Molecular cloning of Chinese hamster 1q31 chromosomal fragile site DNA that is important to mdr1 gene amplification reveals a novel gene whose expression is associated with spermatocyte and adipocyte differentiation.

DNA amplification plays important roles in the development of drug resistance and tumor progression. One mechanism of DNA amplification involves the breakage-fusion-bridge (BFB) cycle. We previously reported that in Chinese hamster ovary (CHO) cell line, breakage at fragile site 1q31 was associated with mdr1 gene amplification through the BFB mechanism. To elucidate the molecular basis of BFB-mediated DNA amplification, we cloned 1q31 fragile site DNA from a Chinese hamster cell line containing an integrated neomycin-resistance marker. Sequence analyses revealed many characteristics similar to those in other common fragile sites. Moreover, this fragile site contains an evolutionarily conserved novel gene, designated fragile site-associated (FSA) gene. FSA encodes a approximately 16-kb mRNA, from which an unusually large open reading frame (orf) of 5005 amino acids can be deduced. The C-terminal portion of FSA shares a striking sequence similarity to that of Caenorhabditi elegans lipid depleted-3 (lpd-3) gene whose function has been demonstrated to involve in lipid storage. We also demonstrated that expression of FSA is associated with the developmental programs of spermatogenesis and adipogenesis. Our results suggest that the Chinese hamster 1q31 fragile site has many important functions including regulation of mdr1 amplification and differentiation of adipocytes and spermatocytes.

Differential phosphorylation and dephosphorylation of beta2-adrenoceptor sites Ser262 and Ser355,356.

Activated beta2-adrenoceptors are rapidly desensitized by phosphorylation of Ser262 by protein kinase A (PKA) and of Ser355,356 by G-protein-coupled receptor kinase (GRK). We sought to determine whether the phosphorylation and subsequent dephosphorylation of these sites had similar kinetics and requirements for receptor endocytosis. The phosphorylation of the PKA and GRK sites were measured using antibodies that recognize phosphoserine 262 and phosphoserine 355,356. Endocytosis in stably transfected HEK293 cells was blocked by inducible expression of dominant-negative dynamin-1 K44A or by treatment with hypertonic sucrose. The phosphorylation of the GRK site Ser355,356 during a 10 microM isoprenaline treatment rapidly reached a steady state, and the extent of kinetics of phosphorylation were unaffected by dynamin-1 K44A expression, and minimally by hypertonic sucrose. In contrast, phosphorylation of the PKA site Ser262 during a 10 microM isoprenaline treatment peaked after 2 min and then rapidly declined, while inhibition of endocytosis enhanced and prolonged phosphorylation. Treatment with 300 pM isoprenaline, a concentration too low to provoke endocytosis, also resulted in prolonged PKA site phosphorylation. The dephosphorylation of these sites was measured after removal of agonist. Significant dephosphorylation of phosphoserines 262 and 355,356 was observed under conditions of very low endocytosis, however dephosphorylation of the GRK site was greater if antagonist was present after removal of agonist. The results indicate that the kinetics of beta2-adrenoceptor GRK and PKA site phosphorylation are distinct and differently affected by endocytosis, and that receptor dephosphorylation can occur either at the plasma membrane or in internal compartments.

Endosome sorting of beta 2-adrenoceptors is GRK5 independent.

1. We have investigated the role of G protein-coupled receptor kinase 5 (GRK5) in the regulation of endosome sorting of human beta(2)-adrenoceptors. 2. Expressing GRK5 at a high level significantly increased the extent of internalization of wild-type beta(2)-adrenoceptors and of an internalization-defective mutant receptor, and increased receptor phosphorylation at serines 355 and 356 in the cytoplasmic tail. 3. Overexpressing GRK5 did not alter beta(2)-adrenoceptor recycling as assessed by immunofluorescence microscopy and radioligand binding assays nor was there any change in receptor downregulation. 4. These data indicate that GRK5 does not regulate the sorting of beta(2)-adrenoceptors in the endocytic pathway.

Role of the G protein-coupled receptor kinase site serine cluster in beta2-adrenergic receptor internalization, desensitization, and beta-arrestin translocation.

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.

Delayed mechanism for induction of gamma-glutamylcysteine synthetase heavy subunit mRNA stability by oxidative stress involving p38 mitogen-activated protein kinase signaling.

