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1.
Cloning and characterization of human SHIP, the 145-kD inositol 5- phosphatase that associates with SHC after cytokine stimulation 总被引:4,自引:0,他引:4
We recently cloned and sequenced a cDNA encoding a 145-kD protein from the murine hematopoietic cell line B6SUtA, that becomes tyrosine phosphorylated and associated with Shc after cytokine stimulation. Based on its domains and enzymatic activity, we named this protein SHIP for SH2-containing inositol phosphatase (Damen et al, Proc Natl Acad Sci USA 93:1689, 1996). We describe here the cloning of the human homologue of murine SHIP (mSHIP) from a human megakaryocytic cell line (MO7e) lambda gt11 cDNA library using two nonoverlapping mSHIP cDNA fragments as probes. Northern blot analysis suggests that human SHIP (hSHIP) is expressed as a 5.3-kb mRNA in human bone marrow and a wide variety of other tissues. Sequence analysis of this cDNA predicts a protein of 1188 amino acids exhibiting 87.2% overall sequence identity with mSHIP. Contained within the defined open reading frame is an N- terminal, group l src homology 2 (SH2) domain; three NXXY motifs that, if phosphorylated, could be bound by phosphotyrosine binding (PTB) domains; a C-terminal proline-rich region; and two centrally located inositol polyphosphate 5-phosphatase motifs. Fluorescence in situ hybridization, using the full-length hSHIP cDNA as a probe, mapped hSHIP to the long arm of chromosome 2 at the border between 2q36 and 2q37. 相似文献
2.
Isolation and heterologous expression of a cDNA encoding bovine inositol polyphosphate 1-phosphatase. 总被引:6,自引:5,他引:6
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J D York P W Majerus 《Proceedings of the National Academy of Sciences of the United States of America》1990,87(24):9548-9552
Inositol polyphosphate 1-phosphatase, an enzyme of the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1-position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. The protein was isolated from calf brain and digested with trypsin or CNBr, and the amino acid sequence of several peptides was determined. Degenerate oligonucleotide primers were designed from amino acid sequence and used to synthesize an 80-base-pair (bp) fragment by the polymerase chain reaction. This product was used to isolate a 1.6-kbp cDNA with an open reading frame of 400 amino acids, 185 bp of 5' untranslated region, and 171 bp of 3' untranslated region followed by a putative poly(A) tail. The coding region of the cDNA was inserted into an expression vector that was used to obtain the recombinant protein from Escherichia coli cells. The recombinant enzyme (44 kDa) had a specific activity and other properties similar to those of native bovine brain inositol polyphosphate 1-phosphatase. It hydrolyzed both inositol phosphate substrates and was inhibited by lithium ions. The enzyme shows minimal sequence similarity to inositol monophosphate phosphatase, the other enzyme inhibited by lithium ions in the signaling pathway. 相似文献
3.
Dynamics of erythropoietin receptor expression on erythropoietin-responsive murine cell lines 总被引:1,自引:0,他引:1
We examined erythropoietin receptor expression in two murine cell lines, B6SUtA and DA-1, that respond to erythropoietin in different ways. While B6SUtA cells undergo erythroid differentiation with limited proliferation after addition of erythropoietin, DA-1 cells show only a proliferative response. Equilibrium binding experiments with 125I-erythropoietin revealed that both B6SUtA and DA-1 cells express a single class of erythropoietin receptors. In the absence of erythropoietin, B6SUtA cells exhibited 145 receptors per cell with a dissociation constant (kd) of 380 pmol/L. Six days after induction with erythropoietin, the B6SUtA cells displayed 310 receptors per cell without a change in binding affinity; exposure to erythropoietin also increased cellular hemoglobin content. DA-1 cells adapted to erythropoietin-dependent growth over a period of months and exhibited a progressive increase in erythropoietin receptor expression, from 85 per cell (kd = 540) to 550 per cell (kd = 530), although the cells remained uniformly benzidine-negative. We interpret the data with B6SUtA cells to indicate that early erythroid differentiation stages are attended by an increase in erythropoietin receptor display, coordinate with the initiation of expression of erythroid-specific genes. In contrast, the results with DA-1 cells are most compatible with clonal selection as the mechanism underlying enhanced receptor expression. Thus, display of the erythropoietin receptor is dynamic and can be modulated during the course of erythropoietin-induced differentiation. 相似文献
4.
