Phosphorylation by CaM kinase or PKA did not alter the Vmax of Ca2+ uptake in SERCA2a expressed in HEK-293 cells, either in the presence or absence of phospholamban.
The study challenges earlier reports by demonstrating that CaM kinase II phosphorylation does not stimulate the Vmax of SERCA2a activity, highlighting the need for adequate controls.
Earlier studies (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198-31206) have suggested that the Vmax of Ca2+ uptake is enhanced up to 2-fold through phosphorylation of Ser38 in the cardiac Ca2+-ATPase (SERCA2a) by calmodulin-dependent protein kinase (CaM kinase). It is difficult, however, to determine whether stimulation is caused by phosphorylation of the Ca2+-ATPase or by phosphorylation of phospholamban in cardiac microsomes. We have expressed SERCA2a in HEK-293 cells in the presence or absence of phospholamban and measured the effects on Ca2+ uptake activity of phosphorylation of microsomal proteins by CaM kinase or protein kinase A (PKA). We found no effect on the Vmax of Ca2+ uptake following phosphorylation by CaM kinase or PKA in either the presence or absence of phospholamban. The K0.5 for Ca2+ dependence of Ca2+ transport, however, was shifted following phosphorylation by either CaM kinase or PKA in those microsomes containing both SERCA2a and phospholamban, but not in those expressing only SERCA2a. Thus, we cannot confirm earlier reports of stimulation of SERCA2a activity by CaM kinase II phosphorylation of Ser38. Our studies, however, emphasize the need for adequate controls for measurement of Vmax.
Odermatt et al. (Sat,) reported a other. Phosphorylation by CaM kinase or PKA vs. Unphosphorylated state was evaluated on Vmax of Ca2+ uptake. Phosphorylation by CaM kinase or PKA did not alter the Vmax of Ca2+ uptake in SERCA2a expressed in HEK-293 cells, either in the presence or absence of phospholamban.