Major Phosphorylation Site (Ser55) of Neurofilament L by Cyclic AMP-Dependent Protein Kinase in Rat Primary Neuronal Culture

2000 ◽  
Vol 74 (3) ◽  
pp. 949-959 ◽  
Author(s):  
Yu Nakamura ◽  
Ryota Hashimoto ◽  
Yujiro Kashiwagi ◽  
Saburo Aimoto ◽  
Eriko Fukusho ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Phosphorylation Site ◽  
Neuronal Culture ◽  
Primary Neuronal Culture ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Major Phosphorylation Site

scholarly journals Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

1991 ◽  
Vol 279 (3) ◽  
pp. 727-732 ◽  
Author(s):  
G B Sala-Newby ◽  
A K Campbell
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Specific Activity ◽  
Phosphorylation Site ◽  
Live Cells ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Ph Optimum ◽  
Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

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.

scholarly journals An active twenty-amino-acid-residue peptide derived from the inhibitor protein of the cyclic AMP-dependent protein kinase

1985 ◽  
Vol 231 (3) ◽  
pp. 655-661 ◽  
Author(s):  
H C Cheng ◽  
S M van Patten ◽  
A J Smith ◽  
D A Walsh
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Amino Acid Residue ◽  
Phosphorylation Site ◽  
Inhibitor Protein ◽  
Inhibitory Peptide ◽  
Dependent Protein Kinase ◽  
Inhibitory Peptides ◽  
Dependent Protein

Digestion with Staphylococcus aureus V8 proteinase of the inhibitor protein of the cyclic AMP-dependent protein kinase results in the sequential formation of three active inhibitory peptides. The smallest active peptide has the sequence Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp . This 20-amino-acid-residue peptide has 20-40% of the activity of the native molecule and a Ki of 0.2 nM. Inhibition, as a minimum, appears to be based upon the inhibitor protein containing the recognition sequences that dictate protein-substrate-specificity. This inhibitory peptide also has sequence homology with the phosphorylation site for a protein kinase other than the cyclic AMP-dependent enzyme.

scholarly journals Cyclic AMP-dependent protein kinase inhibits ADH2 expression in part by decreasing expression of the transcription factor gene ADR1.

1997 ◽  
Vol 17 (3) ◽  
pp. 1450-1458 ◽  
Author(s):  
K M Dombek ◽  
E T Young
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Phosphorylation Site ◽  
Mrna Levels ◽  
Dependent Protein Kinase ◽  
Adh2 Promoter ◽  
Dependent Protein ◽  
Mutant Cells

In Saccharomyces cerevisiae, the unregulated cyclic AMP-dependent protein kinase (cAPK) activity of bcy1 mutant cells inhibits expression of the glucose-repressible ADH2 gene. The transcription factor Adr1p is thought to be the primary target of cAPK. Here we demonstrate that the decreased abundance of Adr1p in bcy1 mutant cells contributes to the inhibition of ADH2 expression. Activation of ADH2 transcription was blocked in bcy1 mutant cells, and UAS1, the Adr1p binding site in the ADH2 promoter, was sufficient to mediate this effect. Concurrent with this loss of transcriptional activation was an up to 30-fold reduction in the level of Adr1p. Mutating the strong cAPK phosphorylation site at serine 230 did not suppress this effect. Analysis of ADR1 mRNA levels and ADR1-lacZ expression suggested that decreased ADR1 transcription was responsible for the reduced protein level. In contrast to the ADH2 promoter, however, deletion analysis suggested that cAPK does not act through a discrete DNA element in the ADR1 promoter. The amount of Adr1p found in bcy1 mutant cells should have been sufficient to support 23% of the wild-type level of ADH2 expression. Since no ADH2 expression was detectable in bcy1 mutant cells, cAPK must also act by other mechanisms. Overexpression of Adr1p only partially restored ADH2 expression, indicating that some of these mechanisms may impinge upon events at or subsequent to the ADR1-dependent step in ADH2 transcriptional activation.

scholarly journals Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals

2004 ◽  
Vol 381 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Jing XUE ◽  
Peter J. MILBURN ◽  
Bernadette T. HANNA ◽  
Mark E. GRAHAM ◽  
John A. P. ROSTAS ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Nerve Terminals ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Phosphoamino Acid Analysis ◽  
The Brain ◽  
Major Phosphorylation Site

The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons.

scholarly journals Alteration of a cyclic AMP-dependent protein kinase phosphorylation site in the c-Fos protein augments its transforming potential

1992 ◽  
Vol 12 (3) ◽  
pp. 998-1006
Author(s):  
I Tratner ◽  
R Ofir ◽  
I M Verma
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Phosphorylation Site ◽  
Dependent Protein Kinase ◽  
Fos Protein ◽  
Dependent Protein ◽  
Cell Growth And Differentiation ◽  
Kinase Phosphorylation

We have studied the phosphorylation of the nuclear oncoprotein Fos by cyclic AMP-dependent protein kinase (PKA). We demonstrate that the human c-Fos protein, phosphorylated either in vitro with purified PKA or in vivo in JEG3 cells following treatment with forskolin, has similar phosphotryptic peptide maps. Serine 362, which constitutes part of a canonical PKA phosphorylation site (RKGSSS), is phosphorylated both in vivo and in vitro. A mutant of Fos protein in which serine residues 362 to 364 have been altered to alanine residues is not efficiently phosphorylated in vitro. Furthermore, Fos protein in which serines 362 to 364 have been altered to alanine shows increased transforming potential. We propose that phosphorylation of Fos by PKA is an important regulatory step in controlling its activity in normal cell growth and differentiation.

Sign in / Sign up

Export Citation Format

Share Document