Identification of electrostatic interactions that determine the phosphorylation site specificity of the cAMP-dependent protein kinase

Biochemistry ◽  
1991 ◽  
Vol 30 (22) ◽  
pp. 5329-5334 ◽  
Author(s):  
Craig S. Gibbs ◽  
Mark J. Zoller
Keyword(s):  
Protein Kinase ◽  
Electrostatic Interactions ◽  
Phosphorylation Site ◽  
Site Specificity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

2000 ◽  
Vol 345 (3) ◽  
pp. 673-680 ◽  
Author(s):  
Sean P. COLLINS ◽  
Junewai L. REOMA ◽  
David M. GAMM ◽  
Michael D. UHLER
Keyword(s):  
Protein Kinase ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Multiple Cancer ◽  
Dependent Protein Kinase ◽  
Rnase Protection ◽  
Camp Dependent Protein Kinase ◽  
Increased Risk ◽  
Dependent Protein

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif (Cys433-Lys-Gln-Gln436) directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site (Arg428-Arg-Leu-Ser431). The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys433 to an alanine residue, but not phosphorylation by PKA, blocked membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1.

Calmodulin-dependent myosin light chain kinases from cardiac and smooth muscle: a comparative study

1980 ◽  
Vol 58 (4) ◽  
pp. 299-308 ◽  
Author(s):  
Michael P. Walsh ◽  
Jean-Claude Cavadore ◽  
Bernard Vallet ◽  
Jacques G. Demaille
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phosphorylation Site ◽  
Dependent Protein Kinase ◽  
Proteolytic Fragment ◽  
Cardiac Enzyme ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Various properties of cardiac and smooth muscle calmodulin-dependent myosin light chain kinases (MLCKs) have been compared. The enzymes exhibit the same isoelectric point (6.5) but differ markedly in molecular weight (Mr = 72 000 for both canine and bovine cardiac MLCK, and Mr = 130 000 for smooth muscle MLCK). Comparison of the tryptic peptide maps of bovine cardiac and turkey gizzard MLCKs indicates that the cardiac enzyme is a fragment of a protein homologous to the smooth muscle kinase. While the smooth muscle kinase can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, the same is not true for either bovine or canine cardiac MLCK. Controlled tryptic hydrolysis of phosphorylated smooth muscle MLCK, followed by affinity chromatography on a column of calmodulin–Sepharose, enables separation of a phosphopeptide (Mr = 22 000) from a mixture of peptides of Mr = 50 000 and 24 000 which are bound to the column in the presence of Ca2+ and eluted with ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The phosphorylation site, therefore, is distinct from the calmodulin-binding site. It appears that cardiac MLCK is proteolyzed during the isolation procedure. The purified cardiac enzyme represents a proteolytic fragment which retains Ca2+ and calmodulin dependence but only a fraction of the specific activity of the native enzyme, and has lost the site of phosphorylation by cAMP-dependent protein kinase. A protease is shown to exist in myocardium which is capable of digesting smooth muscle MLCK rapidly at low temperature, and which is resistant to classical antiproteases.

scholarly journals A protonated histidine residue in a phosphorylation site for cyclic AMP-dependent protein kinase. Comparison of a synthetic peptide with the exposed linking region in the multienzyme polypeptide CAD

1992 ◽  
Vol 287 (3) ◽  
pp. 791-795 ◽  
Author(s):  
E A Carrey
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Synthetic Peptide ◽  
Phosphorylation Site ◽  
Intact Protein ◽  
Histidine Residue ◽  
Dependent Protein Kinase ◽  
Ph Values ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

The multienzyme polypeptide CAD is phosphorylated at two sites by cyclic AMP (cAMP)-dependent protein kinase. Site 2 has two interesting features: it is located in a ‘linking region’ between two discretely folded enzyme domains, and a histidine, instead of the more usual arginine, is found three positions N-terminal to the phosphorylated serine. A synthetic peptide corresponding to the sequence around site 2 has an extended or random structure in solution, and the proton n.m.r. chemical shift of the histidine residues can be titrated against pH in the range 6.0-8.0. The peptide is phosphorylated more rapidly by cAMP-dependent protein kinase at lower pH values, indicating that the protonated histidine side chain corresponds to the arginine in the consensus recognition sequence for the kinase. Kemptide, a specific synthetic substrate for the kinase, was phosphorylated with a higher affinity and at a similar rate at all pH values. CAD was a better substrate than the synthetic peptide, and labelling was not affected by the pH of the incubation conditions. The results indicate that the phosphorylation site in the interdomain linker is sufficiently exposed to the solvent to ensure accessibility to the kinase, but that secondary or tertiary structure in the intact protein allows the histidine residue to remain protonated at physiological pH and enhances recognition of the phosphorylatable serine residue.

Identification and characterization of cAMP-dependent protein kinase and its possible direct interactions with protein phosphatase-1 in marine dinoflagellates

1996 ◽  
Vol 74 (4) ◽  
pp. 559-567 ◽  
Author(s):  
John F. Dawson ◽  
Kathy He Wang ◽  
Charles F. B. Holmes
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Phosphorylation Site ◽  
Protein Phosphatase 1 ◽  
Dependent Protein Kinase ◽  
Prorocentrum Lima ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

We have examined the nature of signal transduction involving reversible protein phosphorylation in marine Prorocentrale species. Of particular interest is the marine dinoflagellate Prorocentrum lima in which the tumour promoter okadaic acid is produced and may interfere with signal transduction. We have identified cAMP-dependent protein kinase (PKA) activity in P. lima, P. micans, and P. minimum. The P. lima enzyme was characterized biochemically and appears to consist of two different isoforms in the R2C2 configuration. Whole cell extracts of P. micans and P. minimum treated with the specific PKA inhibitor peptide PKI (5–24) or cAMP demonstrated altered intensities of phosphopeptide 32P labeling, most likely involving regulation of a protein phosphatase via PKA activity. A primary candidate for PKA regulation is protein phosphatase-1 (PP-1), which in P. lima possesses a classical PKA consensus phosphorylation site. We demonstrate that a peptide fragment of PP-1 from P. lima corresponding to this PKA phosphorylation site can be effectively phosphorylated by PKA and dephosphorylated by calcineurin. We speculate that PP-1 activity among several lower eukaryotes may be mediated directly by reversible phosphorylation. Higher eukaryotes may have developed inhibitor proteins to provide more complex regulation of protein phosphatase activity.Key words: cAMP-dependent protein kinase, protein phosphatase-1, dinoflagellates, Prorocentrum lima, okadaic acid.

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