EC:3.4.21.69 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a central role in virus assembly by binding the nucleocapsid core to the viral envelope during the budding process. A small percentage of M protein molecules are phosphorylated in vivo, but the role of phosphorylation in M protein function is unknown. Using limited proteolysis, we previously determined the sites of in vivo phosphorylation for VSV M protein to be Thr 31 (and possibly Ser 32) and a site N-terminal to position 19 (Ser 2, Ser 3, or Ser 17) (P. E. Kaptur et al., J. Virol. 66, 5384-5392, 1992). M protein mutants were constructed using site-directed mutagenesis by substituting Ala for Ser or Thr at these sites in the M gene of the San Juan strain of VSV. One mutant had substitutions at the major in vivo phosphorylation site(s) at positions 31 and 32 (M31.32) while two others had additional substitutions at positions 2 and 3 (M2.3.31.32) or at position 17 (M17.31.32). Mutant M proteins were expressed in BHK cells using the vaccinia/T7 system, radiolabeled with 32Pi, and then analyzed for 32P content by PAGE and autoradiography. The data show that the site of phosphorylation near the N-terminus is at Ser 2 or 3 and not Ser 17. Further, Ser 38 was not phosphorylated. Mutation of the major phosphorylation site enhanced phosphorylation at alternative sites in the M protein C-terminal to amino acid 43 and at Ser residues 2 and 3. Mutant M proteins were tested for their ability to complement growth of the temperature-sensitive M protein mutant virus tsO23 at the nonpermissive temperature. Mutant M2.3.31.32 was further tested for its ability to assemble into VSV-defective interfering (DI) particles, using a replication system in which the DI genome and all five VSV proteins were expressed from plasmid DNA. Assembly of tsO23 virions or DI particles in the presence of mutant M proteins was similar to that observed for wild-type M proteins. These data indicate that phosphorylation of M protein at the major in vivo sites is not essential for virus assembly.
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PMID:Assembly functions of vesicular stomatitis virus matrix protein are not disrupted by mutations at major sites of phosphorylation. 785 2

The molecular basis of protein C deficiency was studied in three type I and three type II heterozygotes. Three probands showed thrombotic complications. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining by the polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis, and DNA sequencing of the PCR-amplified fragments. Six missense mutations were identified, including three novel ones. One was located in exon II, in which the initiating translation codon (ATG) encoding for Met at position -42 was replaced by ACG encoding for Thr. The other five were located in exon IX, and included TAC(Tyr399)-->CAC(His), CCG(Pro327)-->CTG(Leu), GAC(Asp359)-->AAC(Asn) in two cases, and GGG(Gly350)-->AGG(Arg). Four of the six missense mutations occurred in CG dinucleotide. Sequence analysis of the other exons excluded additional mutations. By restriction enzyme analysis, co-segregation of the mutation with protein C deficiency was observed in four families. The other two mutations at amino acid positions -42 and 350 were also considered to be associated with protein C deficiency due to the absence of these mutations in 50 normal individuals. A structural model of the protease domain of mutant activated protein C was constructed by the chimeric modelling method, and the resultant model suggested conformational changes due to each missense mutation identified in protein C deficiency. The present data also provide some evidence regarding the genetic heterogeneity of protein C deficiency.
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PMID:Six missense mutations associated with type I and type II protein C deficiency and implications obtained from molecular modelling. 786 74

The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor-derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC-10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm2), PC-10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC-10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC-10. The APC-10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By 125I-EGF binding analysis and [35S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC-10 was not decreased relative to PC-10. No difference between PC-10 and APC-10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC-10 was 8-10-fold lower than that of PC-10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC-10 and APC-10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC-10 cells was consistently 2-3-fold lower than that seen in PC-10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity.
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PMID:Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity. 810 61

Thrombomodulin is an anticoagulant protein cofactor that modulates the substrate specificity of thrombin and promotes the cleavage of protein C. The structure-function relationships of the thrombin-thrombomodulin interaction have been explored by recombinant DNA and protein chemistry methods. Thrombomodulin binds to thrombin at an anion-binding exosite on the carboxyl-terminal side of the substrate binding cleft. This interaction interferes with the recognition and cleavage of fibrinogen, factor V, and the platelet thrombin receptor. Binding to thrombomodulin also protects thrombin from inhibition by heparin cofactor II. The major thrombin binding site on thrombomodulin consists of EGF-like domains 5 and 6. In addition, EGF-like domain 4 is required for thrombomodulin to accelerate the activation of protein C. Some thrombomodulin molecules contain a chondroitin sulfate moiety attached to a Ser/Thr-rich domain adjacent to the cell membrane. This modification is not required for the cofactor activity of thrombomodulin, but appears to contribute to 'direct anticoagulant' activity--the ability of thrombomodulin to inhibit fibrinogen clotting, factor V activation, and platelet activation. The chondroitin sulfate moiety of thrombomodulin also can affect the rate of thrombin inhibition by antithrombin III, possibly by competing with heparin for the heparin binding site on thrombin. Detailed understanding of these interactions could lead to new strategies for the treatment of bleeding or thrombotic disorders.
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PMID:Structure-function relationships of the thrombin-thrombomodulin interaction. 838 51