Cloning, heterologous expression, and chromosomal localization of human inositol polyphosphate 1-phosphatase.
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J D York R A Veile H Donis-Keller P W Majerus 《Proceedings of the National Academy of Sciences of the United States of America》1993,90(12):5833-5837
Inositol polyphosphate 1-phosphatase, an enzyme in the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1 position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. We used a cDNA that encodes bovine inositol polyphosphate 1-phosphatase as a probe to isolate the human counterpart by low-stringency hybridization. The 1.74-kb human cDNA has 341 bp of 5' untranslated region, 180 bp of 3' untranslated region, poly(A)32, and predicts a protein of 399 amino acids. Human and bovine inositol polyphosphate 1-phosphatases show 84% amino acid sequence identity. Northern blot analysis from a variety of human tissues demonstrates that a 1.9-kb mRNA is ubiquitously expressed with highest levels in pancreas and kidney. Several higher molecular weight mRNAs also are expressed in brain, muscle, heart, and liver. We have confirmed the functional identity of the human cDNA by heterologous expression in NIH 3T3 fibroblasts, COS-7 cells and Escherichia coli. Polymerase chain reaction assay of a panel of human-rodent somatic cell hybrid DNA using human inositol polyphosphate 1-phosphatase-specific DNA primers resulted in amplification of a specific product using chromosome 2 DNA as template. Fluorescence in situ hybridization of metaphase chromosomes localizes the gene to chromosome 2 band q32. The identification of the human inositol polyphosphate 1-phosphatase gene locus provides a target for linkage analysis to identify defects in patients with inherited psychiatric disorders that respond to lithium ions, an inhibitor of the enzyme. 相似文献
5.
A L Mui R J Kay R K Humphries G Krystal 《Proceedings of the National Academy of Sciences of the United States of America》1992,89(22):10812-10816
The murine interleukin 3 receptor (mIL-3R) is a heterodimer consisting of a 70-kDa alpha subunit and one of two alternative 120-kDa beta subunits termed beta IL-3 and beta c. beta IL-3 (originally called Aic2A) is capable of binding mIL-3 by itself, whereas beta c (Aic2B) does not bind any ligand on its own but increases the affinity of mIL-3, murine granulocyte/macrophage-colony-stimulating factor, and mIL-5 for their respective alpha subunits. Interestingly, although the mIL-3R does not possess tyrosine kinase activity, its beta IL-3 subunit does become tyrosine phosphorylated upon binding mIL-3. To further investigate the properties of this subunit, we have purified it from the cell line B6SUtA1, which expresses a high level of mIL-3R. Intriguingly, studies comparing the stability of the 140-kDa, tyrosine-phosphorylated form of this subunit with its 120-kDa, non-tyrosine-phosphorylated form reveal that the former is far less stable and is rapidly degraded to a 70-kDa fragment. Mixing experiments demonstrate that the differential stability of the two forms is due to an intrinsic difference in protease susceptibility. Phosphatase studies indicate that the higher protease susceptibility of the tyrosine-phosphorylated beta IL-3 is due to the presence of both phosphotyrosine and phosphoserine residues. Western analyses using an anti-N-terminal mIL-3R beta IL-3 chain antibody reveal that this proteolytic cleavage also occurs rapidly in intact cells following stimulation with mIL-3 and occurs at the cell surface, since it takes place within minutes at 37 degrees C, is observed with purified plasma membranes, and is not inhibited by chloroquine. This degradative step may play an important role in the mechanism of action of mIL-3. 相似文献
6.
Erythropoietin changes the globin program of an interleukin 3-dependent multipotential cell line. 总被引:2,自引:3,他引:2
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T Enver B Nakamoto J Karlinsey B Josephson J Greenberger T Papayannopoulou 《Proceedings of the National Academy of Sciences of the United States of America》1988,85(23):9091-9095
B6SUtA is a factor-dependent murine cell line of adult origin displaying the functional properties of a multipotent hematopoietic stem cell. We analyzed the globin programs of B6SUtA cells undergoing erythroid differentiation in both suspension and clonal cultures. In the absence of added erythropoietin, a small number of hemoglobinized cells were present, and these expressed predominantly embryonic globin. Addition of erythropoietin increased the number and maturation of hemoglobinized cells and led to a preferential augmentation of adult globin. Analysis of individual B6SUtA erythroid bursts showed that embryonic and adult globin can be expressed in cells derived from a single progenitor. Furthermore, by studying globin expression in cultured cells from mouse embryos, we found that the globin programs of B6SUtA cells are similar to those of erythroid progenitors at the period of transition from yolk sac to fetal liver erythropoiesis. Since B6SUtA cells are derived from adult bone marrow and they have the capacity to express embryonic globin, we speculate that the globin locus is not irreversibly modified during development and that adult cells at early stages of erythroid differentiation can transiently express ontogenetically primitive globin programs. 相似文献
7.
