Calmodulin effect on purified rat cortical plasma membrane Ca(2+)-ATPase in different phosphorylation states.
The plasma membrane Ca(2+)-ATPase in neuronal tissue plays an important role in fine tuning of the intracellular Ca(2+) concentration. The enzyme exhibits a high degree of tissue specificity and is regulated by several mechanisms. Here we analysed the relationship between separate modes of Ca(2+)-ATPase regulation, i.e., reversible phosphorylation processes mediated by protein kinases A and C, protein phosphatases PP1 and PP2A, and stimulation by calmodulin. The activity of PKA- or PKC-phosphorylated Ca(2+)-ATPase was influenced by the further addition of calmodulin, and this effect was more pronounced for PKC-phosphorylated calcium pump. In both cases the fluorescence study revealed the increased calmodulin binding, and for PKA-mediated phosphorylation it was correlated with a higher affinity of calcium pump for calmodulin. The incubation of Ca(2+)-ATPase with CaM prior to protein kinases action revealed that CaM presence counteracts the stimulatory effect of PKA and PKC. Under the in vitro assay cortical Ca(2+)-ATPase was a substrate for PP1 and PP2A. Protein phosphatases decreased both the basal activity of Ca(2+)-ATPase and its affinity for calmodulin. Fluorescence analysis confirmed the lowered ability of dephosphorylated Ca(2+)-ATPase for calmodulin binding. These results may suggest that interaction of CaM with calcium pump and its stimulatory action could be a partly separate phenomenon that is dependent on the phosphorylation state of Ca(2+)-ATPase.
Gromadzinska
E
E
Department of Biochemistry, Medical University, Lodz, Poland.
Lachowicz
L
L
Walkowiak
B
B
Zylinska
L
L
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D002147:Calmodulin / Q000494:pharmacology*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002540:Cerebral Cortex / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines; D015398:Signal Transduction
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D002147:Calmodulin; D005456:Fluorescent Dyes; D010761:Phosphorus Radioisotopes; D012235:Rhodamines; D000252:Calcium-Transporting ATPases