scholarly journals Mutation-deletion analysis of a Ca2+-dependent phospholipid binding (CaLB) domain within p120 GAP, a GTPase-activating protein for p21 ras

1995 ◽  
Vol 307 (2) ◽  
pp. 487-491 ◽  
Author(s):  
D J Gawler ◽  
L J W Zhang ◽  
M F Moran
Keyword(s):  
Amino Acids ◽  
Sequence Similarity ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Binding Activity ◽  
Membrane Phospholipids ◽  
Gtpase Activating Protein ◽  
Phospholipid Binding ◽  
P21 Ras

p120 GAP is a GTPase activating protein for p21 ras. It is a multidomain protein which exhibits sequence similarity with other GTPase-activating proteins, src, pleckstrin and a central portion of the protein kinase C conserved region 2 domain known as CaLB (Ca(2+)-dependent phospholipid-binding). The presence of this CaLB motif has led to the speculation that p120 GAP may be a member of a family of structurally related proteins containing a Ca(2+)-dependent membrane/lipid-binding domain. Here we have studied the in vitro Ca(2+)-dependent phospholipid-binding properties of the isolated proposed CaLB sequence in human GAP and deduce that a phospholipid-binding sequence is indeed located between amino acids 606 and 648. Binding of phosphatidylserine and phosphatidylinositol, but not phosphatidylcholine, within this sequence is Ca(2+)-dependent, with an estimated EC50 for Ca2+ of approx. 1 microM. Using deletion-mutation analysis we have further defined the minimal boundaries for this in vitro phospholipid-binding activity. p120 GAP amino acids 612-643 exhibit full phospholipid-binding activity, but further deletion of either amino acids 612-617 or amino acids 633-648 significantly decreased or abolished phospholipid binding. These studies establish that amino acids 612-643 of p120 GAP indeed constitute a functional CaLB domain and thereby imply a role for Ca2+ in the regulation of p120 GAP association with cellular (membrane) phospholipids.

scholarly journals Identification of a talin binding site in the cytoskeletal protein vinculin.

1989 ◽  
Vol 109 (6) ◽  
pp. 2917-2927 ◽  
Author(s):  
P Jones ◽  
P Jackson ◽  
G J Price ◽  
B Patel ◽  
V Ohanion ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Cytoskeletal Protein ◽  
Translation System ◽  
Cos Cells ◽  
Cell Matrix ◽  
Do So

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

scholarly journals Inhibitor of Apoptosis Proteins Physically Interact with and Block Apoptosis Induced by Drosophila Proteins HID and GRIM

1998 ◽  
Vol 18 (6) ◽  
pp. 3300-3309 ◽  
Author(s):  
Domagoj Vucic ◽  
William J. Kaiser ◽  
Lois K. Miller
Keyword(s):  
Amino Acids ◽  
Cell Line ◽  
Sequence Similarity ◽  
Binding Activity ◽  
Inhibitor Of Apoptosis ◽  
Physical Interaction ◽  
Antiapoptotic Proteins ◽  
Transient Overexpression ◽  
Induced Apoptosis ◽  
Apoptosis Proteins

ABSTRACT Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183–10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of theseDrosophila proapoptotic proteins.

scholarly journals Exophilin4/Slp2-a Targets Glucagon Granules to the Plasma Membrane through Unique Ca2+-inhibitory Phospholipid-binding Activity of the C2A Domain

2007 ◽  
Vol 18 (2) ◽  
pp. 688-696 ◽  
Author(s):  
Miao Yu ◽  
Kazuo Kasai ◽  
Kazuaki Nagashima ◽  
Seiji Torii ◽  
Hiromi Yokota-Hashimoto ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Secretory Granules ◽  
Cell Types ◽  
Binding Activity ◽  
Effector Proteins ◽  
Membrane Phospholipids ◽  
Β Cells ◽  
Phospholipid Binding ◽  
Lysosome Related Organelles ◽  
C2a Domain

Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic β cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic α cells, in contrast to another effector, granuphilin, in β cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca2+ concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca2+-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca2+-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.

scholarly journals Identification of the hydrophobic strand in the A–B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists

2005 ◽  
Vol 391 (2) ◽  
pp. 221-230 ◽  
Author(s):  
Leonora Niv-Spector ◽  
Dana Gonen-Berger ◽  
Isabelle Gourdou ◽  
Eva Biener ◽  
Eugene E. Gussakovsky ◽  
...  
Keyword(s):  
Amino Acids ◽  
Large Scale ◽  
Gel Filtration ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Activity ◽  
Hydrophobic Cluster Analysis ◽  
Leptin Receptors ◽  
Subsequent Activation

Interaction of leptin with its receptors resembles that of interleukin-6 and granulocyte colony-stimulating factor, which interact with their receptors through binding sites I–III. Site III plays a pivotal role in receptors' dimerization or tetramerization and subsequent activation. Leptin's site III also mediates the formation of an active multimeric complex through its interaction with the IGD (immunoglobulin-like domain) of LEPRs (leptin receptors). Using a sensitive hydrophobic cluster analysis of leptin's and LEPR's sequences, we identified hydrophobic stretches in leptin's A–B loop (amino acids 39–42) and in the N-terminal end of LEPR's IGD (amino acids 325–328) that are predicted to participate in site III and to interact with each other in a β-sheet-like configuration. To verify this hypothesis, we prepared and purified to homogeneity (as verified by SDS/PAGE, gel filtration and reverse-phase chromatography) several alanine muteins of amino acids 39–42 in human and ovine leptins. CD analyses revealed that those mutations hardly affect the secondary structure. All muteins acted as true antagonists, i.e. they bound LEPR with an affinity similar to the wild-type hormone, had no agonistic activity and specifically inhibited leptin action in several leptin-responsive in vitro bioassays. Alanine mutagenesis of LEPR's IGD (amino acids 325–328) drastically reduced its biological but not binding activity, indicating the importance of this region for interaction with leptin's site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented that the transient FRET signalling occurring upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin's site III.

scholarly journals Cloning of the Schizosaccharomyces pombe gene encoding diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) asymmetrical hydrolase: sequence similarity with the histidine triad (HIT) protein family

1995 ◽  
Vol 312 (3) ◽  
pp. 925-932 ◽  
Author(s):  
Y Huang ◽  
P N Garrison ◽  
L D Barnes
Keyword(s):  
Amino Acids ◽  
Schizosaccharomyces Pombe ◽  
Sequence Similarity ◽  
Enzymic Activity ◽  
Protein Family ◽  
Partial Purification ◽  
Binding Motif ◽  
Local Similarity ◽  
Multicopy Plasmid

Diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap4A hydrolase from S. pombe [Robinson, de la Peña and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Séraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn(2+)-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.

scholarly journals Characterization and Antifungal Properties of Wheat Nonspecific Lipid Transfer Proteins

2008 ◽  
Vol 21 (3) ◽  
pp. 346-360 ◽  
Author(s):  
Jin-Yue Sun ◽  
Denis A. Gaudet ◽  
Zhen-Xiang Lu ◽  
Michele Frick ◽  
Byron Puchalski ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Spore Germination ◽  
Sodium Dodecyl ◽  
Membrane Integrity ◽  
Lipid Binding ◽  
Binding Activity ◽  
Lipid Transfer ◽  
Lipid Transfer Proteins ◽  
Fatty Acid Binding

This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZα and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 μM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.

scholarly journals Assembly of flagellar radial spoke proteins in Chlamydomonas: identification of the axoneme binding domain of radial spoke protein 3.

1993 ◽  
Vol 123 (1) ◽  
pp. 183-190 ◽  
Author(s):  
D R Diener ◽  
L H Ang ◽  
J L Rosenbaum
Keyword(s):  
Amino Acids ◽  
Binding Activity ◽  
Binding Domain ◽  
Flagellar Motility ◽  
Radial Spoke ◽  
Acid Protein ◽  
Radial Spokes ◽  
Carboxy Terminal ◽  
Amino Acid Protein

