MEMBRANE TRANSPORT STRUCTURE FUNCTION AND BIOGENESIS
Plasma Membrane Ca2+ATPase Isoform 4b Is Cleaved and Activated by Caspase-3 during the Early Phase of Apoptosis*

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The plasma membrane Ca2+ pump (PMCA) is an essential element in the complex of mechanisms that maintain low intracellular Ca2+ 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 (1077DEID1080) 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 Asp1080 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 the1077DEID1080 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 (1077AEID1080,1077DEIA1080, and1077AEIA1080 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 Ca2+ concentration than the autoinhibited form, may have an important role in regulating intracellular Ca2+ concentration in the apoptotic cell.

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Published, JBC Papers in Press, December 20, 2001, DOI 10.1074/jbc.M109548200

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This work was supported by Hungarian Academy of Sciences Grants OTKA T034536, OTKA M36200, and OM Mu-00350/2000 (to A. E.), by OTKA Postdoctoral Fellowship D38465 and Bolyai Janos Research Fellowship BO/00245/01 (to K. P.), and by National Institutes of Health Grant GM28835 (to J. T. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.