cAMP response element-binding protein monomers cooperatively assemble to form dimers on DNA

We have analyzed the properties of cAMP response element-binding protein (CREB) in solution with emphasis on dimerization and effects of phosphorylation. Using a purified CREB fusion protein, a novel dye-label technique, and sedimentation equilibrium analysis, we directly and conclusively demonstrate that, unlike Jun and Fos, CREB dimerization is DNA-dependent. CREB exists primarily as a monomer in solution and cooperatively assembles on DNA to form dimers. Sedimentation equilibrium analysis also indicates that dimerization is unaffected by cAMP-dependent protein kinase-phosphorylation or by the symmetry of the cAMP-responsive element binding site. Filter binding assays reveal that CREB binding is unaffected by phosphorylation regardless of the symmetry of the cAMP-responsive element binding site. Our results suggest that structurally similar members of the same bZIP superfamily may differ significantly in their regulation at the level of dimerization.     

Occurrence and nuclear localization of cAMP response element-binding protein in the post-natal development of the rat submandibular gland

Cyclic AMP response element-binding protein (CREB) is a 43-kDa polypeptide that binds a cAMP response element located at the 5' promoter region of cAMP regulatory genes. The spatial and temporal distribution of CREB in the postnatal development of the rat submandibular gland was investigated using immunohistochemistry with a specific antibody. At birth, cells of the terminal tubules and ducts in the submandibular gland showed a nuclear CREB immunoreactivity of moderate intensity. At 1-2 weeks after birth, an intense CREB immunoreactivity was localized primarily to acinar cells. When the beta-adrenergic agonist isoproterenol was administered to 2-week-old rats, a twofold transient increase in the number of immunoreactive acinar cells was induced. Beginning 3 weeks after birth, CREB immunoreactivity shifted from acini to the duct system and showed a clear localization in the cells of the intercalated ducts and distal portions of striated ducts, where the granular convoluted tubule develops after 4 weeks. Immunopositive materials were localized exclusively in the nuclei of both acinar and ductal immunoreactive cells. After the development of the granular convoluted tubules, CREB immunoreactivity was absent in the tubule cells and was gradually reduced in intensity over the entire gland. In order to examine a hypothesis that CREB is involved in the initial differentiation of the granular convoluted tubular cells, testosterone was administered to hypophysectomized adult rats. Whereas the tubular cells of hypophysectomized rats showed a complete regression, and no CREB immunoreactivity was found in any acinar or duct cells, administration of testosterone for a few days induced an intense CREB immunoreactivity in the nuclei of duct cells, followed by their differentiation into the granular convoluted tubular cells. These results suggested that CREB is involved not only in the growth and differentiation of acinar cells that are regulated by beta-adrenergic nerves but also in those of the duct system, and especially in the androgen-regulated differentiation of the granular convoluted tubular cells, during the post-natal development of the rat submandibular gland.     

Phosphorylation and activation of cGMP-dependent protein kinase by Src

Naive CD8 T cells can be polarized into effectors producing the type 1 cytokines IFN-gamma and IL-2 or the type 2 cytokines IL-4, IL-5, and IL-10, respectively. To study whether the polarized cytokine phenotype of the effectors is stable, we generated highly cytotoxic hemagglutinin (HA) peptide-specific CD8 Tc1 and Tc2 (cytotoxic CD8 T cells producing type 1 or type 2 cytokines) effectors from Clone-4 TCR-transgenic mice, which were adoptively transferred into syngeneic adult thymectomized irradiated and bone marrow-reconstituted recipients. The highly activated blast-size, CD25+ Tc1 and Tc2 effectors gave rise to homogeneous resting CD25- CD44(high) Ly6C(high) Ag-specific populations, which persisted for at least 13 wk after adoptive transfer. These memory CD8 T cells, recovered 13 wk after transfer of Tc1 or Tc2 effectors, still produced either the type 1 or type 2 cytokines, i.e., IFN-gamma, or IL-4 and IL-5, respectively, upon restimulation with APCs loaded with the HA peptide, but not in the absence of Ag. The amounts of IL-2 detected in the supernatants of Tc1 and Tc2 memory populations were comparable to that in memory CD4 cells, and both Tc1 and Tc2 memory cells became cytotoxic upon restimulation. Thus, cytokine-polarized CD8 memory T cells are a source of a variety of cytokines, which were classically considered helper cytokines, opening new perspectives on their function as regulatory cells in an immune response.     

