Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The complete amino acid sequence of bovine brain DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein, which is a potent and specific inhibitor of the catalytic subunit of protein phosphatase-1, has been determined. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage by endoproteinase Lys-C, endoproteinase Arg-C, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease, and to chemical cleavage by cyanogen bromide. The overlapping sets of peptides were purified by high performance liquid chromatography and subjected to amino acid sequencing by automated Edman degradation to deduce the complete sequence. The protein consists of a single NH2-terminal blocked polypeptide chain of 202 residues, with a calculated molecular mass of 22,591 daltons, excluding the unidentified NH2-terminal blocking group. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or hydrodynamic measurements. The threonine residue that is phosphorylated by cyclic AMP-dependent protein kinase (Hemmings, H. C., Jr., Williams, K. R., Konigsberg, W. H., and Greengard, P. (1984) J. Biol. Chem. 259, 14486-14490), and that must be phosphorylated for the expression of inhibitory activity, is located at position 34. The molecule contains only 1 cysteine residue and 1 tryptophan residue, at positions 72 and 161, respectively. DARPP-32 is very hydrophilic, and contains a stretch of 16 consecutive acidic residues from position 119 to 134. The predicted secondary structure suggests the presence of 47% alpha-helix, 7% beta-sheet, and 46% random coil, with 11 beta-turns. Comparison of the complete amino acid sequence of bovine DARPP-32 with that of rabbit skeletal muscle protein phosphatase inhibitor-1 revealed a significant amount of sequence identity in the NH2-terminal regions of these two proteins. The active region of inhibitor-1 has been localized to an NH2-terminal fragment (Aitken, A., and Cohen, P. (1982) FEBS Lett. 147, 54-58), the part of the molecule that is most similar to DARPP-32. These data suggest that these two protein phosphatase inhibitors may share a common structural basis for their inhibitory activity and may be related by a common ancestral gene.
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PMID:DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein. Primary structure and homology with protein phosphatase inhibitor-1. 351 Oct 54

The complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-1, has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Staphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e.g. 1-49, 49-92, 67-101, 108-134, 142-182 and 163-197) are inactive. Digestion with clostripain generated a peptide comprising residues 25-114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-1.
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PMID:The protein phosphatases involved in cellular regulation. Primary structure of inhibitor-2 from rabbit skeletal muscle. 351 70

Glycogen synthase kinase-3 was isolated from rabbit skeletal muscle by an improved procedure. The purification was estimated to be 67000-fold and 0.2 mg of enzyme was isolated from 5000 g muscle, corresponding to an overall yield of 7%. The preparation was homogeneous by ultracentrifugal and electrophoretic criteria. The enzyme had a relative molecular mass of 47 kDa by sedimentation equilibrium centrifugation and 51 kDa by SDS-polyacrylamide gel electrophoresis. These values demonstrate that glycogen synthase kinase-3 is monomeric. The Stokes radius of 37 nm suggests the molecule to be asymmetric. The activating factor of the Mg-ATP dependent form of protein phosphatase-1 coeluted with glycogen synthase kinase-3 activity at the final step, establishing that these two activities reside in the same protein. Glycogen synthase kinase-3 phosphorylates glycogen synthase at sites-3, while casein kinase-II phosphorylates site-5, just C-terminal to sites-3 (Picton, C., Aitken, A., Bilham, T. and Cohen, P. (1982) Eur. J. Biochem. 124, 37-45). The basis for the substrate specificities of these protein kinases was investigated using chymotryptic peptides that contain the sites phosphorylated by each enzyme. These studies showed that efficient phosphorylation of sites-3, required the presence of phosphate in site-5 and a region of polypeptide more than 20 residues C-terminal to site-5. In contrast, efficient phosphorylation by casein kinase-II does not require this C-terminal region, and the results are consistent with the view that the enzyme recognises acidic residues immediately C-terminal to site-5.
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PMID:Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5). 608 11

