scholarly journals Binding of cGMP to both allosteric sites of cGMP-binding cGMP-specific phosphodiesterase (PDE5) is required for its phosphorylation

1998 ◽  
Vol 329 (3) ◽  
pp. 505-510 ◽  
Author(s):  
V. Illarion TURKO ◽  
H. Sharron FRANCIS ◽  
D. Jackie CORBIN
Keyword(s):  
Protein Kinase ◽  
Cyclic Nucleotide ◽  
Binding Sites ◽  
Catalytic Subunit ◽  
Site Directed Mutagenesis ◽  
Nucleotide Receptors ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Allosteric Sites ◽  
Dependent Protein

cGMP-binding phosphodiesterases contain two homologous allosteric cGMP-binding sites (sites and ) that are arranged in tandem; they constitute a superfamily of mammalian cyclic nucleotide receptors distinct from the cyclic nucleotide-dependent protein kinases/cation channels family. The functional role of each of these two sites in the phosphodiesterases is not known. The cGMP-binding sites of one of these phosphodiesterases, the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE, PDE5), have been analysed by using site-directed mutagenesis. Mutations that affect cGMP binding to either one or both allosteric sites do not influence cGMP hydrolysis in the catalytic site under the conditions used. However, compared with wild-type enzyme, the D289A, D478A and D289A/D478A mutants, which are defective in cGMP binding to either site or site , or both allosteric sites, require much higher cGMP concentrations for the allosteric stimulation of phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. The cGMP effect is on the cGB-PDE rather than on the catalytic subunit of the protein kinase because the latter enzyme does not require cGMP for activity. The D289N mutant, which has higher binding affinity for cGMP than does the wild-type enzyme, is phosphorylated at lower concentrations of cGMP than is the wild-type enzyme. It is concluded that cGMP binding to the allosteric sites of cGB-PDE does not directly affect catalysis, but binding to both of these sites regulates phosphorylation of this enzyme.

scholarly journals A relative of the catalytic subunit of cyclic AMP-dependent protein kinase in Aplysia spermatozoa.

1990 ◽  
Vol 10 (12) ◽  
pp. 6775-6780 ◽  
Author(s):  
S Beushausen ◽  
H Bayley
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Nucleotide ◽  
Aplysia Californica ◽  
Catalytic Subunit ◽  
Spermatogenic Cells ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Mature Spermatozoa ◽  
Kinase Subfamily

Transcripts encoding CAPL-B, an apparent member of the cyclic-nucleotide-regulated kinase subfamily in Aplysia californica, are found exclusively in the ovotestis and are concentrated in meiotic and postmeiotic spermatogenic cells. The CAPL-B polypeptide is present in mature spermatozoa, suggesting that the kinase plays a part in regulating events associated with fertilization.

scholarly journals Consequences of DNA-Dependent Protein Kinase Catalytic Subunit Deficiency on Recombinant Adeno-Associated Virus Genome Circularization and Heterodimerization in Muscle Tissue

2003 ◽  
Vol 77 (8) ◽  
pp. 4751-4759 ◽  
Author(s):  
Dongsheng Duan ◽  
Yongping Yue ◽  
John F. Engelhardt
Keyword(s):  
Protein Kinase ◽  
Muscle Tissue ◽  
Southern Blotting ◽  
Catalytic Subunit ◽  
Inverted Terminal Repeat ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Adeno Associated Virus ◽  
Dependent Protein

ABSTRACT Circular concatemerization of the recombinant adeno-associated virus (rAAV) genome has been suggested as the predominant process facilitating long-term rAAV transduction in muscle. A recent study (S. Song, P. J. Laipis, K. I. Berns, and T. R. Flotte, Proc. Natl. Acad. Sci. USA 98:4084-4088, 2001) with SCID mice, which are defective in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), has suggested that DNA-PKcs regulates the removal of free rAAV vector ends in muscle tissue. In the present study, we have sought to evaluate whether a lack of DNA-PKcs activity reduces circularization of rAAV genomes in SCID muscle and whether such a reduction alters the directivity of heterodimerization. Consistent with the previous report, linear rAAV genomes and free vector ends were detected only in DNA-PKcs-deficient muscle by Southern blotting. Appreciable amounts of circular rAAV genomes were detected in both DNA-PKcs-deficient and wild-type muscle samples by Southern blotting and bacterial trapping experiments. The existence of double-D inverted terminal repeat circular intermediates in SCID and wild-type muscles was also supported by their sensitivity to T7 endonuclease I digestion. However, DNA-PKcs-deficient muscle did demonstrate a ∼50% reduction in the abundance of rescued circular genomes, despite equivalent levels of single rAAV transduction seen in wild-type animals. Dual trans-splicing lacZ vectors were used to functionally evaluate directional head-to-tail intermolecular viral genome concatamerization in vivo. Although AAV genomes are processed differently in SCID and wild-type muscles, a comparable level of trans-splicing-mediated β-galactosidase expression was observed in both strains, suggesting that both circular and linear AAV concatemers may have contributed to the trans-splicing-mediated transgene expression. In summary, we have shown that SCID skeletal muscle retains a fairly high capacity to form circular genomes, despite a significant increase in linear vector genomes. Furthermore, the alteration in equilibrium between circular and linear concatemer genomes caused by the lack of DNA-PKcs activity does not appear to significantly affect the efficiency of dual-vector gene expression from head-to-tail linear and/or circular heterodimers.

A relative of the catalytic subunit of cyclic AMP-dependent protein kinase in Aplysia spermatozoa

1990 ◽  
Vol 10 (12) ◽  
pp. 6775-6780
Author(s):  
S Beushausen ◽  
H Bayley
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Nucleotide ◽  
Aplysia Californica ◽  
Catalytic Subunit ◽  
Spermatogenic Cells ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Mature Spermatozoa ◽  
Kinase Subfamily

Transcripts encoding CAPL-B, an apparent member of the cyclic-nucleotide-regulated kinase subfamily in Aplysia californica, are found exclusively in the ovotestis and are concentrated in meiotic and postmeiotic spermatogenic cells. The CAPL-B polypeptide is present in mature spermatozoa, suggesting that the kinase plays a part in regulating events associated with fertilization.

scholarly journals A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation

2020 ◽  
Vol 295 (10) ◽  
pp. 3316-3329
Author(s):  
Tung O. Chan ◽  
Roger S. Armen ◽  
Santosh Yadav ◽  
Sushrut Shah ◽  
Jin Zhang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Catalytic Subunit ◽  
Site Directed Mutagenesis ◽  
Airway Smooth Muscle Cells ◽  
Activation Loop ◽  
Allosteric Sites ◽  
And Migration ◽  
Kinase A ◽  
In Cells

Phosphorylation of specific residues in the activation loops of AGC kinase group (protein kinase A, G, and C families) is required for activity of most of these kinases, including the catalytic subunit of PKA (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it “constitutively” phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FXXF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FXXF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FXXW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate the phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.

Mapping of the two intrachain cyclic nucleotide binding sites of adenosine cyclic 3',5'-phosphate dependent protein kinase I

Biochemistry ◽  
1983 ◽  
Vol 22 (5) ◽  
pp. 1094-1101 ◽  
Author(s):  
Stein O. Doeskeland ◽  
Dagfinn Oegreid ◽  
Roald Ekanger ◽  
Priscilla A. Sturm ◽  
Jon P. Miller ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Nucleotide ◽  
Binding Sites ◽  
Nucleotide Binding ◽  
Dependent Protein Kinase ◽  
Cyclic Nucleotide Binding ◽  
Nucleotide Binding Sites ◽  
Dependent Protein
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