Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: CAS:1783-96-6 (aspartic acid)
 12,748 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism by which cAMP-dependent protein kinase-catalyzed phosphorylation modulates the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was examined after site-specific mutation of the cAMP-dependent phosphorylation site (Ser32) to aspartic acid or alanine. The mutant and wild-type enzymes were overexpressed in Escherichia coli in a rich medium to levels as high as 30 mg/liter and were then purified to homogeneity. The kinetic properties of the Ser32-Ala mutant were identical with the dephosphorylated wild-type bifunctional enzyme. Mutation of Ser32 to aspartic acid mimicked several effects of cAMP-dependent phosphorylation: there was an increase in the Km for fructose 6-phosphate for 6-phosphofructo-2-kinase and an increase in the maximal velocity of fructose-2,6-bisphosphatase. Fructose-2,6-bisphosphatase activity of the Ser32-Asp mutant was 75% that of the phosphorylated wild-type enzyme, the mutant's kinase reaction had an identical dependence on fructose 6-phosphate, while its maximum velocity was only 60% that of the phosphorylated wild-type enzyme over a wide pH range. Furthermore, catalytic subunit-catalyzed in vitro phosphorylation of the Ser32-Ala mutant on Ser33 increased the Km for fructose 6-phosphate by 4-fold for the 6-phosphofructo-2-kinase. The results support the hypothesis that Ser32 is an important residue in the regulation of the activities of the bifunctional enzyme and that phosphorylation of Ser32 can be functionally substituted by aspartic acid. The results suggest a role for negative charge in the effect of phosphorylation.
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PMID:Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis. 133 50

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.
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PMID:Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase. 142 Mar 67

In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.
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PMID:Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts. 207 Aug 29

The traffic of ions through the kainic acid (KA) receptor/channels present on chick cerebellar glia is modulated by intracellular events likely to involve phosphorylation reactions. The protein carrying the KA binding sites has been isolated from chick cerebellum and shown to be composed of a 49-kDa polypeptide. Its primary structure, established via cDNA cloning, shows the presence of two putative phosphorylation sites. We report here that this 49-kDa polypeptide is a substrate of the cAMP-dependent protein kinase which catalyzes the incorporation of up to 2 mol of phosphate/mol of KA binding site. KA prevents this phosphorylation reaction in a concentration range similar to that needed to activate the KA receptor/channels but higher by 3 orders of magnitude than that needed to saturate the KA binding sites. Kainatergic ligands produce similar effects to those of KA, but 1 mM N-methyl-D-aspartic acid and 1 mM quisqualic acid have no effect. However, 0.01 mM quisqualic acid prevents the inhibitory action of KA. These results raise the possibility that the phosphorylation of KA receptor/channels in their cellular environment is negatively regulated by KA.
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PMID:Phosphorylation of the 49-kDa putative subunit of the chick cerebellar kainate receptor and its regulation by kainatergic ligands. 217 71

cDNAs coding for a plasma membrane Ca2+ pump were isolated from a human teratoma library and sequenced. The translated sequence contained 1,220 amino acids with a calculated molecular weight of 134,683. All regions of functional importance known from other ion-transporting ATPases could be identified. The translated sequence also contained, near the carboxyl terminus, the calmodulin-binding domain and two domains which are very rich in glutamic acid and aspartic acid. These two domains resemble calmodulin somewhat and one of them may play a role in the binding of Ca2+. The enzyme also contains domains rich in serine and threonine, one of which has a sequence matching those of good cAMP-dependent protein kinase substrates. The carboxyl-terminal region is important for regulation by calmodulin, proteolysis, and phosphorylation. Near the amino terminus are two domains which are very rich in lysine and glutamic acid, as well as two domains resembling EF hands, one of which also has some resemblance to calmodulin. Comparison of the cloned sequence with peptide sequences from the erythrocyte Ca2+ pump showed that the two proteins have a very high proportion of identical residues but are not 100% identical, indicating that they represent different isozymes.
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PMID:Complete primary structure of a human plasma membrane Ca2+ pump. 284 59

The transforming gene product encoded by Moloney murine sarcoma virus clone 124, p37mos, contains a lysine residue (lysine-121) that is conserved among all members of the protein kinase family. This lysine has been shown to be part of a conserved ATP-binding site in both the catalytic subunit of the cAMP-dependent protein kinase and p60v-src. We wished to determine whether this lysine is required for the transforming activity of p37mos. Two site-specific mutations were therefore constructed, which result in the substitution of an aspartic acid or arginine codon in place of the codon for lysine-121. Both mutations abolished the ability of the mos gene to transform cells. These results show that lysine-121 is required for the ability of p37mos to transform cells and provide evidence for an ATP-binding site in p37mos. Furthermore, these results suggest that the conserved lysine residue is specifically involved in the catalytic activity of protein kinases in general.
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PMID:Lysine residue 121 in the proposed ATP-binding site of the v-mos protein is required for transformation. 299 82