An essential role for an inositol polyphosphate multikinase, Ipk2, in mouse embryogenesis and second messenger production
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Frederick JP Mattiske D Wofford JA Megosh LC Drake LY Chiou ST Hogan BL York JD 《Proceedings of the National Academy of Sciences of the United States of America》2005,102(24):8454-8459
Phospholipase C and several inositol polyphosphate kinase (IPK) activities generate a branched ensemble of inositol polyphosphate second messengers that regulate cellular signaling pathways in the nucleus and cytoplasm. Here, we report that mice deficient for Ipk2 (also known as inositol polyphosphate multikinase), an inositol trisphosphate and tetrakisphosphate 6/5/3-kinase active at several places in the inositol metabolic pathways, die around embryonic day 9.5 with multiple morphological defects, including abnormal folding of the neural tube. Metabolic analysis of Ipk2-deficient cells demonstrates that synthesis of the majority of inositol pentakisphosphate, hexakisphosphate and pyrophosphate species are disrupted, although the presence of 10% residual inositol hexakisphosphate indicates the existence of a minor alternative pathway. Agonist induced inositol tris- and bis-phosphate production and calcium release responses are present in homozygous mutant cells, indicating that the observed mouse phenotypes are a result of failure to produce higher inositol polyphosphates. Our data demonstrate that Ipk2 plays a major role in the synthesis of inositol polyphosphate messengers derived from inositol 1,4,5-trisphosphate and uncovers a role for their production in embryogenesis and normal development. 相似文献
8.
Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line 总被引:9,自引:0,他引:9
The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hematopoietic cell line, B6SUtA1. Murine granulocyte- macrophage colony-stimulating factor (mGM-CSF) was as potent as mIL-3 in stimulating these cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-O-tetradecanoyl- phorbol-13-acetate (TPA), a known activator of protein kinase C, also stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA1 cells exposed for brief periods to mIL-3, mGM-CSF, and TPA were analyzed for changes in phosphorylation patterns using metabolic 32P-labeling and antibodies to phosphotyrosine. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68-Kd cytosolic protein, whereas all three agents stimulated the serine-specific phosphorylation of a 68-Kd membrane protein. Furthermore, mIL-3 stimulated tyrosine phosphorylation of the 68-Kd membrane protein, as well as of 140-, 90-, 55, and 40-Kd proteins. The 90-Kd protein was also tyrosine phosphorylated in response to mGM-CSF. These phosphotyrosine containing proteins were not detected in TPA-treated cells. These results indicate that protein phosphorylations on tyrosine and serine residues occur in B6SUtA1 cells following short-term incubation with mIL-3 or mGM-CSF and that most of these phosphorylation events are mediated by kinases other than protein kinase C (PkC). 相似文献
9.
We have found that treatment of B6SUtA1 cells with 0.01% glutaraldehyde transformed them into mechanically resistant spheres, thereby making it possible to use these high interleukin 3 (IL-3) receptor-bearing cells as a solid phase reagent suitable for the large scale purification of murine IL-3 (mIL-3). Using this technique, mIL-3 was purified from serum-free pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCCM) approximately 16,000-fold using absorption to B6SUtA1 cells, Sephadex G75 superfine chromatography, and reverse phase high performance liquid chromatography on a C18 column. The overall yield was 16%. The final product consisted of two proteins with molecular weights of 19.5 and 16.5 kd. Both species possessed mIL-3-like activity. N-glycanase treatment of the purified preparation converted all of the 19.5-kd material into the lower molecular weight species, suggesting that the two species represented different glycosylated states of mIL-3 produced by activated T cells. This was confirmed by competition studies that showed that excess pure Escherichia coli-derived recombinant mIL-3, but not granulocyte-macrophage colony-stimulating factor (GM-CSF), could prevent the binding of both species of the PWM-SCCM-derived material to B6SUtA1 cells. 相似文献
10.
Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases. 总被引:5,自引:1,他引:5
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P D Burbelo A Utani Z Q Pan Y Yamada 《Proceedings of the National Academy of Sciences of the United States of America》1993,90(24):11543-11547
We have cloned a gene encoding a DNA-binding protein by Southwestern screening of a murine cDNA library with a double-stranded oligonucleotide containing the sequence from the bidirectional promoter of the alpha 1 and alpha 2 collagen IV genes. The middle portion of this 1131-amino acid protein has a region homologous to bacterial DNA ligases, and the more carboxyl portion contains several domains homologous to p40, p38, p37, and p36.5 subunits of activator 1 (A1, also called replication factor C), a human replication protein complex. Western blotting revealed that antiserum generated against part of the recombinant protein reacted specifically with the 145-kDa component of the purified human A1 complex, indicating that it is the murine counterpart of the A1 p145. Characterization of the DNA-binding activity of the recombinant fusion protein by gel mobility-shift assay revealed that it had a preference for a run of pyrimidines on one strand. Deletion analysis using recombinant proteins revealed that the DNA ligase-like domain was required for DNA-binding activity. The finding that the region required for the binding of murine A1 p145 to DNA has similarity to a domain found in DNA ligases suggests that this region may be utilized by both proteins in recognizing DNA. 相似文献
11.
Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells. 总被引:3,自引:4,他引:3
M Chen Z Q Pan J Hurwitz 《Proceedings of the National Academy of Sciences of the United States of America》1992,89(12):5211-5215
The elongation of primed DNA templates by DNA polymerase delta and DNA polymerase epsilon requires the action of two accessory proteins, proliferating cell nuclear antigen and activator 1 (A1, also called replication factor C). A1 is an enzyme that contains five different subunits (145, 40, 38, 37, and 36.5 kDa). In this paper, we describe the isolation of the gene encoding the 37-kDa subunit from HeLa cells. This gene was cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence shows a high degree of homology to the 40-kDa subunit of A1; they both contain the identical ATP-binding motif, but in contrast to the bacterial expressed 40-kDa protein, the 37-kDa expressed protein did not bind ATP. Both the 37- and 40-kDa proteins share substantial homology with the phage T4 gene 44 protein and to a lesser extent with the tau and gamma subunits of the E. coli DNA polymerase III holoenzyme. Polyclonal antibodies against the bacterially expressed 37- and 40-kDa proteins do not crossreact and are specific in their interaction. Antibodies against the 37-kDa protein maximally inhibited (by 50%) the A1-dependent synthesis of DNA by DNA polymerase delta; antibodies against the 40-kDa protein quantitatively inhibited the same reaction. When A1-dependent synthesis of DNA was partially inhibited by antibodies against the 40-kDa subunit, the addition of antibodies against the 37-kDa subunit inhibited DNA synthesis to a greater extent than the anti-37-kDa antibody alone. These results suggest that both the 37- and 40-kDa subunits of A1 are required for the biological role of A1 and that they may function differently in this process. 相似文献
12.
p21 ras proteins and guanine nucleotides modulate the phosphorylation of 36- and 17-kilodalton mitochondria-associated proteins. 总被引:1,自引:1,他引:1
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J M Backer I B Weinstein 《Proceedings of the National Academy of Sciences of the United States of America》1986,83(17):6357-6361
We have found that, when isolated rat liver mitochondria are incubated with [gamma-32P]ATP, there is phosphorylation of 36- and 17-kDa proteins. These proteins together with their protein kinase(s) are released as a complex by incubation of the isolated rat liver mitochondria at 20 degrees C for 30 min with 10 mM glucose 6-phosphate, 0.5 mM inositol phosphate, or 0.01 mM inositol triphosphate. Phosphorylation of the 36- and 17-kDa proteins in this soluble protein fraction is modulated by p21 proteins encoded by ras oncogenes and synthesized in Escherichia coli via recombinant DNA methods. A normal p21 ras protein stimulates phosphorylation of the 36-kDa protein and inhibits phosphorylation of the 17-kDa protein, whereas two transforming p21 ras proteins inhibit phosphorylation of both the 36- and 17-kDa proteins. Although GDP and 5'-guanylyl imidodiphosphate also influence the phosphorylation of these proteins, we present evidence that the effects of p21 ras protein are not simply due to their bound GDP. This novel system may be useful for further studies on the biochemical functions of the p21 ras proteins. 相似文献
13.
