scholarly journals Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

1980 ◽  
Vol 185 (1) ◽  
pp. 89-99 ◽  
Author(s):  
O G Issinger ◽  
H Beier ◽  
N Speichermann ◽  
V Flokerzi ◽  
F Hofmann
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Ribosomal Proteins ◽  
Small Subunit ◽  
The Other ◽  
Dependent Protein Kinase ◽  
Ribosomal Subunits ◽  
Dependent Protein ◽  
Ascites Tumour Cells

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.

Chemically Induced, Activity-Independent LTD Elicited by Simultaneous Activation of PKG and Inhibition of PKA

1999 ◽  
Vol 82 (3) ◽  
pp. 1577-1589 ◽  
Author(s):  
Linda Santschi ◽  
Magali Reyes-Harde ◽  
Patric K. Stanton
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Long Term Potentiation ◽  
Dependent Protein Kinase ◽  
Schaffer Collateral ◽  
Chemically Induced ◽  
Bath Application ◽  
Dependent Protein

Although it is widely agreed that cyclic AMP is necessary for the full expression of long-term potentiation of synaptic strength, it is unclear whether cyclic AMP or cyclic AMP-dependent protein kinase (PKA) play roles in the induction of long-term depression (LTD). We show here that two PKA inhibitors, H-89 (10 μM) and KT5720 (1 μM), are unable to block induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices in vitro. Rather, H-89 enhanced the magnitude of LTD induced by submaximal low-frequency stimulation. Raising [cGMP] with zaprinast (20 μM), a selective type V phosphodiesterase inhibitor, reversibly depressed synaptic potentials. However, coapplication of H-89 plus zaprinast converted this to a robust LTD that depended critically on activation of cyclic GMP-dependent protein kinase (PKG). Chemically induced LTD is activity-independent because it could be induced without stimulation and in tetrodotoxin (0.5 μM). Additionally, chemical LTD did not require activation of N-methyl-d-aspartate or GABA receptors and could be reversed by LTP. Stimulus-induced LTD occluded chemical LTD, suggesting a common expression mechanism. In contrast to bath application, postsynaptic infusion of H-89 into CA1 pyramidal neurons did not enhance LTD, suggesting a presynaptic site of action. Further evidence for a presynaptic locus was supplied by experiments where H-89 applied postsynaptically along with bath application of zaprinast was unable to produce chemical LTD. Thus simultaneous presynaptic generation of cyclic GMP and inhibition of PKA is sufficient to induce LTD of synaptic transmission at Schaffer collateral-CA1 synapses.

scholarly journals Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells.

1980 ◽  
Vol 87 (2) ◽  
pp. 336-345 ◽  
Author(s):  
C L Browne ◽  
A H Lockwood ◽  
J L Su ◽  
J A Beavo ◽  
A L Steiner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Cyclic Gmp ◽  
Cyclic Nucleotides ◽  
Mitotic Spindle ◽  
Cyclic Nucleotide ◽  
Dependent Protein Kinase ◽  
Mitotic Apparatus ◽  
Dependent Protein

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide-dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.

scholarly journals Phosphoproteins associated with cyclic nucleotide stimulation of ciliary motility in Paramecium

1990 ◽  
Vol 95 (2) ◽  
pp. 219-230
Author(s):  
N.M. Bonini ◽  
D.L. Nelson
Keyword(s):  
Molecular Weight ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Permeabilized Cells ◽  
Ciliary Motility ◽  
Dependent Protein

