scholarly journals Rat liver fructose-1,6-bisphosphatase. Identification of serine 338 as a third major phosphorylation site for cyclic AMP-dependent protein kinase and activity changes associated with multisite phosphorylation in vitro.

1987 ◽  
Vol 262 (34) ◽  
pp. 16699-16703
Author(s):  
KN Ekdahl
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Phosphorylation Site ◽  
Dependent Protein Kinase ◽  
Multisite Phosphorylation ◽  
Fructose 1,6 Bisphosphatase ◽  
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 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 Effects of phosphorylation on the kinetic properties of rat liver fructose-1,6-bisphosphatase

1984 ◽  
Vol 222 (1) ◽  
pp. 125-130 ◽  
Author(s):  
D W Meek ◽  
H G Nimmo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Kinetic Properties ◽  
Purification Procedure ◽  
Dependent Protein Kinase ◽  
Fructose 1,6 Bisphosphatase ◽  
Dependent Protein ◽  
A Subunit ◽  
Fructose 1,6 Bisphosphate

A new purification procedure for rat liver fructose-1,6-bisphosphatase that involves use of Procion Red-Sepharose is described. The purified enzyme was homogeneous, had a subunit Mr of 40 000-41 000 and seemed to be undegraded. The enzyme could be phosphorylated by cyclic AMP-dependent protein kinase with a stoicheiometry of one per subunit. Phosphorylation caused a 2-fold decrease in the Km of the enzyme for fructose 1,6-bisphosphate, but did not affect its allosteric responses to AMP, Mg2+ and fructose 2,6-bisphosphate.

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.

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.

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