Role of magnesium-ATP in the activation and reassociation of cAMP-dependent protein kinase I: consequences of replacing the essential arginine in cAMP-binding site A

Biochemistry ◽  
1991 ◽  
Vol 30 (3) ◽  
pp. 733-739 ◽  
Author(s):  
James J. Neitzel ◽  
Wolfgang R. G. Dostmann ◽  
Susan S. Taylor
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals Role of the Glycine Triad in the ATP-binding Site of cAMP-dependent Protein Kinase

1997 ◽  
Vol 272 (27) ◽  
pp. 16946-16954 ◽  
Author(s):  
Wolfram Hemmer ◽  
Maria McGlone ◽  
Igor Tsigelny ◽  
Susan S. Taylor
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Atp Binding ◽  
Dependent Protein Kinase ◽  
Atp Binding Site ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals Role of the N-terminal region in covalent modification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: comparison of phosphorylation and ADP-ribosylation

1995 ◽  
Vol 309 (1) ◽  
pp. 119-125 ◽  
Author(s):  
J L Rosa ◽  
J X Pérez ◽  
F Ventura ◽  
A Tauler ◽  
J Gil ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Covalent Modification ◽  
Terminal Region ◽  
Bifunctional Enzyme ◽  
Dependent Protein Kinase ◽  
Adp Ribosylation ◽  
Camp Dependent Protein Kinase ◽  
Arginine Residues ◽  
Dependent Protein

The effect of cyclic AMP (cAMP)-dependent phosphorylation and ADP-ribosylation on the activities of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), was investigated in order to determine the role of the N-terminus in covalent modification of the enzyme. The bifunctional enzyme was demonstrated to be a substrate in vitro for arginine-specific ADP-ribosyltransferase: 2 mol of ADP-ribose was incorporated per mol of subunit. The Km values for NAD+ and PFK-2/FBPase-2 were 14 microM and 0.4 microM respectively. A synthetic peptide (Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln) corresponding to the site phosphorylated by cAMP-dependent protein kinase was ADP-ribosylated on all three arginine residues. Analysis of ADP-ribosylation of analogue peptides containing only two arginine residues, with the third replaced by alanine, revealed that ADP-ribosylation occurred predominantly on the two most C-terminal arginine residues. Sequencing of the ADP-ribosylated native enzyme also demonstrated that the preferred sites were at Arg-29 and Arg-30, which are just N-terminal to Ser-32, whose phosphorylation is catalysed by cAMP-dependent protein kinase (PKA). ADP-ribosylation was independent of the phosphorylation state of the enzyme. Furthermore, ADP-ribosylation of the enzyme decreased its recognition by liver-specific anti-bifunctional-enzyme antibodies directed to its unique N-terminal region. ADP-ribosylation of PFK-2/FBPase-2 blocked its phosphorylation by PKA, and decreased its PFK-2 activity, but did not alter FBPase-2 activity. In contrast, cAMP-dependent phosphorylation inhibited the kinase and activated the bisphosphatase. These results demonstrate that ADP-ribosylation of arginine residues just N-terminal to the site phosphorylated by PKA modulate PFK-2 activity by an electrostatic and/or steric mechanism which does not involved uncoupling of N- and C-terminal interactions as seen with cAMP-dependent phosphorylation.

scholarly journals A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors

1996 ◽  
Vol 318 (1) ◽  
pp. 241-245 ◽  
Author(s):  
Hedley A COPPOCK ◽  
Ali A OWJI ◽  
Stephen R BLOOM ◽  
David M SMITH
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Binding Site ◽  
Binding Sites ◽  
Calcitonin Gene Related Peptide ◽  
Dependent Protein Kinase ◽  
Related Peptide ◽  
Camp Dependent Protein Kinase ◽  
Calcitonin Gene ◽  
Dependent Protein

We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22±0.04 nM (mean±S.E.M.) and a concentration of binding sites (Bmax) of 0.95±0.19 pmol/mg of protein (mean±S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8–37) competed weakly at this site (IC50 > 10 and 601±298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13±0.01 nM; Bmax = 0.83±0.10 pmol/mg of protein) where both CGRP(8–37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15±0.12 and 8.68±0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site–ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8–37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.

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