I have isolated glucose-6-phosphate dehydrogenase from rabbit liver microsomes and determined its complete amino acid sequence. Sequence determination was achieved by automated Edman degradation of peptides generated by chemical and enzymatic cleavages. The microsomal enzyme consists of 763 residues and is quite dissimilar from the previously characterized cytosolic enzymes. The N terminus of the microsomal enzyme is blocked by a pyroglutamyl residue. Carbohydrate is attached at Asn-138 and Asn-263, implying that the bulk of the protein is oriented on the lumenal side of the endoplasmic membrane. The amino acid sequence of the microsomal protein shows limited homology to the extensively sequenced cytosolic glucose-6-phosphate dehydrogenases. Clusters of up to six identical residues can be identified in four regions: peptide segments at residues 10-21, 154-163, and 173-261. In addition, another array of identical residues, requiring a 100-residue deletion in the sequence of the microsomal enzyme, spans residues 436-462 and corresponds to residues 348-373 of the cytosolic protein. Two segments with a Gly-Xaa-Gly-Xaa-Xaa-Gly motif, related to a coenzyme binding fold, were identified at Gly-399 and Gly-491. In the cytosolic enzymes, a variation of this sequence motif occurs at Gly-37 and Gly-241. The 300-residue C-terminal segment of the microsomal enzyme is unique and has no counterpart in the cytosolic or the bacterial enzymes. An unexpected finding with regard to the microsomal enzyme is that it lacks an identifiable membrane-spanning region or the lumenal-protein C-terminal consensus sequences Lys-Asp-Glu or His-Ile/Thr-Glu-Leu. Thus, the mode of transport and retention of this protein in the lumen of endoplasmic reticulum remains to be determined.
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PMID:Isolation and the complete amino acid sequence of lumenal endoplasmic reticulum glucose-6-phosphate dehydrogenase. 850 77

The APC gene is mutated in familial adenomatous polyposis and sporadic colorectal tumors. The product of this gene is a 300 kDa cytoplasmic protein and its overexpression results in the block of cell cycle progression from the G0/G1 to the S phase. In the present study, we studied the expression and phosphorylation of the APC protein through the cell cycle. The APC protein was found to be constantly expressed and phosphorylated at serine and threonine residues. Moreover, the APC protein immunoprecipitated from cells arrested in the M phase by nocodazole treatment migrated in SDS-PAGE more slowly than those from the G1 and S phases. Phosphatase treatment abolished this M phase-specific retarded migration, suggesting that APC is transiently hyperphosphorylated in the M phase.
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PMID:The tumor suppressor gene product APC is hyperphosphorylated during the M phase. 860 42

The importance of the P2 residue in determining serpin specificity was examined by making a series of substitutions in the P2 position of recombinant alpha 1-antichymotrypsin that contained an arginine P1 residue. The importance of the P2 residue in governing the association rate constant (Kon) of the serpin varied with the protease examined. For trypsin, the P2 residue played a relatively minor role, whereas the nature of this residue markedly influenced the rates of inhibition of thrombin, factor Xa, and APC. A 1000-fold difference in Kon values was observed between the fastest (P2 proline) and the slowest (P2 threonine) inhibitors of thrombin. Similar differences were observed with factor Xa; the best inhibitor (P2 glycine) displayed a 200-fold higher Kon value than the poorest (P2 threonine). The nature of the P2 residue also affected whether the interaction of the serpin with the protease resulted in inhibition of the protease or cleavage of the serpin; a P2 proline residue increased the rate of cleavage of alpha 1-antichymotrypsin by trypsin. By using mutants of thrombin, it was possible to show that the B-insertion loop, which partially occludes the active site, is important in determining the P2 specificity of this enzyme. Deletion of three amino acids from this loop yielded a protease (des-PPW) that became more like trypsin in its specificity. In addition, it was shown that Glu192 dramatically restricts thrombin's ability to accommodate a threonine in the P2 position. Taken together, the results demonstrated the importance of complementary interactions between the P2 residue of the serpin and the S2 binding site of the protease in regulating the specific interaction between serpin and protease.
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PMID:Role of the P2 residue in determining the specificity of serpins. 878 2