Cardiac myosin binding protein-C (cardiac MyBP-C, cardiac C protein) belongs to a family of proteins implicated in both regulatory and structural functions of striated muscle. For the cardiac isoform, regulatory phosphorylation in vivo by cAMP-dependent protein kinase (PKA) upon adrenergic stimulation is linked to modulation of cardiac contraction. The sequence of human cardiac MyBP-C now reveals regulatory motifs specific for this isoform. Site-directed mutagenesis identifies a LAGGGRRIS loop in the N-terminal region of cardiac MyBP-C as the key substrate site for phosphorylation by both PKA and a calmodulin-dependent protein kinase associated with the native protein. Phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform-specific site. This phosphorylation switch can be mimicked by aspartic acid instead of phosphoserine. Cardiac MyBP-C is therefore specifically equipped with sensors for adrenergic regulation of cardiac contraction, possibly implicating cardiac MyBP-C in cardiac disease. The gene coding for cardiac MyBP-C has been assigned to the chromosomal location 11p11.2 in humans, and is therefore in a region of physical linkage to subsets of familial hypertrophic cardiomyopathy (FHC). This makes cardiac MyBP-C a candidate gene for chromosome 11-associated FHC.
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PMID:Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction? 774 2

Site-directed mutagenesis was used to remove a critical phosphorylation site, Thr-197, near the active site of the catalytic subunit of cAMP-dependent protein kinase. This residue is present in a number of protein kinases, and its phosphorylation largely influences catalytic activity. We changed Thr-197 to aspartic acid and alanine and measured the effects of these substitutions on the kinetic mechanism and inhibitor affinities. The mutants were expressed as the free catalytic subunit and as soluble fusion proteins of glutathione-S-transferase. The values for KATP and Kpeptide for all three mutants are raised by approximately 2 orders of magnitude relative to the wild-type enzyme. Viscosometric measurements indicate that elevations in Kpeptide are the result of reduced rates for phosphoryl transfer and not reduced substrate affinities. This implies that the loop that contains the phosphothreonine, the activation loop, does not reduce access to the substrate site as proposed for the inactive forms of cdk2 kinase [DeBont, H. L., et al. (1993) Nature 363, 595-602] and MAP kinase [Zhang, F., et al. (1994) Nature 367, 704-711]. The mutants associate slowly with the wild-type regulatory subunit, although the cAMP-free wild-type regulatory subunit inhibits the mutants stoichiometrically. A mutant regulatory subunit that binds cAMP poorly and rapidly inhibits the wild-type catalytic subunit does not inhibit the mutant proteins. These data suggest that the phosphothreonine region serves as a docking surface for the regulatory subunit in the holoenzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase. 787 23

Casein kinase-II (CK-II) belongs to the protein kinases recognizing serine/threonine in proximity to acidic residues in protein substrates. Crystallography and mutagenesis studies on the cAMP-dependent protein kinase (PKA) disclosed that glutamic acid-170 (E170), is important for interaction of substrates with the enzyme. At a position corresponding to E170 in PKA most Ser/Thr kinases have an aspartic or glutamic acid, while CK-II has a histidine residue (H160). In order to examine the relevance of this substitution for CK-II substrate specificity, a mutant of the catalytic alpha subunit (H160D), in which H160 was changed to aspartic acid, was made. Our results show that H160 is not primarily involved in canonical substrate recognition, but does interact with an acidic residue located at position -2 with respect to the target Ser/Thr.
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PMID:Substrate recognition by casein kinase-II: the role of histidine-160. 798 80

Many guanine-nucleotide-binding-protein-coupled receptors contain consensus sequences for phosphorylation by cAMP-dependent protein kinase (PKA), often located in the membrane proximal regions critically important for receptor signalling. In the present study, we have evaluated by site-directed mutagenesis the role of the putative PKA phosphorylation sites in the m4 muscarinic acetylcholine receptor (mAChR), i.e. Thr145 in the second cytoplasmic loop and Thr399 in the third cytoplasmic loop, and the influence of PKA on m4 mAChR function and internalization. Antagonist binding was unaltered by any of the mutations studied, while the agonist-binding affinity was either not affected (Thr145 alanine), increased (Thr399 alanine) or decreased (Thr399 serine or aspartic acid). m4 mAChR-mediated inhibition of adenylyl cyclase was unaltered by the mutations, except for an approximately tenfold reduced agonist potency of the Thr399 aspartic acid mutated receptor. Agonist-induced receptor internalization was unaltered with Thr399 serine or aspartic acid mutations of the receptors, but was strongly decreased in its rate and extent upon replacement of Thr399, Thr145 or both of these residues with alanine. These mutational effects could not be reproduced by treatment of wild-type receptor-expressing cells with the PKA inhibitor H-8. Furthermore, maximal stimulation of cellular PKA neither affected receptor internalization nor signalling measured as receptor-mediated Ca2+ mobilization. We conclude that the membrane proximal threonine residues of the m4 mAChR are not required for receptor signalling, but replacement by alanine residues can significantly affect receptor internalization, independently of PKA phosphorylation. Sequence comparisons suggest that threonine residues at corresponding positions may be relevant to internalization of other guanine-nucleotide-binding-protein-coupled receptors.
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PMID:The role of membrane proximal threonine residues conserved among guanine-nucleotide-binding-protein-coupled receptors in internalization of the m4 muscarinic acetylcholine receptor. 853


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