Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P67775 (alpha isoform)
 797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By virtue of their capacity to bind plasminogen activators and plasminogen, to accelerate plasminogen activation and to protect bound plasmin from inactivation by alpha 2 antiplasmin, cells can harness the broad proteolytic activity of plasmin to their surface. Most cells bind plasminogen with a very high capacity, a relatively low affinity (Kd approximately 1 microM) and recognize the lysine binding sites of the molecule. Gangliosides serve as non-protein plasminogen binding sites, and a subset of membrane proteins with carboxy-terminal lysine residues also serve as receptors. The alpha isoform of enolase possesses a carboxy-terminal lysine and is a prominent plasminogen binding protein of cells. Cells of the monocytoid lineage, including peripheral blood monocytes, can markedly upregulate their expression of plasminogen receptors. The capacity to modulate expression of receptors for fibrinolytic components establishes an additional mechanism by which the cell-surface regulates the function of the plasminogen system.
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PMID:Cellular regulation of fibrinolysis. 165 42

Mouse L929 cells were used to study the mechanism of cAMP induction of alkaline phosphatase (AP) activity. Following treatment with 200 microM 8-chlorophenylthio-cAMP (CPT-cAMP), alkaline phosphatase enzyme activity was observed to increase 80-fold after 24 h. The CPT-cAMP dose response of the alkaline phosphatase enzyme activity correlated well with the CPT-cAMP activation of cAMP-dependent protein kinase in L cells. A cDNA clone for the alkaline phosphatase was isolated and used to demonstrate a 10-fold increase in alkaline phosphatase mRNA levels after a 24-h treatment of L cells with CPT-cAMP. Increased mRNA levels were first detected 4-6 h, after CPT-cAMP treatment, and the level of alkaline phosphatase mRNA decreased rapidly after removal of CPT-cAMP. In vitro nuclear transcription studies showed that a 3-fold increase in alkaline phosphatase gene transcription was detectable 6 h after CPT treatment, and this increase was blocked by cycloheximide. In order to determine if the catalytic (C) subunit of cAMP-dependent protein kinase was able to mediate the induction of AP, L cells were transfected with expression vectors containing the metallothionein promoter and coding for the C alpha isoform of the catalytic subunit of cAMP-dependent protein kinase or for a catalytic subunit in which lysine 72 had been mutated to methionine (C alpha K72M). Zinc treatment of stably transfected cells expressing the wild-type C subunit showed an increase in protein kinase activity and an increase in AP activity. Zinc treatment of cells containing the mutant C subunit expression vector produced an increase in the amount of a protein which was recognized by C subunit antibodies on Western blots, but these cells showed no increase in protein kinase activity or in AP activity. We conclude that the C subunit is sufficient for transcriptional induction of the AP gene and that the phosphotransferase activity of the C subunit is required for this induction.
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PMID:Induction of alkaline phosphatase in mouse L cells by overexpression of the catalytic subunit of cAMP-dependent protein kinase. 216 96

Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.
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PMID:Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain. 302 70

The alpha isoform of enolase is a candidate plasminogen receptor on U937 monocytoid cells [Miles, L. A., Dahlberg, C. L., Plescia, J., Felez, J., Kato, K. & Plow, E. F. (1991) Biochemistry 30, 1682-1691]. In the present study, an enolase-related molecule was detected on the surfaces of peripheral blood monocytes and neutrophils by fluorescence-activated cell sorting. A mRNA transcript encoding a unique membrane form of an enolase-related molecule was not detected by Northern-blotting and primer-extension analyses, consistent with the cell-surface protein being authentic alpha-enolase. Both the alpha and beta isoforms of purified enolase, bound plasminogen with an affinity similar to that of the cell surface. Moreover, immunopurified alpha-enolase enhanced plasminogen activation by tissue plasminogen activator and blocked the binding of plasminogen to alpha 2-antiplasmin, mimicking functions arising from the association of plasminogen with cells. The interaction of the enolase isoforms with plasminogen was dependent upon recognition of the C-terminal lysyl residue of the enolases by the lysine-binding sites of plasminogen, as the interaction was blocked by (a) peptides with C-terminal lysine residues and (b) an antibody to the C-terminal aspect of enolase. A monoclonal antibody was developed, characterized and utilized to quantify the enolase molecules present on the surface of U937 cells. A substantial number of molecules, 1.8 x 10(6)/cell, was present, accounting for approximately 10% of the plasminogen-binding capacity of these cells. These studies clearly establish the role of enolase as a cell-surface plasminogen-binding site with profibrinolytic functions.
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PMID:The role of an enolase-related molecule in plasminogen binding to cells. 785 15

Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4, 5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5Kgamma) and the characterization of members of the type I PIP5K family. Type I PIP5Kgamma has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the alpha and beta isoforms. Northern blot analysis revealed that the gamma isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the beta isoform has the greatest Vmax value for the PtdIns(4)P kinase activity and the gamma isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the beta and gamma isoforms were found to confer the greatest Vmax value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the alpha isoform is essential for the PtdIns(4)P kinase activity. As was the case with the alpha isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581), overexpression of either the beta or the gamma isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.
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PMID:Type I phosphatidylinositol-4-phosphate 5-kinases. Cloning of the third isoform and deletion/substitution analysis of members of this novel lipid kinase family. 953 51