It is thought that only a limited number of residues in the extended binding pocket of coagulation proteases are critical for substrate and inhibitor specificity. A candidate residue from the crystal structures of thrombin and factor Xa (FXa) that may be critical for specificity at the S2 subsite is residue 99. Residue 99 is Tyr in FXa and Thr in activated protein C (APC). To determine the role of residue 99 in S2 specificity, a Gla-domainless mutant of protein C (GDPC) was prepared in which Thr99 was replaced with Tyr of FXa. GDPC T99Y bound Ca2+ and was activated by the thrombin-thrombomodulin complex normally. The T99Y mutant, similar to FXa, hydrolyzed the chromogenic substrates with a Gly at the P2 positions. This mutant was also inhibited by antithrombin (AT) (k2 = 4.2 +/- 0.2 x 10(1) M-1 s-1), and heparin accelerated the reaction >350-fold (k2 = 1.5 +/- 0.1 x 10(4) M-1 s-1). The T99Y mutant, however, did not activate prothrombin but inactivated factor Va approximately 2-fold better than wild type. To try to switch the specificity of FXa, both Tyr99 and Gln192 of FXa were replaced with those of APC in the Gla-domainless factor X (GDFX Y99T/Q192E). This mutant was folded correctly as it bound Ca2+ with a similar affinity as GDFX and was also activated by the Russell's viper venom at similar rate, but it cleaved the chromogenic substrates with a Gly at the P2 positions poorly. The mutant, instead, cleaved the APC-specific chromogenic substrates efficiently. The Y99T/Q192E mutant became resistant to inhibition by AT in the absence of heparin but was inhibited by AT almost normally in the presence of heparin (k2 = 3.4 +/- 0.5 x 10(5) M-1 s-1). The Y99T/Q192E mutant did not inactivate factor Va, and prothrombin activation by this mutant was impaired. These results indicate that 1) residue 99 is critical for enzyme specificity at the S2 subsite, 2) a role for heparin in acceleration of FXa inhibition by AT may involve the S2-P2 modulation, and 3) the exchange of residues 99 and 192 in FXa and APC may switch the enzyme specificity with the chromogenic substrates and inhibitors but not with the natural substrates.
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PMID:Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity. 879 9

About 30% of human plasma protein C (PC) is of lower molecular weight than the predominant alpha-form. The minor beta-form arises as a consequence of the lack of glycosylation at Asn329. Although the functional role of Asn329 has been investigated by in vitro mutagenesis, until now no naturally occurring mutations have been reported at this site. We describe here the case of two identical twin sisters compound heterozygous for two novel PC mutations: Cys78-->Stop inherited from the maternal side and Asn329-->Thr inherited from the paternal side, associated with the presence of only the beta-form of PC in plasma. The Cys78-->Stop substitution is predicted to abolish PC synthesis from one allele, whereas the Asn329-->Thr substitution results in the reduced synthesis of a beta PC variant with decreased functional activity. PCN329T from the two monovular twin sisters was purified and its active form APCN329T was assessed for its ability to inactivate factor Va. Whereas no differences were observed between the activation rates of normal PC and PCN329T, APCN329T inactivated human factor Va with a rate slower than the normal APC. This is the first report of a PC defect involving glycosylation of the molecule. This defect results in the presence of only the beta-form of PC in human plasma and is responsible for the reduced anticoagulant activity observed.
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PMID:Compound heterozygous protein C deficiency resulting in the presence of only the beta-form of protein C in plasma. 882 29

Human protein S (HPS) is a vitamin K dependent plasma glycoprotein involved in the regulation of activated protein C and possibly fibrinolysis. Its c-DNA sequence shows three N-glycosylation consensus sequences (Asn-X-Ser/Thr). In order to study influence of N-linked glycosylation on HPS function, set of mutants of HPS was constructed. Mutants were generated, starting from an SV40/Adeno derived pD5HPS2 expression vector, using PCR enabled, site specific methodology. They included single amino acid substitutions at each of three N-glycosylation consensus sequences: Asn458-->Gln, Ser460-->Gly, Asn468-->Gln, Thr470-->Gly, Asn489-->Gln, Thr491-->Gly. Variant HPS were expressed in stable 293 human kidney cell lines in the presence of vitamin K1 (we did not succeed in expressing variant Asn489-->Gln) and purified from conditioned media using pseudoaffinity chromatography on QAE-Sepharose. Variant Asn468-->Gln showed decreased gamma-carboxyglutamate content. All of the mutants were active in a clotting type assay based on factor Va inactivation, and they were compared to wt-HPS and plasma HPS. In conclusion, we have constructed, expressed and purified set of HPS mutants useful in studying the role of N-glycosylation in HPS function.
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PMID:Construction, expression and preliminary characterization of glycosylation mutants of human protein S. 911 51

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