scholarly journals Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility.

1986 ◽  
Vol 103 (2) ◽  
pp. 649-655 ◽  
Author(s):  
J S Tash ◽  
H Hidaka ◽  
A R Means
Keyword(s):  
Protein Kinase ◽  
De Novo ◽  
Protein Kinase Activity ◽  
Dependent Manner ◽  
Flagellar Motility ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Heat Stable ◽  
Protein Kinase N ◽  
Dependent Protein

Using a selective inhibitor of cAMP-dependent protein kinase, N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the requirement for cAMP-dependent phosphoproteins in the initiation of dog sperm flagellar motility was examined. H-8 inhibited motility of live as well as reactivated sperm in a dose-dependent manner. The half-maximal inhibition of reactivated motility (32 microM) paralleled the inhibition of pure catalytic subunit of cAMP-dependent protein kinase (50 microM) measured under the same conditions. H-8 inhibited protein phosphorylation both in whole models and in isolated Nonidet P-40 (NP-40) extracts of sperm. Axokinin, the heat-stable NP-40-soluble protein whose phosphorylation is required for flagellar reactivation, represented 97% of the de novo phosphate incorporation in the NP-40 extract after stimulation by cAMP. 500 microM H-8 inhibited axokinin phosphorylation by 87%. When sperm were reactivated in the presence of up to 5 mM H-8 with NP-40 extract that had been prephosphorylated with cAMP-dependent protein kinase, then neither cAMP nor cAMP-dependent protein kinase activity was required for full flagellar reactivation. If sperm were rendered completely immotile by pretreatment with H-8, then the resulting model remained immotile in the continued presence of H-8 unless prephosphorylated axokinin was added. These results suggest that phosphorylated axokinin is not only required for flagellar reactivation but is sufficient as well.

scholarly journals Disruption of the A-Kinase Anchoring Domain in Flagellar Radial Spoke Protein 3 Results in Unregulated Axonemal cAMP-dependent Protein Kinase Activity and Abnormal Flagellar Motility

2006 ◽  
Vol 17 (6) ◽  
pp. 2626-2635 ◽  
Author(s):  
Anne R. Gaillard ◽  
Laura A. Fox ◽  
Jeanne M. Rhea ◽  
Branch Craige ◽  
Winfield S. Sale
Keyword(s):  
Protein Kinase ◽  
Physiological Role ◽  
Binding Domain ◽  
Cell Phenotype ◽  
Protein Kinase Activity ◽  
Flagellar Motility ◽  
Dependent Protein Kinase ◽  
Radial Spoke ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility.

Analytical subcellular distribution of cAMP-dependent protein kinase activity in bull sperm

1984 ◽  
Vol 10 (4) ◽  
pp. 433-444 ◽  
Author(s):  
Claude C. Pariset ◽  
Jacqueline S. Weinman ◽  
Francoise T. Escaig ◽  
Michele Y. Guyot ◽  
Francine C. Iftode ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Subcellular Distribution ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Bull Sperm

Involvement of cAMP-dependent protein kinase in the regulation of heart contractile force. II

1979 ◽  
Vol 236 (1) ◽  
pp. H84-H91
Author(s):  
S. L. Keely ◽  
A. Eiring
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Activity Ratio ◽  
Contractile Force ◽  
Protein Kinase Activity ◽  
Phosphorylase Activity ◽  
Dependent Protein Kinase ◽  
Guinea Pig Heart ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

The effects of histamine on heart cAMP-dependent protein kinase activity, cAMP levels, phosphorylase activity, and contractile force was investigated in the perfused guinea pig heart. To accurately determine the protein kinase activity ratio in guinea pig heart, it was necessary to measure kinase activity in whole homogenates immediately after homogenization of the tissue. Histamine produced a rapid dose-dependent increase in cAMP and the protein kinase activity ratio followed by increased in contractile force and phosphorylase activity. There was a good correlation between the degree of protein kinase activation and the increase in phosphorylase and force. The beta-adrenergic blocking agent propranolol did not reduce the effects of histamine, but metiamide, a potent H2-receptor antagonist, greatly attenuated all the effects of histamine. The data support the hypothesis that increases in heart cAMP-dependent protein kinase activity produce corresponding increases in contractile force and phosphorylase activity.

scholarly journals Imaging of cAMP-dependent protein kinase activity in living neural cells using a novel fluorescent substrate

FEBS Letters ◽  
1997 ◽  
Vol 414 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Hideyoshi Higashi ◽  
Kazuki Sato ◽  
Atsuko Ohtake ◽  
Akira Omori ◽  
Sachiyo Yoshida ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Neural Cells ◽  
Dependent Protein Kinase ◽  
Fluorescent Substrate ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.

1994 ◽  
Vol 13 (5) ◽  
pp. 1065-1072 ◽  
Author(s):  
A.C. Chao ◽  
F.J. de Sauvage ◽  
Y.J. Dong ◽  
J.A. Wagner ◽  
D.V. Goeddel ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Cl Channel ◽  
Camp Dependent Protein Kinase ◽  
Heat Stable ◽  
Dependent Protein

scholarly journals Characterization of ATM Expression, Localization, and Associated DNA-dependent Protein Kinase Activity

1998 ◽  
Vol 9 (9) ◽  
pp. 2361-2374 ◽  
Author(s):  
Dennis P. Gately ◽  
James C. Hittle ◽  
Gordon K. T. Chan ◽  
Tim J. Yen
Keyword(s):  
Dna Damage ◽  
Protein Kinase ◽  
Ataxia Telangiectasia ◽  
Kinase Activity ◽  
Human Fibroblasts ◽  
Protein Kinase Activity ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Atm Protein

Ataxia telangiectasia–mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA–dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.

Relaxing effect of pharmacologic interventions increasing cAMP in rat heart

1981 ◽  
Vol 240 (4) ◽  
pp. H441-H447
Author(s):  
L. Vittone ◽  
A. Grassi ◽  
L. Chiappe ◽  
M. Argel ◽  
H. E. Cingolani
Keyword(s):  
Protein Kinase ◽  
Rat Heart ◽  
Kinase Activity ◽  
Isolated Rat Heart ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
High Calcium ◽  
Dependent Protein ◽  
Camp Levels

The relationship between cAMP and relaxation was studied in the isolated rat heart beating at constant rate and perfused at constant coronary flow. After treatment during 1 min with different positive inotropic interventions, cyclic nucleotide levels (cAMP and cGMP) and cAMP-dependent protein kinase activity were determined in heart homogenates. Glucagon, norepinephrine, and isoproterenol increased cAMP from 0.503 +/- 0.025 pmol/mg wet wt to 1.051 +/- 0.099, 0.900 +/- 0.064, and 0.982 +/- 0.138, respectively. Simultaneously glucagon, norepinephrine, and isoproterenol increased cAMP-dependent protein kinase activity ratio from 0.21 +/- 0.02 to 0.45 +/- 0.04, 0.33 +/- 0.02, and 0.34 +/- 0.02, respectively. The ratio between maximal velocities of contraction and relaxation (+T/-T) was significantly decreased by these interventions, whereas time to peak tension (TTP) was shortened by norepinephrine and isoproterenol. High calcium, ouabain, and paired stimulation did not affect cAMP levels, TTP, or +T/-T. A striking correlation was found between cAMP-dependent protein kinase activity and relaxation induces, i.e., TTP, -T, or +T/-T (r = +/- 0.7 to -0.9). Results suggest that inotropic interventions increasing cAMP levels might be primarily affecting intracellular mechanisms causing relaxation.

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