The activity and allosteric properties of plant phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved serine residue near the N-terminus. This up/down-regulation of PEPC is catalyzed by a dedicated and highly regulated serine/threonine (Ser/Thr) kinase (PEPC-kinase) and an opposing type-2A Ser/Thr phosphatase (PP2A). In marked contrast to PEPC-kinase, the PP2A holoenzyme from photosynthetic tissue has been virtually unstudied to date. In the present investigation, we have partially purified and characterized the native form of this PP2A from illuminated leaves of maize (Zea mays L.), a C4 plant, using maize [32P]PEPC as substrate. Various conventional chromatographic matrices, together with thiophosphorylated C4 PEPC-peptide and microcystin-LR affinity-supports, were exploited for the enrichment of this PP2A from soluble leaf extracts. Biochemical and immunological results indicate that the C4-leaf holoenzyme is analogous to other eukaryotic PP2As in being a approximately 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately 38- and approximately 65-kDa subunits, respectively) complexed with a putative, approximately 74-kDa B-type regulatory/targeting subunit. This heterotrimer lacks any strict substrate specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro. This activity is independent of free Me2+, insensitive to levamisole and the Inhibitor-2 protein that targets PP1, activated by several polycations such as protamine and poly-L-lysine, and highly sensitive to inhibition by microcystin-LR and okadaic acid (IC50 approximately 30 pM), all of which are diagnostic features of yeast and mammalian PP2As. In addition, this C4-leaf PP2A holoenzyme (i) is inhibited in vitro by physiological concentrations of certain C4 PEPC-related metabolites (L-malate, PEP, glucose 6-phosphate, but not the activator glycine) when either 32P-labeled maize PEPC or rabbit muscle phosphorylase a is used as substrate, suggesting a direct effect on this Ser/Thr phosphatase; and (ii) displays, at best, only modest light/dark effects in vivo on its apparent molecular mass, component core subunits and activity against C4 PEPC, in marked contrast to the opposing activity of PEPC-kinase in C4 and Crassulacean acid metabolism leaves. This report represents one of the few studies of a heteromeric PP2A holoenzyme from photosynthetic tissue that dephosphorylates a known target enzyme in plants, such as PEPC, sucrose-phosphate synthase or nitrate reductase.
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PMID:Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosophoenolpyruvate carboxylase. 1150 60

The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr(886) and Lys(890) of the PI3Kgamma isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the beta isoform (Glu(858) and Asp(862)) and those in the alpha isoform (His(855) and Gln(859)) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of beta-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the alpha-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kbeta activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the alpha-isoform-selectivity of the compounds studied.
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PMID:Dissecting isoform selectivity of PI3K inhibitors: the role of non-conserved residues in the catalytic pocket. 1848 60

Antithrombin III (ATIII) is the main inhibitor of the coagulation proteases like factor Xa and thrombin. Anticoagulant activity of ATIII is increased by several thousand folds when activated by vascular wall heparan sulfate proteoglycans (HSPGs) and pharmaceutical heparins. ATIII isoforms in human plasma, alpha-ATIII and beta-ATIII differ in the amount of glycosylation which is the basis of differences in their heparin binding affinity and function. Crystal structures and site directed mutagenesis studies have mapped the heparin binding site in ATIII, however the hydrogen bond switch and energetics of interaction during the course of heparin dependent conformational change remains largely unclear. An analysis of heparin bound conformational states of ATIII using PEARLS software showed that in heparin bound intermediate state, Arg 47 and Arg 13 residues make hydrogen bonds with heparin but in the activated conformation Lys 11 and Lys 114 have more hydrogen bond interactions. In the protease bound-antithrombin-pentasaccharide complex Lys 114, Pro 12 and Lys 125 form important hydrogen bonding interactions. The results showed that A-helix and N-terminal end residues are more important in the initial interactions but D-helix is more important during the latter stage of conformational activation and during the process of protease inhibition. We carried out the residue wise Accessible Surface Area (ASA) analysis of alpha and beta ATIII native states and the results indicated major differences in burial of residues from Ser 112 to Ser 116 towards the N-terminal end. This region is involved in the P-helix formation on account of heparin binding. A cavity analysis showed a progressively larger cavity formation during activation in the region just adjacent to the heparin binding site towards the C-terminal end. We hypothesize that during the process of conformational change after heparin binding beta form of antithrombin has low energy barrier to form D-helix extension toward N and C-terminal end as compared to alpha isoform.
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PMID:Energetics of hydrogen bond switch, residue burial and cavity analysis reveals molecular basis of improved heparin binding to antithrombin. 2187 53

WNK1 (with no lysine [K] kinase 1) is an atypical kinase protein ubiquitously expressed in humans and mice. A mutation in its encoding gene causes hypertension in humans, which is associated with abnormal ion homeostasis. WNK1 is critical for in vitro decidualization in human endometrial stromal cells, thereby demonstrating its importance in female reproduction. Using a mouse model, WNK1 was ablated in the female reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine morphology, causing endometrial epithelial hyperplasia, adenomyotic features, and a delay in embryo implantation, ultimately resulting in compromised fertility. Combining transcriptomic, proteomic, and interactomic analyses revealed a potentially novel regulatory pathway whereby WNK1 represses AKT phosphorylation through protein phosphatase 2A (PP2A) in endometrial cells from both humans and mice. We show that WNK1 interacted with PPP2R1A, the alpha isoform of the PP2A scaffold subunit. This maintained the levels of PP2A subunits and stabilized its activity, which then dephosphorylated AKT. Therefore, loss of WNK1 reduced PP2A activity, causing AKT hypersignaling. Using FOXO1 as a readout of AKT activity, we demonstrate that there was escalated FOXO1 phosphorylation and nuclear exclusion, leading to a disruption in the expression of genes that are crucial for embryo implantation.
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PMID:WNK1 regulates uterine homeostasis and its ability to support pregnancy. 3304 43