Plasma membrane Ca2+ATPase isoform 4b is cleaved and activated by caspase-3 during the early phase of apoptosis

The plasma membrane Ca(2+) pump (PMCA) is an essential element in the complex of mechanisms that maintain low intracellular Ca(2+) concentration in the living cell. This pump is tightly regulated by calmodulin through binding to a high affinity calmodulin-binding domain at the C terminus that also serves as an autoinhibitor of the enzyme. Inspection of the C terminus of hPMCA4b, the most widely distributed form of PMCA, revealed a caspase-3 consensus sequence ((1077)DEID(1080)) just a few residues upstream of the calmodulin-binding domain. We demonstrate here that, in the early phase of apoptosis, hPMCA4b is cleaved at aspartic acid Asp(1080) in hPMCA4b-transfected COS-7 cells or in HeLa cells that naturally express this protein. This cleavage of hPMCA4b produces a single 120-kDa fragment that is fully active in the absence of calmodulin, because the whole inhibitory region downstream of the (1077)DEID(1080) sequence is removed. Our experiments show that caspase-3 or a caspase-3-like protease is responsible for the formation of the constitutively active 120-kDa PMCA4b fragment: 1) Pretreatment of the cells with the caspase-3 inhibitor Z-DEVD-FMK (benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone) was able to block the production of the 120-kDa fragment. 2) In vitro treatment of hPMCA4b with recombinant caspase-3 also generated a 120-kDa cleavage product, consistent with that seen in cells undergoing apoptosis. 3) Mutants in which the caspase-3 consensus sequence was altered ((1077)AEID(1080), (1077)DEIA(1080), and (1077)AEIA(1080) mutants) were resistant to proteolysis. Based on these data, we conclude that hPMCA4b is a newly identified, natural caspase-3 substrate. We suggest that a constitutively active form of this protein, responding much faster to an increase in Ca(2+) concentration than the autoinhibited form, may have an important role in regulating intracellular Ca(2+) concentration in the apoptotic cell.