The phosphatidylinositol-3 kinase (PI3K) pathway plays a central role in regulating numerous biologic processes, including survival, adhesion, migration, metabolic activity, proliferation, differentiation, and end cell activation through the generation of the potent second messenger PI-3,4,5-trisphosphate (PI-3,4,5-P(3)). To ensure that activation of this pathway is appropriately suppressed/terminated, the ubiquitously expressed 54-kDa tumor suppressor PTEN hydrolyzes PI-3,4,5-P(3) to PI-4,5-P(2), whereas the 145-kDa hematopoietic-restricted SH2-containing inositol 5'-phosphatase SHIP (also known as SHIP1), the 104-kDa stem cell-restricted SHIP sSHIP, and the more widely expressed 150-kDa SHIP2 break it down to PI-3,4-P(2). In this review, we focus on the properties of these phospholipid phosphatases and summarize recent data showing that the activities of these negative regulators often are modulated by simply altering their protein levels. We also highlight the critical role that SHIP plays in lipopolysaccharide-induced macrophage activation and in endotoxin tolerance. 相似文献
14.
Subunits of purified calcium channels: a 212-kDa form of alpha 1 and partial amino acid sequence of a phosphorylation site of an independent beta subunit. 总被引:4,自引:4,他引:4
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K S De Jongh D K Merrick W A Catterall 《Proceedings of the National Academy of Sciences of the United States of America》1989,86(21):8585-8589
Antibodies prepared against peptides CP2, CP4, and CP5, which occur within the first 1522 amino acid residues of the alpha 1 subunit of dihydropyridine-sensitive skeletal muscle calcium channels, specifically recognized a 175-kDa form of the alpha 1 subunit in immunoblots and immunoprecipitation experiments. In contrast, antibodies prepared against peptide CP1, which represents the C-terminal 18 amino acid residues predicted by cloning and sequence analysis of the alpha 1 subunit, recognized a minor, previously undescribed 212-kDa protein, which is the size predicted for the full length of the alpha 1 subunit from cDNA cloning [Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1987) Nature (London) 328, 313-318]. Both the 175-kDa and 212-kDa forms were phosphorylated by cAMP-dependent protein kinase and both were present in isolated transverse tubule membranes. The 175-kDa form may arise from posttranslational proteolytic cleavage of the C terminus of the 212-kDa form of the alpha 1 subunit predicted by cDNA cloning and sequence analysis. Partial amino acid sequencing of the 54-kDa beta subunit of the calcium channel indicated this protein was not derived from the proteolytically cleaved C terminus of the alpha 1 subunit. This analysis identified a threonine residue in the sequence (Lys/Arg)-Arg-Pro-Thr-Pro of the beta subunit that was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of this residue in the beta subunit may play a role in modulation of calcium channel function. Separate functional roles of the 175-kDa form of the alpha 1 subunit in excitation-contraction coupling and of the 212-kDa form in ion conductance are proposed. 相似文献
15.
16.
Interleukin-3 down-regulates its own receptor 总被引:4,自引:0,他引:4
To gain insight into the mechanisms involved in regulating murine interleukin-3 (mIL-3) receptor expression, we have examined the effects of mIL-3 and murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) on mIL-3 receptor internalization and re-expression and studied the relationship between mIL-3 cell surface receptor density and growth factor sensitivity. As a source of cells for our studies, we used a B6SUtA clone, B6SUtA1, which grows equally well in mIL-3 or mGM- CSF when supplemented with 20% fetal calf serum (FCS) in RPMI 1640. Intracellular processing studies carried out in the presence and absence of methylamine suggested that mIL-3 is cleaved at two specific sites before its complete digestion within lysosomes. However, unlike its ligand, cycloheximide studies indicated that internalized mIL-3 receptors are recycled to the cell surface. When B6SUtA1 cells were continuously passaged in mIL-3, cell populations allowed to exhaust the mIL-3 in the medium (high density cells) expressed more than ten times (ie, approximately 100,000/cell) the mIL-3 receptor number of those growing exponentially at low cell concentrations (low density cells). Since the high density cells were no larger than the low density cells, the marked increase in mIL-3 receptor number per cell reflects a true up-regulation of receptor expression. A kinetic analysis of this up- regulation revealed that it begins within one hour of mIL-3 exhaustion. Moreover, proliferation assays with these two cell populations, using 3H-thymidine incorporation, suggested that the high density cells were 30-fold more responsive to mIL-3. However, when B6SUtA1 cells were passaged in mGM-CSF, there was no difference in mIL-3 receptor number between high density and low density cells (ie, approximately 100,000/cell). Identical studies carried out with another mIL-3 dependent cell line, 32D C3, demonstrated that this phenomenon was not unique to B6SUtA1 cells. 相似文献
17.
Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases 总被引:1,自引:0,他引:1
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Stevenson-Paulik J Bastidas RJ Chiou ST Frye RA York JD 《Proceedings of the National Academy of Sciences of the United States of America》2005,102(35):12612-12617
Phytate (inositol hexakisphosphate, IP6) is a regulator of intracellular signaling, a highly abundant animal antinutrient, and a phosphate store in plant seeds. Here, we report a requirement for inositol polyphosphate kinases, AtIPK1 and AtIPK2beta, for the later steps of phytate synthesis in Arabidopsis thaliana. Coincident disruption of these kinases nearly ablates seed phytate without accumulation of phytate precursors, increases seed-free phosphate by 10-fold, and has normal seed yield. Additionally, we find a requirement for inositol tetrakisphosphate (IP4)/inositol pentakisphosphate (IP5) 2-kinase activity in phosphate sensing and root hair elongation. Our results define a commercially viable strategy for the genetic engineering of phytate-free grain and provide insights into the role of inositol polyphosphate kinases in phosphate signaling biology. 相似文献
18.
Inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate receptor proteins: isolation and characterization from rat brain. 总被引:2,自引:2,他引:2
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A B Theibert V A Estevez C D Ferris S K Danoff R K Barrow G D Prestwich S H Snyder 《Proceedings of the National Academy of Sciences of the United States of America》1991,88(8):3165-3169
High-affinity, membrane-associated inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6) binding proteins were solubilized and isolated utilizing a heparin-agarose resin followed by an IP4 affinity resin. The IP6 receptor comprises a protein complex of 115-, 105-, and 50-kDa subunits, all of which comigrate under native conditions. The Kd of the receptor for IP6 is 12 nM, whereas inositol 1,3,4,5,6-pentakisphosphate (IP5), IP4, and inositol 1,4,5-trisphosphate (IP3) are 50%, 30%, and 15%, respectively, as potent. Two protein complexes copurify with the IP4 receptor fraction. A 182/123-kDa complex elutes first from the affinity column followed by a 174/84-kDa protein complex, which elutes at higher salt. Both complexes show high affinity for IP4 (Kd = 3-4 nM). IP5, IP6, and IP3 display approximately 25%, 10%, and 0.1%, respectively, the affinity of IP4. Ligand binding to IP6 and IP4 receptors is inhibited 50% by heparin at 0.1 microgram/ml. IP4 receptor proteins are stoichiometrically phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, whereas negligible phosphorylation is observed for the IP6 receptor. 相似文献
19.
J M Huibregtse M Scheffner S Beaudenon P M Howley 《Proceedings of the National Academy of Sciences of the United States of America》1995,92(7):2563-2567
E6-AP is a 100-kDa cellular protein that interacts with the E6 protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor protein for ubiquitin-mediated proteolysis. E6-AP is an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of protein sequences over an approximately 350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of ubiquitin thioester formation. Two of the E6-AP-related proteins, a rat 100-kDa protein and a yeast 95-kDa protein (RSP5), both of previously unknown function, are shown here to form thioesters with ubiquitin. Mutation of the conserved cysteine residue of these proteins destroys their ability to accept ubiquitin. These data strongly suggest that the rat 100-kDa protein and RSP5, as well as the other E6-AP-related proteins, belong to a class of functionally related E3 ubiquitin-protein ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain). 相似文献
20.
cDNA and amino acid sequences of the cell adhesion protein receptor recognizing vitronectin reveal a transmembrane domain and homologies with other adhesion protein receptors. 总被引:36,自引:2,他引:36
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S Suzuki W S Argraves R Pytela H Arai T Krusius M D Pierschbacher E Ruoslahti 《Proceedings of the National Academy of Sciences of the United States of America》1986,83(22):8614-8618