Expression of the gamma-glutamylcysteine synthetase heavy subunit (gamma-GCSh), which encodes the rate-limiting enzymes for glutathione biosynthesis, is regulated by many cytotoxic agents. Moreover, gamma-GCSh mRNA expression is elevated in colorectal cancer, but how gamma-GCSh expression is regulated is not completely understood. By using actinomycin D, which inhibits new RNA synthesis, we showed that treatment of human colorectal cancer cells with the prooxidant sulindac increased the half-life of gamma-GCSh mRNA. By using a tetracycline-regulated gamma-GCSh mRNA assay system, we systematically dissected the cis-acting sequence and trans-acting factors that regulate the stability of gamma-GCSh by cytotoxic prooxidants. We demonstrated that a HuR recognition sequence, AUUUA, in the 3'-untranslated region is responsible for the decay of gamma-GCSh mRNA. Oxidative stress enhanced cytoplasmic content of HuR. Overexpression of HuR by transfection stabilized gamma-GCSh mRNA, whereas overexpression of a dominant-negative HuR mutant suppressed the induced stability. Furthermore, prooxidant-induced gamma-GCSh mRNA stabilization and HuR binding were blocked by p38 mitogen-activated protein kinase inhibitors. We provide the first evidence that reduction-oxidation regulation of gamma-GCSh expression, itself a reduction-oxidation sensor and regulator, is mediated at least in part by the p38 mitogen-activated protein kinase signaling through the HuR RNA-binding protein.

The NucE and NucD lysis proteins are not essential for secretion of the Serratia marcescens extracellular nuclease.

The nuclease of Serratia marcescens is an extracellular protein encoded by the nucA gene. Pre-nuclease carries a typical 21-amino-acid N-terminal signal sequence that interacts with the Sec machinery to allow the translocation of nuclease to the periplasm. In Escherichia coli the nuclease remains in the periplasm; however, S. marcescens has the capacity to secrete nuclease extracellularly. The nucC operon carrying the nucEDC genes of S. marcescens has been identified previously. NucC is a transcriptional activator necessary for expression of nuclease as well as the extracellular bacteriocin 28b. NucE resembles and can act as a bacteriophage holin, whereas NucD has homology to bacteriophage lysozyme-like proteins. When present on a multicopy plasmid, the nucC operon, and specifically the nucED genes, appeared to allow extracellular secretion of nuclease from E. coli. Here experiments are reported which demonstrate that, when the nucC operon was placed in the E. coli chromosome in single copy, nuclease secretion was lost and nuclease remained periplasmic. The converse experiment, deletion of the nucE and nucD genes from the chromosome of S. marcescens, likewise had no effect on nuclease secretion by S. marcescens. It is concluded therefore that NucD and NucE are not necessary for nuclease secretion.

Rab11 regulates the recycling and lysosome targeting of beta2-adrenergic receptors.

The pericentriolar recycling endosome (RE) may be an alternative compartment through which some beta2-adrenergic receptors (beta2ARs) recycle from early endosomes to the cell surface during prolonged exposure to agonist. For the transferrin receptor, CXCR2, and the M4-muscarinic acetylcholine receptor, trafficking through the RE and receptor recycling is regulated by the small GTPase rab11. The precise role of the RE and rab11 in regulating the cellular trafficking of the beta2AR is not understood. We therefore monitored trafficking of beta2ARs in HEK293 cells following the modulation of rab11 activity. Expression of a rab11 mutant deficient in GTP binding (as a fusion between enhanced green fluorescent protein (EGFP) and the rab11S25N mutant) significantly slowed receptor recycling to the cell surface from dispersed transferrin-positive peripheral vesicles following a brief exposure to agonist. The agonist was applied at a time when receptors have undergone only one or two rounds of endocytosis and recycling. In cells overexpressing wild-type rab11, beta2ARs localized to a rab11-positive compartment and the rate of beta2AR recycling to the cell surface was reduced, but only after prolonged exposure to agonist and multiple rounds of receptor endocytosis and recycling. This effect was associated with impaired beta2AR trafficking to lysosomes and receptor proteolysis, whereas the sorting of low-density lipoprotein from transferrin-positive vesicles to late endosomes and lysosomes was not affected. These data highlight a pivotal role for rab11 in regulating the traffic of a G protein-coupled receptor at the level of the RE, where modulation of rab11 activity dictates the balance between receptor recycling and downregulation during prolonged exposure to agonist.