[6] Catalytic subunit of cAMP-dependent protein kinase

Reimann, Erwin M.; Beham, Richard A.

doi:10.1016/0076-6879(83)99039-0

Cited by 211 publications

(72 citation statements)

References 7 publications

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“…Site-directed mutations were introduced by oligonucleotide-directed mutagenesis of a uracil-containing single-stranded Kunkel template (37). The translation of the coding sequences was carried out in the TNT-coupled transcription/translation rabbit reticulocyte expression system (Promega), as recommended by the manufacturer, and per-formed in the presence of [ 35 …”

Section: Construction Of Mutants and Their Translation In A Cell-freementioning

confidence: 99%

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

Chestukhin¹,

Litovchick²,

Muradov

et al. 1997

Journal of Biological Chemistry

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The kinase splitting membranal proteinase (KSMP) is a metalloendopeptidase that inactivates the catalytic (C) subunit of protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at Glu 332 -Glu 333 , within the cluster of acidic amino acids (Asp 328 -Glu 334 ) of the kinase. The K m values of KSMP and of meprin ␤ (which reproduces KSMP activity) for the C-subunit are below 1 M. The K m for peptides containing a stretch of four Glu residues are in the micromolar range, illustrating the significant contribution of this cluster to the substrate recognition of meprin ␤. This conclusion is supported by a systematic study using a series of the C-subunit mutants with deletions and mutations in the cluster of acidics. Hydrophobic amino acids vicinal to the cleavage site increase the K cat of the proteinase. These studies unveil a new specificity for meprin ␤, suggesting new substrates that are 1-2 orders of magnitude better in their K m and K cat than those commonly used for meprin assay. A search for substrates having such a cluster of acidics and hydrophobics, which are accessible to meprin under physiological conditions, point at gastrin as a potential target. Indeed, meprin ␤ is shown to cleave gastrin at its cluster of five glutamic acid residues and also at the M-D bond within its WMDF-NH 2 sequence, which is indispensable for all the known biological activities of gastrins. The latter meprin cleavage will lead to the inactivation of gastrin and thus to the control of its activity.The presence of a kinase splitting membranal proteinase (KSMP) 1 in the brush-border membranes of the rat small intestine was demonstrated as early as 1979 (1). This proteinase was shown to clip the catalytic (C) subunit of PKA, yielding a distinct cleavage product (CЈ) that was found to be devoid of the kinase activity. The biochemical characterization of KSMP as a proteinase revealed that it is an intriguing enzyme with a combination of the following unique features. (a) KSMP cleaves the C-subunit when it is free but not when inhibited by its regulatory (R) subunits, as in the R 2 C 2 complex (1, 2). (b) This cleavage could not be simulated by other proteinases (trypsin, chymotrypsin, clostripain, and papain (2)), suggesting that its specificity is not due merely to an interdomain exposure in C. (c) The proteinase was found to single out and selectively cleave the C-subunit in the presence of the large number of other proteins found in crude extracts of different tissues (brain, liver, or muscle) (3). (d) KSMP was found to cleave the Csubunit in its native conformation but not if the kinase is pre-denatured (2). (e) It distinguishes between the "open" and "closed" conformations of the C-subunit (4) that were recently identified by x-ray crystallography of this kinase (5).The cleavage of the C-subunit by KSMP leads to the removal of the carboxyl terminus tail of this kinase and seemed to occur at a distinct site (6 -8). Interestingly, two other kinases, the EGF-and the insulin-receptor ki...

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Section: Construction Of Mutants and Their Translation In A Cell-freementioning

confidence: 99%

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

Chestukhin¹,

Litovchick²,

Muradov

et al. 1997

Journal of Biological Chemistry

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“…The catalytic subunit of CAMP-dependent protein kinase was purified from bovine heart [19], phosphorylase [20] (prepared by Dr. C. MacKintosh), phosphorylase kinase 1211 (Mr F. B. Caudwell), glycogen synthase [22] (Miss S. Nakielny), inhibitor-1 and inhibitor-2 [23] (Dr M . J. Hubbard) from rabbit skeletal muscle and calmodulin from sheep brain [24] (Mr F. B. Caudwell) were prepared at Dundee by the members of the laboratory indicated in parentheses.…”

