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.
Pászty
Katalin
K
National Institute of Haematology and Immunology, Daroczi ut 24, Budapest 1113, Hungary.
Verma
Anil K
AK
Padányi
Rita
R
Filoteo
Adelaida G
AG
Penniston
John T
JT
Enyedi
Agnes
A
D000595:Amino Acid Sequence; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000378:metabolism*; D004396:Coloring Agents / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D053499:Plasma Membrane Calcium-Transporting ATPases; D017354:Point Mutation; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D016324:Sequence Tagged Sites; D013997:Time Factors; D014162:Transfection
D017304:Annexin A5; D002147:Calmodulin; D027682:Cation Transport Proteins; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D020033:Protein Isoforms; D011994:Recombinant Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium