Physiological role of phosphorylation of the cyclic AMP response element binding protein in rat cardiac nuclei

We determine whether the cyclic AMP signal transduction pathway affects phosphorylation of cyclic AMP response element binding protein and increases muscle gene expression in the heart. Elevation of cyclic AMP results in phosphorylation of the binding protein which is detected using an antibody specific for the phosphorylated, but not the unphosphorylated, form. The protein is present, but not phosphorylated, within the nuclei of myocytes in intact neonatal rat hearts and in high-density cultures. It is not expressed in low-density cultures. Increasing the amount of phosphorylated cyclic AMP with either isoproterenol or forskolin also increases the frequency and force of the beating. The phosphorylated form of the response element binding protein is visible in the nuclei by 10 min and persists for 2 h of drug treatment. A 1.5-fold increase in skeletal α-actin and α-myosin gene expression is detected after 48 h of isoproterenol treatment. However, blockage of beating with a calcium channel blocker (verapamil) in the presence of cyclic AMP results in a similar increased gene expression. This suggests that muscle gene expression can be regulated directly by the cyclic AMP pathway, probably via phosphorylation of the cyclic AMP response element binding protein but independent of contractile activity. Received: 1 December 1995 / Accepted: 2 May 1996     

Requirements of class II-mediated B cell differentiation for class II cross-linking and cyclic AMP.

Cells of the mouse B cell clone, CH12.LX, receive Ag-dependent differentiative signals through their surface membrane class II molecules. The present study was performed to determine the role of class II cross-linking and cAMP in the successful delivery of these signals. Delivery of differentiative signals by anti-Ek mAb was increased by further cross-linking with a secondary anti-isotype antibody. Intact or (Fab')2, but not Fab forms of anti-Ek successfully delivered the Ag-dependent differentiative signal. Inability of monovalent Fab fragments to deliver the signal could not be attributed to an inability to adequately bind Ek molecules. The requirement for cAMP for class II-mediated signaling was also examined, because previous studies have implicated elevated cAMP levels as necessary for class II signaling. Both Ag-dependent, Ek-mediated differentiation and the Ek-mediated inhibition of Ag-independent LPS-induced differentiation were inhibited by the adenyl cyclase inhibitor 2'5'ddA, although elevation of cAMP was not in itself sufficient to deliver the differentiative signal. Inhibition of LPS-induced differentiation could be mediated by mAb binding to either Ek, Abk, or Abb on CH12.LX or an Ab-bearing transfectant, CH12.ABB1. This inhibition was abrogated by 2'5'ddA in the case of Ek or Abb, both of which deliver Ag-dependent differentiative signals to CH12.LX cells. In the case of Abk, which does not deliver such signals to CH12.LX, 2'5'ddA did not abrogate anti-Abk-mediated inhibition of the LPS response. The effects of 2'5'ddA were reversed by the cAMP analog, dibutyryl cAMP, and Ag-dependent-induced differentiation of CH12.LX or CH12.ABB1 was accompanied by an increase in cAMP levels.     

Cyclic AMP and mitogen-activated protein kinases are required for glutamate-dependent cyclic AMP response element binding protein and Elk-1 phosphorylation in the dorsal striatum in vivo

Dopaminergic and glutamatergic signalling cascades are integrated in striatal medium spiny neurones by cyclic AMP response-element binding protein and Elk-1 phosphorylation. Phosphorylated cyclic AMP response-element binding protein and phosphorylated Elk-1 contribute to c-fos expression by binding to the calcium and cyclic AMP response-element and the serum response element, respectively, in the c-fos promoter. The role of cyclic AMP and mitogen-activated protein kinase signalling cascades in glutamate-induced cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression was investigated using semiquantitative immunocytochemistry in vivo. Intracerebroventricular infusion of the sodium channel blocker, tetrodotoxin, decreased the glutamate-induced increase in phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Intracerebroventricular infusion of the mitogen-activated and extracellular signal-regulated kinase inhibitor, PD98059, the p38 mitogen-activated protein kinase inhibitor, SB203580, or the cyclic AMP inhibitor, Rp-8-Br-cAMPS, decreased glutamate-induced phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Simultaneous infusion of glutamate and Sp-8-Br-cAMPS, a cyclic AMP analogue, augmented induction of Fos immunoreactivity but not phosphorylated cyclic AMP response-element binding protein or phosphorylated Elk-1 immunoreactivity. These data indicate that cyclic AMP and mitogen-activated protein kinase signalling cascades are necessary for glutamate to induce cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression in the striatum. Furthermore, neuronal activity plays an important role in glutamate-induced signalling cascades in vivo.     

