Type A allatostatins from Drosophila melanogaster and Diplotera puncata activate two Drosophila allatostatin receptors, DAR-1 and DAR-2, expressed in CHO cells

The type-A allatostatins A (AST-A) are a group of insect peptides with a common C-terminal motif Y/FXFGL-NH(2). The existence of at least four putative type A Drosophila melanogaster ASTs (called type A drostatins or DST-As) has been predicted from the sequence of a recently cloned DST-A preprohormone [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 126-1131]. SRPYSFGL-NH(2), (DST-3A), the only DST isolated from Drosophila so far, activated the first cloned DST-A GPCR (DAR-1) [N. Birgül et al. (1999) EMBO J. 18, 5892-5900]. A newly cloned orphan Dm GPCR, which shares 47% overall and 60% transmembrane region sequence identity with DAR-1, was classified as a second putative Dm DST-A receptor (DAR-2) [C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 273, 571-577]. Although activation of DAR-2 by DSTs has been postulated, no experimental evidence for that has been presented to date. In this study, we expressed both DAR-1 and DAR-2 in CHO cells and used a GTPgammaS and a Ca(2+) mobilization assay for pharmacological evaluation of the receptors. Synthetically prepared DST-As, as well as selected Diplotera punctata (cockroach) ASTs, activated DAR-1 and DAR-2 in both functional assays indicating ligand redundancy and cross species activity. Cell pretreatment with pertussis toxin led to some differences in the nature and magnitude of signaling pathways at the DAR-1 and DAR-2 receptors, suggesting possible differential coupling to cellular effector system(s) and distinct biological functions of each receptor in vivo.     

Role of atypical protein kinase C in estradiol-triggered G1/S progression of MCF-7 cells

Expression of a dominant negative atypical protein kinase C (aPKC), PKCzeta, prevents nuclear translocation of extracellular regulated kinase 2 (ERK-2), p27 nuclear reduction, and DNA synthesis induced by estradiol in human mammary cancer-derived MCF-7 cells. aPKC action upstream of these events has been analyzed. In hormone-stimulated NIH 3T3 and Cos cells ectopically expressing human estrogen receptor alpha (hERalpha), aPKC is activated by phosphatidylinositol 3-kinase (PI 3-kinase) and, in turn, controls the Ras/MEK-1/ERK cascade. In MCF-7 and Cos cells stimulated by hormone, PI 3-kinase activates PKCzeta by Thr410 phosphorylation. Serine phosphorylation of PKCzeta is simultaneously induced. PKCzeta activation leads to recruitment of Ras to a multimolecular complex that also includes hERalpha, Src, PI 3-kinase, and aPKC. We propose that PKCzeta pushes Ras and the signaling complex close together in such a way that it facilitates the Src-dependent Ras activation. This activation is crucial for the interplay between estradiol-triggered signaling and cell cycle machinery.     

Loss of the amino-terminal helix-loop-helix domain of the vav proto-oncogene activates its transforming potential.

vav, a novel human oncogene, was originally generated in vitro by replacement of its normal 5' coding sequences with sequences from pSV2neo DNA, cotransfected as a selectable marker (S. Katzav, D. Martin-Zanca, and M. Barbacid, EMBO J. 8:2283-2290, 1989). The vav proto-oncogene is normally expressed in cells of hematopoietic origin. To determine whether the 5' rearrangement of vav or its ectopic expression in NIH 3T3 cells contributes to its transforming potential, we isolated murine and human proto-vav cDNA clones as well as human genomic clones corresponding to the 5' end of the gene. Normal proto-vav was poorly transforming in NIH 3T3 cells, whereas truncation of its 5' end greatly enhanced its transforming activity. The relative failure of full-length proto-vav cDNA clones to transform NIH 3T3 cells indicates that the transforming activity of vav is not simply due to ectopic expression. Analysis of the predicted amino terminus of the vav proto-oncogene shows that it contains a helix-loop-helix domain and a leucine zipper motif similar to that of myc family proteins, though it lacks a basic region that is usually found adjacent to helix-loop-helix domains. Loss of the helix-loop-helix domain of proto-vav, either by truncation or by rearrangement with pSV2neo sequences, activates its oncogenic potential.     

Vesl/Homer proteins regulate ryanodine receptor type 2 function and intracellular calcium signaling

Cellular signaling proteins such as metabotropic glutamate receptors, Shank, and different types of ion channels are physically linked by Vesl (VASP/Ena-related gene up-regulated during seizure and LTP)/Homer proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851]. Vesl/Homer proteins have also been implicated in differentiation and physiological adaptation processes [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348]. Here we provide evidence that a Vesl/Homer subtype, Vesl-1L/Homer-1c (V-1L), reduces the function of the intracellular calcium channel ryanodine receptor type 2 (RyR2). In contrast, Vesl-1S/Homer-1a (V-1S) had no effect on RyR2 function but reversed the effects of V-1L. In live cells, in calcium release studies and in single-channel electrophysiological recordings of RyR2, V-1L reduced RyR2 activity. Important physiological functions and pharmacological properties of RyR2 are preserved in the presence of V-1L. Our findings demonstrate that a protein-protein interaction between V-1L and RyR2 is not only necessary for organizing the structure of intracellular calcium signaling proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23(2000)80; J. Cell Sci. 113 (2000) 1851; Nat Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348; Nature 386 (1997) 284], but that V-1L also directly regulates RyR2 channel activity by changing its biophysical properties. Thereby it may control cellular calcium homeostasis. These observations suggest a novel mechanism for the regulation of RyR2 and calcium-dependent cellular functions.     

