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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ras GTPase activating protein (ras GAP), a regulator of Ras activity, has two isoforms; ras GAP 120 and ras GAP 100. The latter, whose molecular size is about 100 kDa, is generated alternative splicing from the ras GAP 120 gene and is considered placenta-specific, while the former is expressed ubiquitously. As point mutations of ras are frequently observed in human tumors, we investigated the expression of ras GAP in several human cancer cell lines and samples of human colon cancer using immunoprecipitation and immunoblot analysis with an anti-GAP monoclonal antibody, B4F8, as well as reverse transcription-polymerase chain reaction (RT-PCR). ras GAP 100 protein was detected in 4 of 9 colonic, 1 of 6 gastric and 1 of 4 lung cancer cell lines as well as ras GAP 120, but not in colon cancer specimens. In contrast, ras GAP 100 mRNA was present in all tested cell lines and colon cancer specimens. Then, we investigated ras GAP 100 expression in normal tissues, ras GAP 100 protein was not detected in human normal tissues except placenta. Contrary, ras GAP 100 message was expressed in normal tissues derived from liver, stomach, colon and lymphocyte although the level of which was smaller than that in placenta. These findings demonstrate that ras GAP 100, reportedly placenta-specific, is distributed in other normal tissues at least at mRNA level and its expression is augmented in some cancer cell lines.
Mol Cell Biochem 1997 Oct
PMID:Expression of the placenta-specific, 100 kDa ras GTPase activating protein in several human cancer cell lines and normal human tissues. 935 52

A normal mode and energy minimization of ras p21 is used to determine the flexibility of the protein and the origin of the conformational differences between GTP and GDP-bound forms. To preserve the integrity of the structures, a hydration shell of water molecules was included as part of the system. Certain low-frequency modes were found to have high involvement coefficients with the conformational transition between the GTP and GDP-bound structures; the involvement coefficients of some of the modes increase when the gamma-phosphate group is removed. Two unstable modes that appear in the GTP-bound structure upon deletion of the gamma-phosphate group were determined and shown to have dominant contributions in the regions of switch I and switch II; there was also a significant displacement of loop 1. The initial motion in these regions is predicted by the modes to be approximately perpendicular to the direction of the transition from the GTP-bound state to the GDP-bound state. The overall conformational change in the switch I and II regions involves rearrangements of the protein backbone within these regions, rather than rigid body motion. Differences in the low-frequency modes of the GTP and GDP-bound forms appear to play a role in ligand binding. A coupling between the helix alpha3 position and the deletion of the gamma-phosphate group may be involved in the interaction with GAP. The oncogenic mutation G12D leads to a global increase in the rigidity of the protein. Thus, the mutant is likely to have a higher barrier for the conformational change to the inactive form; this would slow the transition and could be related to its oncogenic properties.
J Mol Biol 1997 Nov 21
PMID:Ligand-induced conformational changes in ras p21: a normal mode and energy minimization analysis. 939 20

The complementary DNAs (cDNA) encoding the [Trp7,Leu8]-gonadotrophin-releasing hormone (salmon-type GnRH; sGnRH:GeneBank accession no. u60667) and the [His5,Trp7,Tyr8]-GnRH (chicken-II-type GnRH; cGnRH-II: GeneBank accession no. u60668) precursor in the roach (Rutilus rutilus) were isolated and sequenced following reverse transcription and rapid amplification of cDNA ends (RACE). The sGnRH and cGnRH-II precursor cDNAs consisted of 439 and 628 bp, and included open reading frames of 282 and 255 bp respectively. The structures of the encoded peptides were the same as GnRHs previously identified in other vertebrates. The sGnRH and cGnRH-II precursor cDNAs, including the non-coding regions, had 88.6 and 79.9% identity respectively, to those identified in goldfish (Carassius auratus). However, significant similarity was not observed between the non-coding regions of the GnRH cDNAs of Cyprinidae and other fish. The presumed third exon, encoding partial sGnRH associated peptide (GAP) of roach, demonstrated significant nucleotide and amino acid similarity with the appropriate regions in the goldfish, but not with other species, and this may indicate functional differences of GAP between different families of fish. cGnRH-II precursor cDNAs from roach had relatively high nucleotide similarity across this GnRH variant. Cladistic analysis classified the sGnRH and cGnRH-II precursor cDNAs into three and two groups respectively. However, the divergence between nucleotide sequences within the sGnRH variant was greater than those encoding the cGnRH-II precursors. Consistent with the consensus developed from previous studies, Northern blot analysis demonstrated that expression of sGnRH and cGnRH-II was restricted to the olfactory bulbs and midbrain of roach respectively. This work forms the basis for further study on the mechanisms by which the tapeworm, Ligula intestinalis, interacts with the pituitary-gonadal axis of its fish host.
J Mol Endocrinol 1997 Dec
PMID:Isolation and characterisation of mRNA encoding the salmon- and chicken-II type gonadotrophin-releasing hormones in the teleost fish Rutilus rutilus (Cyprinidae). 946 Jun 54

