Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. II. Effect of cAMP-dependent protein kinase

1990 ◽  
Vol 258 (6) ◽  
pp. C1086-C1091 ◽  
Author(s):  
P. Volpe ◽  
B. H. Alderson-Lang
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Catalytic Subunit ◽  
Scatchard Analysis ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Plot Analysis ◽  
Receptor Sites ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Protein

The effect of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) on Ca2+ loading, inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release, and [3H]IP3 binding of canine cerebellar membrane fractions was investigated. PKA in the presence of cAMP and the catalytic subunit of PKA did not change Ca2+ loading yet increased the extent of IP3-induced Ca2+ release by approximately 35%. Hill plot analysis indicated that the catalytic subunit of PKA increased the apparent Michaelis constant of IP3-induced Ca2+ release twofold, from 0.3 to 0.7 microM IP3. The protein kinase inhibitor reversed these changes. cAMP affected neither Ca2+ loading nor IP3-induced Ca2+ release. The catalytic subunit of PKA did not appreciably affect the maximum binding and dissociation constant of [3H]IP3 binding, as judged by Scatchard analysis. Thus the catalytic subunit of PKA influences the opening of Ca2+ channels by IP3 without interfering with the binding of IP3 to its receptor sites.

The protein kinase A catalytic subunit Cβ2: molecular characterization and distribution of the splice variant

2000 ◽  
Vol 351 (1) ◽  
pp. 123-132 ◽  
Author(s):  
Sandra THULLNER ◽  
Frank GESELLCHEN ◽  
Stefan WIEMANN ◽  
Walter PYERIN ◽  
Volker KINZEL ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Splice Variant ◽  
Cardiac Tissue ◽  
Kinase Inhibitor ◽  
Morphological Changes ◽  
Affinity Purification ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Cβ2, a 46 kDa splice variant of the Cβ isoform, is the largest isoform so far described for catalytic subunits from cAMP-dependent protein kinase in mammals. It differs from Cβ in the first 15 N-terminal residues which are replaced with a 62-residue domain with no similarity to other known proteins. The Cβ2 protein was identified in cardiac tissue by MS, microsequencing and C-subunit-isoform-selective antibodies. The Cβ2 protein has a very low abundance of about 2% of total affinity-purified C subunits from bovine cardiac tissue. This, and the similarity of its biochemical properties to Cα and Cβ, are probably some of the reasons why the Cβ2 protein has escaped detection so far. The abundance of the Cβ2 protein differs dramatically between tissues, with most protein detected in heart, liver and spleen, and the lowest level in testis. Cβ2 protein shows kinase activity against synthetic substrates, and is inhibited by the protein kinase inhibitor peptide PKI(5-24). The degree of Cβ2 removal from tissue extracts by binding to PKI(5-24) depends on the cAMP level, i.e. on the dissociation state of the holoenzyme. Two sites in the protein are phosphorylated: Thr-244 in the activation segment and Ser-385 close to the C-terminus. By affinity purification and immunodetection Cβ2 was found in cattle, pig, rat, mouse and turkey tissue and in HeLa cells. In the cAMP-insensitive CHO 10260 cell line, which has normal Cβ but is depleted of Cα, stable transfection with Cβ2 restored most of the cAMP-induced morphological changes. Cβ2 is a ubiquitously expressed protein with characteristic properties of a cAMP-dependent protein kinase catalytic subunit.

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