cotransfection of one of the three NF-I proteins purified from bovine brain, NF-I/Red1, together with a proenkephalin/bacterial chloramphenicol acetyl transferase (CAT) fusion gene repressed protein kinase A or forskolin stimulated CAT expression.