Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca2+-ATPase by calmodulin and protein kinase A.
The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.
Valverde
Rafael H F
RH
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil.
Tortelote
Giovane G
GG
Lemos
Thiago
T
Mintz
Elisabeth
E
Vieyra
Adalberto
A
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004593:Electrophorus / Q000378:metabolism / Q000502:physiology*; D019811:Hydroxylamine / Q000737:chemistry / Q000494:pharmacology; D015151:Immunoblotting; D007700:Kinetics; D008956:Models, Chemical; D010766:Phosphorylation; D011485:Protein Binding; D013449:Sulfonamides / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D000096926:Benzenesulfonamides
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D001596:Benzylamines; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000252:Calcium-Transporting ATPases; D002147:Calmodulin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019811:Hydroxylamine; D013449:Sulfonamides; D019284:Thapsigargin; C072105:KN 93; D000096926:Benzenesulfonamides