The 'native' Mg-ATP-dependent protein phosphatase was isolated from rabbit skeletal muscle by a procedure that avoided the use of organic solvents or heating at 90-100 degrees C. The purified enzyme was composed of two major proteins (molecular mass 37 kDa and 31 kDa) that were present in a 1:1 molar ratio, and accounted for 70-80% of the material. The 37-kDa component comigrated with the catalytic subunit of protein phosphatase-1, and its identity with this protein was established by peptide mapping, and by its cleavage to the characteristic 34-kDa and 33-kDa fragments following incubation with chymotrypsin. The 31-kDa protein comigrated with inhibitor-2, and its identity with this protein was established by its heat stability, ability to inhibit protein phosphatase-1 at nanomolar concentrations, and its phosphorylation on a threonine residue by glycogen synthase kinase 3. It is therefore concluded that the 'native' Mg-ATP-dependent protein phosphatase is composed of the catalytic subunit of protein phosphatase-1 (37 kDa) and inhibitor-2 (31 kDa) in a 1:1 molar ratio. The 'native' Mg-ATP-dependent protein phosphatase had virtually identical properties to the enzyme reconstituted from inhibitor-2 and the 37-kDa catalytic subunit of protein phosphatase-1. Each preparation had a similar specific activity and was inhibited by identical concentrations of inhibitor-1. Both enzymes could be activated by incubation with glycogen synthase kinase-3 and Mg-ATP, or by Mn2+ and trypsin (or chymotrypsin). However, Mn2+ alone, or proteinase digestion in the absence of Mn2+, failed to activate either preparation. Incubation with glycogen synthase kinase-3 and Mg-ATP did not dissociate the 'native' or 'reconstituted' enzymes, whereas treatment with Mn2+ and trypsin decreased their apparent molecular masses from 70 kDa to 35 kDa. Incubation with chymotrypsin converted the 'native' and 'reconstituted' enzymes to forms that required preincubation with glycogen synthase kinase-3, Mg-ATP and inhibitor-2, in order to exhibit catalytic activity. The Mg-ATP-dependent protein phosphatase reconstituted from the 'nicked' 33-kDa catalytic subunit dissociated upon activation, in contrast to the enzyme reconstituted from the undegraded 37-kDa catalytic subunit. The results suggest that a 3-4-kDa fragment at one end of the polypeptide is involved in strengthening interaction between the undegraded 37-kDa catalytic subunit and the phosphorylated form of inhibitor-2.
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PMID:The protein phosphatases involved in cellular regulation. Comparison of native and reconstituted Mg-ATP-dependent protein phosphatases from rabbit skeletal muscle. 609 83

The regional and cellular distribution of G-substrate, a 23,000-dalton protein substrate specific for guanosine 3',5'-cyclic monophosphate-dependent protein kinase, has been examined in mammalian brain using immunoprecipitation, radioimmunoassay, and peptide-mapping techniques. In rabbit brain, G-substrate was found to be highly concentrated in the cerebellum. The concentration of G-substrate in cerebellar cytosol was 27.2 pmol/mg. The concentrations of G-substrate in cortex, hippocampus, and caudate were only 1 to 2% of that found in cerebellum. Studies of neurological mutant mice lacking either Purkinje cells (PCD and nervous) or granule cells (weaver) suggested that, within the cerebellum, G-substrate is localized almost exclusively in Purkinje cells. A phosphoprotein present in noncerebellar brain regions, which co-migrated with G-substrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was shown by peptide mapping to consist predominantly of phosphatase inhibitor-1. Phosphatase inhibitor-1, a potent inhibitor of protein phosphatase-1, is known to share several physicochemical properties with G-substrate. In contrast to the results obtained with G-substrate, the concentration of phosphatase inhibitor-1 was significantly lower in cerebellum than in other major brain regions. These and other data suggest that G-substrate may be a Purkinje cell-specific protein phosphatase inhibitor.
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PMID:Localization in mammalian brain of G-substrate, a specific substrate for guanosine 3',5'-cyclic monophosphate-dependent protein kinase. 609 45

Acetyl-CoA carboxylase has been purified from lactating rat mammary gland using a combination of ammonium sulphate and poly(ethyleneglycol) precipitations. The enzyme was purified from 35--70-fold with a yield of over 50%, the exact figures being difficult to estimate because of activation of the enzyme that occurs during the preparation. The preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis in sodium dodecyl sulphate and had a single subunit of molecular weight 240,000, containing 1.02 +/- 0.04 molecules of biotin and 3.1 +/- 1.7 molecules of alkali-labile phosphate per subunit. The purified enzyme was phosphorylated and inactivated rapidly when incubated in the presence of [gamma 32P]ATP and magnesium ions with the purified catalytic subunit of cyclic-AMP-dependent protein kinase from rabbit skeletal muscle. Both phosphorylation and inactivation are blocked by the heat-stable protein inhibitor of cyclic-AMP-dependent protein kinase, and can be reversed by incubation with purified protein phosphatase-1 from rabbit skeletal muscle. The inactivation by the protein kinase and reactivation by the protein phosphatase correlate with the near-stoichiometric phosphorylation and dephosphorylation of site(s) located in a single tryptic peptide. Phosphorylation does not affect the Km for substrates, but brings about a twofold decrease in V and a twofold increase in the apparent dissociation constant for the allosteric activator, citrate. We also present evidence that the activation of rabbit mammary acetyl-CoA carboxylase by protein phosphatase-1 described previously [Hardie and Cohen (1979) FEBS Lett. 103, 333-338] is due to dephosphorylation at site(s) which are not phosphorylated by either cyclic-AMP-dependent protein kinase or acetyl-CoA carboxylase kinase-2. These results suggest that the rapid inactivation of acetyl-CoA carboxylase, and hence fatty acid synthesis, by adrenaline in adipose tissue, or glucagon in the liver, is due to phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase.
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PMID:Reversible phosphorylation and inactivation of acetyl-CoA carboxylase from lactating rat mammary gland by cyclic AMP-dependent protein kinase. 610 9