Section: Protein Preparationsmentioning

confidence: 99%

The control of protein phosphatase‐1 by targetting subunits

Alessi

MacDougall

Sola

et al. 1992

European Journal of Biochemistry

365

308

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The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130,37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component was the catalytic subunit, while the 130-kDa and 20-kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the /3 isoform of protein phosphatase-1 (PPl) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PPlg with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.Although phosphoserine and phosphothreonine account for more than 99% of phosphorylated amino acids [I] and hundreds of phosphoproteins are present in cells, only four major classes of protein serineithreonine phosphatase (PP) catalytic subunits, termed PP1, PP2A, PP2B and PP2C have been identified (reviewed in [2]). Three of these, namely PP1 , PP2A and PP2C, have broad and overlapping substrate specificities in vitro, accounting for most, if not all, of the phosphatase activity in tissue extracts towards a variety of enzymes that control the major metabolic pathways (reviewed in [3]). Furthermore, studies with the tumour promoter okadaic acid, a potent and specific inhibitor of PP1 and PP2A (reviewed in [4]), and the phenotypes of genetic mutants lacking these enzymes in fungi [5], fission yeast [6] and fruit flies [7, 81, has implicated PP1 and PP2A in the regulation of additional processes, such as the transport of ions and nutrients into cells, gene transcription, muscle contractility and the celldivision cycle. The regulation of many physiological processes by a limited number of phosphatase catalytic subunits, has therefore raised the question of how each of these processes can be regulated independently of one another.A major clue originated from studies on thc control of glycogen metabolism in mammalian skeletal muscle, whereCorrespondence to P. Cohen, MRC Protein Phosphorylation Unit,

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“…The catalytic subunit of CAMP-dependent protein kinase, prepared from bovine heart [9], was a generous gift from Dr Carol MacKintosh. Radiolabelled ATP and sodium bicarbonate were obtained from Amersham International.…”

Section: Experimental Procedures Materialsmentioning

confidence: 99%

Regulation of the mammalian carbamoyl‐phosphate synthetase II by effectors and phosphorylation

Shaw

Carrey

1992

European Journal of Biochemistry

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We have measured the 'core' mammalian carbamoyl-phosphate synthetase I1 (CPSII) activity, using NH4C1 as the nitrogen-donating substrate and trapping carbamoyl phosphate as urea through its reaction with ammonium ions. When ATP and magnesium ion concentrations are close to those found in the cell, the substrate saturation curves for ammonia and bicarbonate are hyperbolic, giving K, (NH,) values of 166 pM at high ATP concentrations and 26 pM at low ATP concentrations, while the K, (bicarbonate) is 1.4 mM at both ATP concentrations used. These values for the K,,, (NH,) are lower than previously reported for CPS 11, and closer to the values for the mitochondria1 counterpart. The K, for ammonia and bicarbonate are not altered by phosphorylation of the multienzyme polypeptide CAD, which contains the first three enzyme activities of pyrimidine biosynthesis. The CPS I1 activity is lower with an excess of either ATP or magnesium ions, causing the apparently sigmoid dependence of activity upon ATP concentration to be enhanced at low concentrations of free magnesium ions. The feedback inhibitor, UTP, acts by stabilising a state with a low affinity for magnesium ions and for ATP. In the presence of the activator, 5-phosphoribosyl diphosphate (PRibPP), the enzyme has a higher affinity for magnesium ions and thus the ATP dependence of the activity is hyperbolic. Phosphorylation of CAD similarly activates the CPS I1 enzyme by increasing the affinity for magnesium ions and by pushing the equilibrium away from the low-affinity UTP-stabilised state. Using our improved assay procedure, we observe a very large activation by PRibPP of carbamoylphosphate synthesis at low concentrations of magnesium ions, and we find that unlike UTP, the activator PRibPP is able to act on the phosphorylated enzyme.Carbamoyl phosphate is synthesised in all organisms by carbamoyl-phosphate synthetase using ammonia, bicarbonate, and two molecules of ATP-Mg, each with a distinct role and binding site. The mammalian urea-cycle enzyme, CPS I, is found in the mitochondria. The pyrimidine-biosynthetic enzyme, CPS 11, is part of the cytoplasmic multienzyme polypeptide CAD [l]. This 240-kDa molecule contains an active glutaminase domain at the amino-terminus, adjacent to the carbamoyl-phosphate synthetase domains; these two activities comprise the glutamine-dependent CPS 11. CAD also contains the aspartate transcarbamylase and dihydroorotase activities, Abbreviations. CAD, multienzyme polypeptide containing the first enzymes dedicated to pyrimidine biosynthesis in higher organisms, i. e. carbamoyl-phosphate synthetase 11, aspartate carbamoyltransferase and dihydroorotase; CPS I, ammonia-dependent carbamoyl-phosphate synthetase from mammalian mitochondria; CPS 11; glutaminedependent carbamoyl-phosphate synthetase, part of the multienzyme polypeptide CAD: PRibPP, 5-phosphoribosyl diphosphate; So.,, the concentration of substrate required for half-maximal activity.Enzymes. Aspartate transcarbamylase, aspartate carbamoyltransferase (EC 2.1.3.2); carbamoyl-phospha...

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[6] Catalytic subunit of cAMP-dependent protein kinase

Cited by 211 publications

References 7 publications

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

The control of protein phosphatase‐1 by targetting subunits

Regulation of the mammalian carbamoyl‐phosphate synthetase II by effectors and phosphorylation

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