Mechanism of the stimulation of calcium ion dependent ATPase of cardiac sarcoplasmic reticulum by adenosine 3',5'-monophosphate dependent protein kinase

Biochemistry ◽  
1980 ◽  
Vol 19 (23) ◽  
pp. 5434-5439 ◽  
Author(s):  
Evangelia Galani Kranias ◽  
Frederic Mandel ◽  
Taitzer Wang ◽  
Arnold Schwartz
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Calcium Ion ◽  
Dependent Protein Kinase ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Dependent Atpase ◽  
Dependent Protein ◽  
Stimulation Of

scholarly journals Translation of Ser16 and Thr17 phosphorylation of phospholamban into Ca2+-pump stimulation

1996 ◽  
Vol 316 (1) ◽  
pp. 201-207 ◽  
Author(s):  
Wayne A. JACKSON ◽  
John COLYER
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Dependent Protein Kinase ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Oligomeric Protein ◽  
Pump Activity ◽  
Dependent Protein ◽  
Indicator Dye ◽  
Phospholamban Phosphorylation ◽  
Stimulation Of

Stimulation of cardiac sarcoplasmic reticulum Ca2+-pump activity is achieved by phosphorylation of the oligomeric protein phospholamban at either Ser16 or Thr17. The altered mobility of phosphorylated forms of pentameric phospholamban has been utilized to demonstrate that the mechanisms of phosphorylation of the two sites differ. Phosphorylation of Ser16 by the AMP-dependent protein kinase proceeds via a random mechanism [Li, Wang and Colyer (1990) Biochemistry 29, 4535–4540], whereas phosphorylation of Thr17 by calmodulin-dependent protein kinase is shown here to proceed via a co-operative mechanism. This co-operative reaction mechanism was unaffected by the phosphorylation status of Ser16. These two mechanisms of phosphorylation generate very different phosphoprotein profiles depending on whether the Ser16 or Thr17 residue is phosphorylated. The translation of these patterns of phosphorylation into Ca2+-pump function was reviewed using a fluorimetric Ca2+-indicator dye, fluo-3, to measure Ca2+ uptake by cardiac sarcoplasmic reticulum vesicles. The rate of Ca2+ accumulation, which parallels Ca2+-pump activity, was stimulated in proportion with the stoichiometry of phospholamban phosphorylation, irrespective of whether phosphorylation was on Ser16 or Thr17.

scholarly journals Effect of cyclic AMP-dependent phosphorylation on the reactivity of sulphydryl groups in cardiac sarcoplasmic reticulum

1980 ◽  
Vol 192 (3) ◽  
pp. 867-872 ◽  
Author(s):  
C J Limas
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Cyclic Amp ◽  
Thiol Groups ◽  
Dependent Protein Kinase ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Dependent Atpase ◽  
Dependent Protein ◽  
Sulphydryl Groups ◽  
Atpase Inhibition

Phosphorylation of cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase results in enhanced Ca2+ transport even though Ca2+-dependent ATPase is not a substrate for the kinase. The mechanisms involved in this enhancement are not clear. In the present study, we used the reactivity of sulphydryl groups in the Ca2+-dependent ATPase as an index of conformational change during the Ca2+ transport cycle and examined the effects of protein kinase-catalysed phosphorylation. N-Ethylmaleimide alkylation allowed the distinction of several thiol groups with variable functional significance for the ATPase. A sulphydryl group involved in the formation of the phosphorylated intermediate (EP) of the Ca2+-dependent ATPase was protected by adenosine 5′-[beta, gamma-imido]triphosphate. Reactivity of an additional thiol group was related to EP dephosphorylation and was dependent on Ca2+. The Ca2+ concentration for change in the reactivity of this sulphydryl group and ATPase inhibition occurred within the range for Ca2+ binding to the high-affinity sites. Phosphorylation of cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase resulted in decreased N-ethyl[1-14C]-maleimide binding and the ATPase inhibition; the thiol groups involved in EP dephosphorylation were selectively protected. The results indicate that protein kinase-catalysed phosphorylation results in conformational changes of the ATPase, which renders certain thiol groups inaccessible to N-ethylmaleimide. This conformational change may facilitate functional movements involved in Ca2+ transport.

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