IDENTIFICATION OF SER(38) AS THE SITE IN CARDIAC SARCOPLASMIC-RETICULUM CA2-ATPASE THAT IS PHOSPHORYLATED BY CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE()

In previous studies (Xu, A., Hawkins, C., and Narayanan, N. (1993) J. Biol. Chem. 268, 8394-8397), the Ca2+-ATPase of cardiac muscle sarcopl asmic reticulum (SERCA2) was shown to be phosphorylated by Ca2+/calmod ulin-dependent protein kinase II (CaM kinase) on a serine residue, lik ely to be either Ser(38), Ser(167), or Ser(531). SERCA2 and SERCA2 mut ants S38A, S167A, and S531A were expressed in HEK-293 cells and tested for phosphorylation with CaM kinase. Mutant S38A was not phosphorylat ed, while mutants S167A and S531A were phosphorylated, suggesting that Ser(38) is the site of CaM kinase phosphorylation in SERCA2. This con clusion was supported by the observation that phosphorylation of SERCA 2 and mutants S167A and S531A by CaM kinase increased the V-max for Ca 2+ transport, while the V-max for Ca2+ transport by mutant S38A was un affected by exposure to a phosphorylation reaction mix. SERCA1, contai ning a potential CaM kinase phosphorylation site at Ser(167) and two S ERCA1 mutants, K35R plus H38S and T532S, in which potential CaM kinase sites were created, were not phosphorylated by CaM kinase, and V-max for Ca2+ transport was unaffected by exposure to a phosphorylation rea ction mix. Thus phosphorylation of Ser(38) in SERCA2 results in a uniq ue activation of V-max for Ca2+ transport, providing a potential regul atory mechanism for Ca2+ removal from cardiac and other tissues in whi ch SERCA2 is expressed.