A chloroplastic RNA-binding protein is a new member of the PPR family

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963–4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621–2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.     

CFTR Activation Raises Extracellular pH of NIH/3T3 Mouse Fibroblasts and C127 Epithelial Cells

Cystic Fibrosis (CF) is caused by mutations in the gene for CFTR, a cAMP-activated anion channel found in apical membranes of wet epithelia. Since CFTR is permeable to HCO⁻ ₃ and changes in extracellular fluid composition may contribute to CF lung disease, we investigated possible differences in extracellular pH (pHo) between CFTR-expressing and control cell lines. The Cytosensor™ Microphysiometer was used to study forskolin-stimulated extracellular acidification rates in CFTR-expressing and control mouse mammary epithelial (C127) and fibroblast (NIH/3T3) cell lines. Forskolin, which activates CFTR via raised cAMP, caused decreased extracellular acidification of CFTR-expressing NIH/3T3 and C127 cells by 15–35%. By contrast, forskolin caused increased extracellular acidification of control cells by 10–20%. Ionomycin, which may activate CFTR via PKC, also elicited this decreased extracellular acidification signal only in cells expressing CFTR. In control experiments, dideoxyforskolin had no effect on the acidification rates and osmotic stimuli were shown to equally stimulate all cell lines. These results suggest a role for CFTR in controlling pHo and complement recent evidence that HCO⁻ ₃ dependent epithelial secretion may be reduced in amount and altered in composition in CF. Received: 20 June 2000/Revised: 13 November 2000     

New cosmid vectors developed for eukaryotic DNA cloning

A series of ColE1 and pSC101 cosmid vectors have been constructed suitable for cloning large stretches of DNA. All contain a single BamHI site allowing cloning of Sau3A, MboI, BglII, BclI , and BamHI-generated fragments. These vectors have the following characteristics: (i) they are relatively small (1.7-3.4 kb); (ii) the BamHI cloning site is flanked by restriction enzyme sites enabling direct cloning of unfractionated insert DNA without generating multiple insert or vector ligation products [ Ish - Horowitz and Burke, Nucl . Acids Res. 9 (1981) 2989-2998]; (iii) two vectors ( pHSG272 and pHSG274 ) contain a hybrid Tn5 KmR/ G418R gene which is selectable in both prokaryotic and eukaryotic cells, making them suitable for transferring DNA into eukaryotic cells, and (iv) the different prokaryotic selectable markers available in the other vectors described facilitate cosmid rescue of the transferred DNA sequences from the eukaryotic cell: CmR, ApR, KmR, ( pHSG429 ), CmR, ( pHSG439 ), colicin E1 immunity ( pHSG250 ), (v) the cosmid pHSG272 was used successfully to construct a shuttle vector based on the BPVI replicon [ Matthias et al., EMBO J. 2 (1983) 1487-1492].     

The proteasome/multicatalytic-multifunctional proteinase. In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes.

Proteinase yscE, the proteasome/multicatalytic-multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555-562]. A well-defined set of proteins degraded via ubiquitin-mediated proteolysis are the substrates of the N-end rule pathway [(1986) Science 234, 179-186; (1989) Science 243, 1576-1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N-end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin-mediated proteolysis.     

The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug,Amantadine

Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important. HCV p7 is a small hydrophobic protein of unknown function, yet necessary for particle infectivity in related viruses [Harada, T. et al., (2000) J. Virol. 74, 9498-9506]. We show that p7 can be cross-linked in vivo as hexamers. Escherichia coli expressed p7 fusion proteins also form hexamers in vitro. These and HIS-tagged p7 function as calcium ion channels in black lipid membranes. This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza [Hay, A.J. et al. (1985) EMBO J. 4, 3021-3024; Duff, K.C. and Ashley, R.H. (1992) Virology 190, 485-489] and has recently been shown to be active in combination with current HCV therapies.     

Multiple domains exist within the upstream activator sequence of the octopine synthase gene.

It is known that a 16-base pair palindrome (ACGTAAGCGCTTACGT) located upstream of the ocs gene can activate a maize adh1 promoter in a transient expression system [Ellis et al. (1987). EMBO J. 6, 11-16; Ellis et al. (1987). EMBO J. 6, 3203-3208]. We have determined that this palindrome is also essential for ocs promoter activity in tobacco calli. In addition, sequences immediately adjacent to this palindrome, both 5' and 3', modulate its activity. The palindrome is sensitive to the differentiated state of the plant cells in which it resides; it is active in calli and the leaves of small shoots but is inactive in the leaves of rooted plants. We have tested upstream sequences from two other T-DNA genes that have homology to this palindrome for their ability to activate the octopine synthase promoter in tobacco calli. The upstream region from the mannopine synthase gene can activate the octopine synthase promoter, but an upstream region from the gene implicated in octopine and nopaline secretion cannot activate the promoter.