scholarly journals Release of Ca2+ by inositol 1,4,5-trisphosphate in platelet membrane vesicles is not dependent on cyclic AMP-dependent protein kinase

1989 ◽  
Vol 257 (3) ◽  
pp. 715-721 ◽  
Author(s):  
F O'Rourke ◽  
G B Zavoico ◽  
M B Feinstein
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Membrane Protein ◽  
Protein Phosphorylation ◽  
Kinase Inhibitor ◽  
Membrane Vesicles ◽  
Platelet Membrane ◽  
Dependent Protein Kinase ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Protein

In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation. IP3-induced release of Ca2+ was also unaffected by the absence of ATP from the medium, supporting the conclusion that Ca2+ release by IP3 does not require the phosphorylation of membrane protein.

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.

scholarly journals Protein kinase activities in rat pancreatic islets of Langerhans

1979 ◽  
Vol 180 (1) ◽  
pp. 219-229 ◽  
Author(s):  
M C Sugden ◽  
S J Ashcroft ◽  
P H Sugden
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Islets Of Langerhans ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitor ◽  
Dependent Protein Kinase ◽  
Rat Islets ◽  
Dependent Protein ◽  
Rat Islets Of Langerhans ◽  
Independent Protein

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637–6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to ‘Type I’ and ‘Type II’ cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218–225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.

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