The amino terminus of Tax is required for interaction with the cyclic AMP response element binding protein.

Tax of human T-cell leukemia virus type 1 was analyzed for interaction with the cyclic AMP response element binding protein (CREB) in vitro with and without Tax response element DNA. Mutations in the carboxy terminus of Tax (L296G and L320G) did not affect binding to CREB and led to supershifts. In contrast, mutants with changes in the amino-terminal cysteine-rich region lost the ability to bind to CREB. The S10A mutant protein bound moderately. Thus, the amino terminus of Tax is essential for Tax-CREB interaction.     

Modulation of sterol regulatory element binding protein-2 in response to rapid follicle development in chickens

We investigated the expression profiles of sterol regulatory element binding proteins (SREBPs) and their related genes in chicken developing follicle membranes, from the small white follicle (SWF) stage to the Follicle 1 (F1) stage. Expression of SREBP-2 was significantly increased in the rapid stages of follicle development, however, no significant change in SREBP-1 mRNA expression was observed during follicle development. Immunoreactive SREBP-2 protein levels isolated from nuclear extracts in rapid growth stages, particularly in Follicle 2, were higher than those in SWF and small yellow follicle (SYF). In contrast, SREBP-1 immunoreactive protein levels were only slightly changed over all stage of follicle development. 3-Hydroxy-3-methylglutaryl CoA reductase (HMGR) mRNA levels significantly increased in the rapid stages of follicle development, suggesting that SREBP-2 controls the biosynthesis of cholesterol in follicles. LDL receptor and LDL receptor related protein 1 mRNA also tended to increase with follicular development, however, expression of LDL receptor relative with eight ligand binding repeats (LR8) was only slightly affected by SREBP-2. Liver X receptor α (LXR α) was expressed in chicken follicles; its expression patterns corresponded with SREBP-2 gene expression. These results suggest that SREBP-2, which might be regulated by LXRα, is involved in the rapid growth stages of follicle development in avian species.     

Phosphorylation of CREB, a cyclic AMP responsive element binding protein, contributes partially to lysophosphatidic acid-induced fibroblast cell proliferation

Lysophospholipids regulate a wide array of biological processes including cell survival and proliferation. In our previous studies, we found that in addition to SRE, CRE is required for maximal c-fos promoter activation triggered by lysophosphatidic acid (LPA). c-fos is an early indicator of various cells into the cell cycle after mitogenic stimulation. However, role of CREB activation in LPA-stimulated proliferation has not been elucidated yet. Here, we investigate how LPA induces proliferation in Rat-2 fibroblast cell via CREB activation. We found that total cell number and BrdU-positive cells were increased by LPA. Moreover, levels of c-fos mRNA and cyclin D1 protein were increased via LPA-induced CREB phosphorylation. Furthermore, LPA-induced Rat-2 cell proliferation was decreased markedly by ERK inhibitor (U0126) and partially by MSK inhibitor (H89). Taken together, these results suggest that CREB activation could partially up-regulate accumulation of cyclin D1 protein level and proliferation of LPA-stimulated Rat-2 fibroblast cells.     

Cloning,expression, characterization,and role in autocrine cell growth of cell surface retention sequence binding protein-1

Cell surface retention sequence binding protein-1 (CRSBP-1) is a cell surface binding protein for the cell surface retention sequence (CRS) motif of the v-sis gene product (platelet-derived growth factor-BB). It has been shown to be responsible for cell surface retention of the v-sis gene product in v-sis-transformed cells (fibroblasts) and has been hypothesized to play a role in autocrine growth and transformation of these cells. Here we demonstrate that the CRSBP-1 cDNA cloned from bovine liver libraries encodes a 322-residue type I membrane protein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain. CRSBP-1 expressed in transfected cells is an approximately 120-kDa disulfide-linked homodimeric glycoprotein and exhibits dual ligand (CRS-containing growth regulators (v-sis gene product and insulin-like growth factor binding protein-3, IGFBP-3) and hyaluronic acid) binding activity. CRSBP-1 overexpression (by stable transfection of cells with CRSBP-1 cDNA) enhances autocrine loop signaling, cell growth, and tumorigenicity (in mice) of v-sis-transformed cells. CRSBP-1 expression also enhances autocrine cell growth mediated by IGFBP-3 in human lung carcinoma cells (H1299 cells), which express very little, if any, endogenous CRSBP-1 and exhibits a mitogenic response to exogenous IGFBP-3, stably transfected with IGFBP-3 cDNA. However, CRSBP-1 overexpression does not affect growth of normal and transformed cells that do not produce these CRS-containing growth regulators. These results suggest that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by growth regulators containing CRS.