RGS-GAIP (Galpha-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate Galphai/Galphaq-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein Galphai3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi- or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.
Mol Biol Cell 1998 May
PMID:RGS-GAIP, a GTPase-activating protein for Galphai heterotrimeric G proteins, is located on clathrin-coated vesicles. 957 Dec 44

Coordination between karyokinesis and cytokinesis in the cell division cycle is fundamental to a precise transmission of duplicated genome into dividing daughter cells. byr4, a previously isolated essential gene, affects the mitotic cell cycle and cytokinesis in S. pombe. Phenotypic analyses of the null alleles and the overexpression of byr4 suggest that byr4 is a dosage-dependent coordinator of karyokinesis and cytokinesis (Song et al., 1996). In this study, the functional mechanisms of byr4 were investigated using a byr4 mutant that exhibits byr4 overexpression phenotypes in thiamine deficient media. Genetic suppression analyses of this byr4 mutant with other cytokinesis regulatory genes in S. pombe, cdc16, cdc7, cdc15, cdc14, and plo1, show that byr4 overexpression phenotypes are suppressed by the overexpression of cdc16 and cdc7, but not by plo1, cdc14, and cdc15. Also, the basal expression of byr4 and cdc7 suppresses the temperature-sensitive cdc16 mutation. However, the basal expression of either byr4 or cdc16 does not suppress the temperature-sensitive cdc7 mutation. The results of these suppression tests suggest that byr4 genetically interacts with cdc16 and cdc7: byr4 functions at the same level with or downstream of cdc16 and upstream of cdc7. In the present study, we also show that Byr4 interacts with Cdc16 and Spg1 in the yeast two-hybrid assays. Recent reports suggest a possible small GTPase pathway to regulate the timing of cytokinesis where Cdc16 functions as a GAP (GTPase activating protein), Spg1 as a GTPase, and Cdc7 as a downstream effector. Combined genetic and two-hybrid analyses of this study strongly suggest that Byr4 directly interacts with this possible small GTPase pathway including Cdc16, Spg1, and Cdc7 to regulate cytokinesis in S. pombe.
Mol Cells 1998 Apr 30
PMID:Byr4, a dosage-dependent regulator of cytokinesis in S. pombe, interacts with a possible small GTPase pathway including Spg1 and Cdc16. 963 58

Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton following growth factor stimulation. A major regulator of Ras, Ras-GAP, contains several structural motifs, including an SH3 domain and two SH2 domains, and there is evidence that they harbor a signalling function. We have previously described a monoclonal antibody to the SH3 domain of Ras-GAP which blocks Ras signalling in Xenopus oocytes. We now show that microinjection of this antibody into Swiss 3T3 cells prevents the formation of actin stress fibers stimulated by growth factors or activated Ras, but not membrane ruffling. This inhibition is bypassed by coinjection of activated Rho, suggesting that the Ras-GAP SH3 domain is necessary for endogenous Rho activation. In agreement, the antibody blocks lysophosphatidic acid-induced neurite retraction in differentiated PC12 cells. Furthermore, we demonstrate that microinjection of full-length Ras-GAP triggers stress fiber polymerization in fibroblasts in an SH3-dependent manner, strongly suggesting an effector function besides its role as a Ras downregulator. These results support the idea that Ras-GAP connects the Ras and Rho pathways and, therefore, regulates the actin cytoskeleton through a mechanism which probably does not involve p190 Rho-GAP.
Mol Cell Biol 1998 Sep
PMID:Ras-GAP controls Rho-mediated cytoskeletal reorganization through its SH3 domain. 971 Jun 40

The protein tyrosine kinase Csk downregulates the activity of the Src family of kinases and has a negative effect on signal transduction through several Src kinase-associated receptors. Because the Src-family kinase Lyn plays a pivotal role in FcepsilonRI-mediated cellular activation, we examined whether Csk is involved in FcepsilonRI signaling events. Using anti-Csk antibodies and recombinant fusion proteins we detected a single tyrosine-phosphorylated protein of 60 kD (herein referred to as 'p60') that associates with the SH2 domain of Csk after stimulation of the FcepsilonRI. p60 phosphorylation reached a maximum within one minute and remained constant while the receptors were aggregated; disaggregation of the receptors resulted in rapid dephosphorylation of p60. The phosphorylation of p60 was only detected after activation by IgE and antigen and not by stimulation with PMA and/or ionomycin. Phosphorylated p60 was associated entirely with the membrane fraction of the cells. A considerable fraction of Csk was associated with the membrane in both unstimulated and stimulated cells, this fraction did not change upon activation. p60 coprecipitated with Csk from both unstimulated and FcepsilonRI stimulated cells and was phosphorylated by the immunocomplex. Total kinase activity of Csk immunoprecipitates increased upon FcepsilonRI stimulation. p60 did not react with antibodies to a number of known signaling molecules, including the recently cloned, GAP-associated protein, p62dok. Our data demonstrate that Csk associates with a membrane-anchored protein complex that is directly involved in FcepsilonRI signal transduction.
Mol Immunol 1998 Mar
PMID:Stimulation of the high-affinity IgE receptor results in the tyrosine phosphorylation of a 60 kD protein which is associated with the protein-tyrosine kinase, Csk. 973 41