Radial spokes of the eukaryotic flagellum extend from the A tubule of each outer doublet microtubule toward the central pair microtubules. In the paralyzed flagella mutant of Chlamydomonas pf14, a mutation in the gene for one of 17 polypeptides that comprise the radial spokes results in flagella that lack all 17 spoke components. The defective gene product, radial spoke protein 3 (RSP3), is, therefore, pivotal to the assembly of the entire spoke and may attach the spoke to the axoneme. We have synthesized RSP3 in vitro and assayed its binding to axonemes from pf14 cells to determine if RSP3 can attach to spokeless axonemes. In vitro, RSP3 binds to pf14 axonemes, but not to wild-type axonemes or microtubules polymerized from purified chick brain tubulin. The sole axoneme binding domain of RSP3 is located within amino acids 1-85 of the 516 amino acid protein; deletion of these amino acids abolishes binding by RSP3. Fusion of amino acids 1-85 or 42-85 to an unrelated protein confers complete or partial binding activity, respectively, to the fusion protein. Transformation of pf14 cells with mutagenized RSP3 genes indicates that amino acids 18-87 of RSP3 are important to its function, but that the carboxy-terminal 140 amino acids can be deleted with little effect on radial spoke assembly or flagellar motility.

scholarly journals Sequence and biochemical similarities between the luciferases of the glow-worm Lampyris noctiluca and the firefly Photinus pyralis

1996 ◽  
Vol 313 (3) ◽  
pp. 761-767 ◽  
Author(s):  
Graciela B. SALA-NEWBY ◽  
Catherine M. THOMSON ◽  
Anthony K. CAMPBELL
Keyword(s):  
Amino Acids ◽  
Sequence Similarity ◽  
Biochemical Properties ◽  
Cdna Expression Library ◽  
Photinus Pyralis ◽  
Expression Library ◽  
Ph Optimum ◽  
Click Beetle ◽  
Light Organs

A full-length clone encoding Lampyris noctiluca (British glow-worm) luciferase was isolated from a complementary DNA (cDNA) expression library constructed with mRNA extracted from light organs. The luciferase was a 547-residue protein, as deduced from the nucleotide sequence. The protein was closely related to those of other lampyrid beetles, the similarity to Photinus pyralis luciferase being 84% and to Luciola 67%. In contrast, Lampyris luciferase had less sequence similarity to the luciferases of the click beetle Pyrophorus, at 48%. Engineering Lampyris luciferase in vitro showed that the C-terminal peptide containing 12 amino acids in Photinus and 9 amino acids in Lampyris was essential for bioluminescence. The pH optimum and the Km values for ATP and luciferin were similar for both Photinus and Lampyris luciferases, although the light emitted by the latter shifted towards the blue and was less stable at 37 °C. It was concluded that the molecular and biochemical properties were not sufficient to explain the glowing or flashing of the two beetles Lampyris and Photinus.

Lipid binding activity of a neuron-specific protein NAP-22 studied in vivo and in vitro

2002 ◽  
Vol 70 (2) ◽  
pp. 172-179 ◽  
Author(s):  
Akira Terashita ◽  
Nobuo Funatsu ◽  
Masato Umeda ◽  
Yukiko Shimada ◽  
Yoshiko Ohno-Iwashita ◽  
...  
Keyword(s):  
Specific Protein ◽  
Lipid Binding ◽  
Binding Activity

scholarly journals Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein.

1986 ◽  
Vol 6 (4) ◽  
pp. 1002-1009 ◽  
Author(s):  
J C Lacal ◽  
S A Aaronson
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Dna Synthesis ◽  
Binding Activity ◽  
Structural Domain ◽  
Serum Stimulation ◽  
Amino Acid Region ◽  
Gtp Binding ◽  
P21 Ras

The p21 products of ras proto-oncogenes are GTP-binding proteins with associated GTPase activity. Recent studies have indicated that ras p21 may be required for the initiation of normal cell DNA synthesis, since microinjection of a monoclonal antibody, Y13-259, blocks serum stimulation of DNA synthesis in quiescent cell cultures (L. S. Mulcahy, M.R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We localized the structural domain within the p21 molecule recognized by the Y13-259 monoclonal antibody. By analysis of a series of bacterially expressed p21 deletion mutants, the monoclonal antibody was found to interact with a region between positions 70 and 89 in the p21 amino acid sequence. By comparison of the coding sequences of different p21 proteins recognized by this monoclonal antibody, a highly conserved amino acid region between positions 70 and 81 was found to be the most likely site for the epitope detected by the Y13-259 antibody. This monoclonal antibody was further shown not to interfere directly with in vitro biochemical functions of the molecule, including GTP binding, GTPase, and autokinase activities.

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