Effect of alpha adrenergic agonist on the rabbit ear artery contraction to serotonin: enhanced response mediated by serotonergic1-like receptors

The effect of methoxamine or phenylephrine (PHE) on the contractile response of the rabbit ear artery to serotonin was assessed by using isolated arterial rings mounted in tissue baths for the measurement of isometric force development. A contractile threshold concentration of methoxamine or PHE (10-30 nM) shifted the serotonin concentration-response curve to the left by approximately 200-fold. Neither mechanical removal of the vascular endothelium nor chemical denervation had any effect on the alpha agonist-amplified response of ear artery to serotonin. Although the response to serotonin in the absence of the alpha agonist was mediated primarily by alpha-1 adrenergic receptors, prazosin did not block the amplified response to serotonin. Ketanserin (10 nM), ritanserin (50 nM) and MDL 72222 (1 microM) also had no effect on the amplified response, ruling out the involvement of serotonergic (5-HT)2 and 5-HT3 receptors. However, methiothepin (3 nM) and 1-(1-naphthyl)piperazine (10 and 100 nM) blocked the PHE-amplified contraction of ear artery to serotonin. When the contractile response of ear artery to 5-carboxamidotryptamine was measured in the presence of a threshold concentration of alpha agonist, the concentration-response curve was shifted 8300-fold to the left. The amplified response to 5-carboxyamidotryptamine was insensitive to 10 nM ketanserin, but was blocked by 3 nM methiothepin. Sumatriptan, a selective 5-HT1 agonist, failed to induce vasoconstriction in the absence of a threshold concentration of alpha agonist. However, in the presence of PHE, sumatriptan induced a concentration-dependent contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation and activation of tryptophan hydroxylase by exogenous protein kinase A

The catalytic subunit of protein kinase A increases brain tryptophan hydroxylase activity. The activation is manifested as an increase in Vmax without alterations in the Km for either tetrahydrobiopterin or tryptophan. The activation of tryptophan hydroxylase by protein kinase A is dependent on ATP and an intact kinase and is inhibited specifically by protein kinase A inhibitors. Protein kinase A also catalyzes the phosphorylation of tryptophan hydroxylase. The extent to which tryptophan hydroxylase is phosphorylated by protein kinase A is dependent on the amount of kinase used and is closely related to the degree to which the hydroxylase is activated. These results suggest that a direct relationship exists between phosphorylation and activation of tryptophan hydroxylase by protein kinase A.     

Inhibitory alpha adrenergic receptors in white adipose tissue of rabbits

Rabbit white adipose tissue, in vitro, may not respond to epinephrine by an increase in the rate of lipolysis although beta adrenergic receptors may be postulated from our experiments with isoproterenol. When basal rate of lipolysis is high, an antilipolytic effect may be shown for low concentrations of epinephrine (0.01 and 0.1 mug/ml). However, epinephrine and an alpha adrenergic blocker, phentolamine (5 mug/ml), acting together, abolish the antilipolytic effect and better still a strong lipolytic effect of epinephrine is unmasked in such a case. The best evidence of these results is that inhibitory alpha, adrenergic receptors are involved in Rabbit white fat cells. These facts partially explain the absence of lipolytic response with epinephrine previously described.     

Association and direct activation of signal transducer and activator of transcription1alpha by platelet-derived growth factor receptor

The binding parameters of [3H]nociceptin were examined in membrane preparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 nM) low capacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10,316 nM) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potency: alpha-neo-endorphin > Dyn A-(2-13) = Dyn A-(3-13) > Dyn A-(5-13) > Dyn A-(1-13) > Dyn A > Dyn B > Dyn A-(6-10) > Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Ki of 4.8 microM as compared with 0.72 microM for Dyn A. The order of potency of the various peptides in inhibiting [3H]nociceptin binding correlated well (r = 0.93) with their ability to complete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mM) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100 microM). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs. Gpp(NH)p and GTP-gamma-S. Nociceptin (1-50 microM) was also shown to inhibit the uptake of [3H]noradrenaline ([3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL1 mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.