The sequences of related proteins can diverge beyond the point where their relationship can be recognised by pairwise sequence comparisons. In attempts to overcome this limitation, methods have been developed that use as a query, not a single sequence, but sets of related sequences or a representation of the characteristics shared by related sequences. Here we describe an assessment of three of these methods: the SAM-T98 implementation of a hidden Markov model procedure; PSI-BLAST; and the intermediate sequence search (ISS) procedure. We determined the extent to which these procedures can detect evolutionary relationships between the members of the sequence database PDBD40-J. This database, derived from the structural classification of proteins (SCOP), contains the sequences of proteins of known structure whose sequence identities with each other are 40% or less. The evolutionary relationships that exist between those that have low sequence identities were found by the examination of their structural details and, in many cases, their functional features. For nine false positive predictions out of a possible 432,680, i.e. at a false positive rate of about 1/50,000, SAM-T98 found 35% of the true homologous relationships in PDBD40-J, whilst PSI-BLAST found 30% and ISS found 25%. Overall, this is about twice the number of PDBD40-J relations that can be detected by the pairwise comparison procedures FASTA (17%) and GAP-BLAST (15%). For distantly related sequences in PDBD40-J, those pairs whose sequence identity is less than 30%, SAM-T98 and PSI-BLAST detect three times the number of relationships found by the pairwise methods.
J Mol Biol 1998 Dec 11
PMID:Sequence comparisons using multiple sequences detect three times as many remote homologues as pairwise methods. 983 38

Graf, the GTPase regulator associated with focal adhesion kinase was previously shown to have GAP activity for &Rgr; A and Cdc42 in vitro (Hildebrand et al 1996 Mol. Cell Biol. 16: 3169-3178). In this study we sought to determine whether Graf acted at the level of Cdc42, Rho, or both in vivo and whether Graf was a signal terminator or transducer for these proteins. Microinjection of Graf cDNA into subconfluent Swiss 3T3 cells (in the presence of serum) has marked effects on cell shape and actin localization. Graf expression causes clearing of stress fibers followed by formation of long actin based filopodial-like extensions. Similar phenotypes were observed following injection of the Rho-inhibitor, C3 into these cells. The Graf response was dependent on GAP activity, since injection of Graf cDNA containing point mutations in the GAP domain (R236Q or N351V) which block enzymatic activity, does not confer this phenotype. Injection of Graf into Swiss 3T3 cells in which Rho has been down-regulated by serum starvation has no effect on cell morphology. Using this system, we demonstrate that Graf blocks sphingosine-1-phosphate (SPP) stimulated (Rho-mediated) stress fiber formation. Conversely, Graf expression does not inhibit bradykinin stimulated (Cdc42-mediated) filopodial extensions. These data indicate that Graf is a GAP for Rho in vivo. To further substantiate these results we examined the effect of Graf over-expression on Rho-mediated neurite retraction in nerve growth factor (NGF)-differentiated PC12 cells. In PC12 cells, which express relatively high levels of endogenous Graf, overexpression of Graf (but not Graf containing the R236Q mutation) enhances SPP-induced neurite retraction. These data indicate the possibility that Graf may be an effector for Rho in certain cell types.
 ...
PMID:Cytoskeletal changes induced by GRAF, the GTPase regulator associated with focal adhesion kinase, are mediated by Rho. 985 76

A unique glycoprotein, called endometriosis protein-I (ENDO-I), has previously been shown to be synthesized and secreted by rat and human endometriotic explants. Furthermore, the N-terminal amino acid sequence analysis showed that ENDO-I shares homology with haptoglobin. The present study was designed to determine this sequence of ENDO-I cDNA from human peritoneal endometriotic lesions and to determine the relative expression of the ENDO-I gene in several human tissues. Poly A-enriched RNA was isolated and reverse-transcribed. To determine the sequence of ENDO-I cDNA, a polymerase chain reaction was performed on cDNA from human endometriotic lesions and a gene-specific primer based on the human haptoglobin sequence. A similar procedure was followed to assess the relative expression of the ENDO-I gene in various tissues. Glyceraldehyde-3-phosphate-dehydrogenase was used as an internal control. ENDO-I gene expression was quantified by densitometry. Sequence analysis of ENDO-I cDNA identified 873 nucleotides that displayed 99.4% homology with the human haptoglobin beta-chain. Relative expression of ENDO-I mRNA by peritoneal endometriotic lesions was 19-fold greater than peritoneum, 28-fold greater than endometrioma and 37-fold greater than eutopic endometrium (P<0.01). Haptoglobin-like ENDO-I may be associated with localized angiogenesis and altered immune response involved with the aetiology/pathophysiology of endometriosis.
Mol Hum Reprod 1999 Jan
PMID:Peritoneal endometriotic lesions differentially express a haptoglobin-like gene. 1005 Jun 65


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