Permeabilized, MgATP-reactivated cells of Paramecium (models) respond to cyclic AMP and cyclic GMP by increasing forward swimming speed. In association with the motile response, cyclic AMP and 8-bromo-cyclic GMP (8-Br-cyclic GMP) stimulated protein phosphorylation. Cyclic AMP addition to permeabilized cells reproducibly stimulated the phosphorylation of 10 proteins, ranging in molecular weight from 15 to 110K (K = 10(3) Mr). 8-Br-cyclic GMP, which selectively activates the cyclic GMP-dependent protein kinase of Paramecium, stimulated the phosphorylation of a subset of the proteins phosphorylated by cyclic AMP. Ca2+ addition caused backward swimming and stimulated the phosphorylation of four substrates, including a 25K target that may also be phosphorylated in response to cyclic nucleotide addition. Ba2+ and Sr2+ also induced backward swimming, but did not cause detectable phosphorylation. To identify ciliary targets of cyclic nucleotide-dependent protein kinase activity, permeabilized cells were deciliated following reactivation of motility with Mg-[gamma-32P]ATP in the presence or absence of cyclic nucleotide. Soluble proteins of the deciliation supernatant were enriched in 15 cyclic AMP-stimulated phosphoproteins, ranging in molecular weight from 15 to 95K. Most of the ciliary substrates were axonemal and could be released by high salt solution. A 29K protein that copurified in sucrose gradients with the 22S dynein, and a high molecular weight protein (greater than 300K) in the 19 S region were phosphorylated when cyclic AMP was added to permeabilized, motile cells. These data suggest that regulation of ciliary motility by cyclic AMP may include phosphorylation of dynein-associated proteins.

Changes in sodium, potassium-ATPase induced by repeated fencamfamine: the roles of cyclic AMP-dependent protein kinase and the nitric oxide–cyclic GMP pathway

2003 ◽  
Vol 45 (8) ◽  
pp. 1151-1159 ◽  
Author(s):  
Carolina Demarchi Munhoz ◽  
Isaias Glezer ◽  
Elisa Mitiko Kawamoto ◽  
Ana Paula Natalini Araújo ◽  
Lucília B Lepscha ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Dependent Protein Kinase ◽  
Sodium Potassium ◽  
Dependent Protein ◽  
Potassium Atpase

scholarly journals Differential responses of cyclic GMP-dependent and cyclic AMP-dependent protein kinases to synthetic peptide inhibitors

1983 ◽  
Vol 213 (1) ◽  
pp. 159-164 ◽  
Author(s):  
D B Glass
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Cyclic Gmp ◽  
Synthetic Peptide ◽  
Peptide Inhibitors ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
Peptide Substrates ◽  
Dependent Protein

The peptide Arg-Lys-Arg-Ala-Arg-Lys-Glu was synthesized and tested as an inhibitor of cyclic GMP-dependent protein kinase. This synthetic peptide is a non-phosphorylatable analogue of a substrate peptide corresponding to a phosphorylation site (serine-32) in histone H2B. The peptide was a competitive inhibitor of cyclic GMP-dependent protein kinase with respect to synthetic peptide substrates, with a Ki value of 86 microM. However, it did not inhibit phosphorylation of intact histones by cyclic GMP-dependent protein kinase under any conditions tested. Arg-Lys-Arg-Ala-Arg-Lys-Glu competitively inhibited the phosphorylation of either peptides or histones by the catalytic subunit of cyclic AMP-dependent protein kinase, with similar Ki values (550 microM) for both of these substrates. The peptide Leu-Arg-Arg-Ala-Ala-Leu-Gly, which was previously reported to be a selective inhibitor of both peptide and histone phosphorylation by cyclic AMP-dependent protein kinase, was a poor inhibitor of cyclic GMP-dependent protein kinase acting on peptide substrates (Ki = 800 microM), but did not inhibit phosphorylation of histones by cyclic GMP-dependent protein kinase. The selectivity of these synthetic peptide inhibitors toward either cyclic GMP-dependent or cyclic AMP-dependent protein kinases is probably based on differences in the determinants of substrate specificity recognized by these two enzymes. It is concluded that histones interact differently with cyclic GMP-dependent protein kinase from the way they do with the catalytic subunit of cyclic AMP-dependent protein kinase.

Sign in / Sign up

Export Citation Format

Share Document