The synthetic phosphohexapeptides Arg-Arg-Ala-Thr(35P)-Val-Ala and Arg-Arg-Ala-Ser(32P)-Val-Ala, phosphorylated by the cAMP-dependent protein kinase and differing only in the nature of the phosphorylated residue, have been used as substrates of a partially purified rat liver protein phosphatase-T, distinct from the multifunctional protein phosphatase-1. While the phosphothreonyl hexapeptide is readily dephosphorylated (exhibiting a Km = 15 microM), the phosphoseryl one is almost unaffected. Such a behavior is not shared by protein phosphatase-1, calf intestine alkaline phosphatase, and potato acid phosphatase, all of which are more active on the phosphoseryl hexapeptide. The NH2-terminal basic residues critical for cAMP-dependent phosphorylation are not required in the dephosphorylation reaction, as both Arg can be removed without impairing the efficiency of protein phosphatase-T toward the phosphothreonyl peptide. On the other hand, the replacement of 2 Pro for the Ala and Val flanking Thr(32P), to give a new phosphohexapeptide reproducing the phosphorylated site of protein phosphatase inhibitor-1, prevents the protein phosphatase-T activity. Moreover, IgG heavy chain 32P labeled in tyrosine is not affected by protein phosphatase-T, while it is dephosphorylated by alkaline phosphatase. These results would indicate that protein phosphatase(s)-T represent a distinct class of protein phosphatases specifically involved in the dephosphorylation of phosphothreonyl residues fulfilling definite structural requirements.
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PMID:Dephosphorylation of synthetic phosphopeptides by protein phosphatase-T, a phosphothreonyl protein phosphatase. 628 35

A Mg-ATP-dependent protein phosphatase has been reconstituted from the catalytic subunit of protein phosphatase-1 and inhibitor-2, and consists of a 1:1 complex between these proteins. Activation of this enzyme by glycogen synthase kinase-3 and Mg-ATP results from the phosphorylation of inhibitor-2 on a threonine residue(s) and is accompanied by the dissociation of the complex. The results prove that protein phosphatase-1 and the Mg-ATP-dependent protein phosphatase contain the same catalytic subunit, and that they are interconvertible forms of the same enzyme.
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PMID:Reconstitution of a Mg-ATP-dependent protein phosphatase and its activation through a phosphorylation mechanism. 629 78

Homogenous preparations of the catalytic subunit of protein phosphatase-1 and inhibitor-2 can be combined to produce an inactive enzyme that consists of a 1:1 complex between these two proteins. This species is indistinguishable from the Mg-ATP-dependent protein phosphatase in that preincubation with glycogen synthase kinase-3 and Mg-ATP is required to generate activity. Activation results from the phosphorylation of inhibitor-2. The molar concentrations of protein phosphatase-1 and inhibitor-2 in rabbit skeletal muscle (0.25-0.5 microM) are similar. Incubation of the reconstituted Mg-ATP-dependent protein phosphatase with chymotrypsin is accompanied by limited proteolysis of inhibitor-2 and the loss of its phosphorylation site(s). This species can be activated by glycogen synthase kinase-3 and Mg-ATP provided that inhibitor-2 is added. This exogenous inhibitor-2 appears to displace the fragments of inhibitor-2 from the enzyme that were generated by chymotryptic digestion. These experiments may explain the report [Yang, S.D., Vandenheede, J.R. and Merlevede, W. (1981) J. Biol. Chem. 256, 10231-10234] that inhibitor-2 can function as an 'activator' as well as an inhibitor of the Mg-ATP-dependent protein phosphatase. Incubation of the catalytic subunit of protein phosphatase-1 with sodium fluoride or sodium pyrophosphate converted the enzyme to an inactive form that could be partially reactivated by manganese ions, but not by glycogen synthase kinase-3 and Mg-ATP. Conversely, the reconstituted Mg-ATP-dependent protein phosphatase could only be activated by glycogen synthase kinase-3 and Mg-ATP, and not by manganese ions. It is concluded that the conversion of protein phosphatase-1 to a manganese-ion dependent form is a quite separate phenomenon from the formation of the Mg-ATP-dependent protein phosphatase. Inhibitor-2 can inactivate protein phosphatase-1 by a second mechanism that is not reversed by preincubation with glycogen synthase kinase-3 and Mg-ATP. This occurs at higher concentrations of inhibitor-2 than those required to form the Mg-ATP-dependent protein phosphatase, and appears to result from the binding of inhibitor-2 to a distinct site on the enzyme.
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PMID:Characterisation of a reconstituted Mg-ATP-dependent protein phosphatase. 630 89

Microinjection of cAMP-dependent protein kinase inhibitor (1.8 microM) increases the cAMP level of Xenopus oocyte. Its effect was observed in full-grown (stage VI) as well as in vitellogenic (stage IV) oocytes. In contrast the inhibitor I1 of protein phosphatase-1 blocks cAMP accumulation. Progesterone (1 microM) decreases the cAMP level in control and in PKI-treated oocytes of both stages. These results show that cAMP concentration is regulated by a cAMP-dependent phosphorylation indicating the presence of a feedback mechanism. The feedback control is disrupted when oocyte is induced to mature by progesterone.
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PMID:cAMP-dependent protein kinase regulates in ovo cAMP level of the Xenopus oocyte: evidence for an intracellular feedback mechanism. 630 83


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