title: The caspase-3 cleavage product of the plasma membrane Ca2+-ATPase 4b is activated and appropriately targeted.
abstract: The calmodulin-activated transporter hPMCA4 (human plasma membrane Ca2+-ATPase isoform 4) is a target for cleavage by caspase-3 during apoptosis. We have demonstrated that caspase-3 generates a 120 kDa fragment of this pump which lacks the complete autoinhibitory sequence [Paszty, Verma, Padanyi, Filoteo, Penniston and Enyedi (2002) J. Biol. Chem. 277, 6822-6829]. In the present study we analysed further the characteristics of the fragment of hPMCA4b produced by caspase-3. We did this by overexpressing the caspase-3 cleavage product of hPMCA4b in COS-7 and MDCKII (Madin-Darby canine kidney II) cells. This technique made it possible to clearly define the properties of this fragment, and we showed that it is constitutively active, as it forms a phosphoenzyme intermediate and has high Ca2+ transport activity in the absence of calmodulin. When this fragment of hPMCA4b was stably expressed in MDCKII cell clones, it was targeted without degradation to the basolateral plasma membrane. In summary, our studies emphasize that the caspase-3 cleavage product of hPMCA4b is constitutively active, and that the C-terminus is not required for proper targeting of hPMCA4b to the plasma membrane. Also, for the first time, we have generated cell clones that stably express a constitutively active PMCA.
authors.affiliation: Membrane Research Group of the Hungarian Academy of Sciences, Nádor u.7., H-1051, Budapest, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Pászty
authors.forename: Géza
authors.initials: G
authors.lastname: Antalffy
authors.forename: Alan R
authors.initials: AR
authors.lastname: Penheiter
authors.forename: László
authors.initials: L
authors.lastname: Homolya
authors.forename: Rita
authors.initials: R
authors.lastname: Padányi
authors.forename: Attila
authors.initials: A
authors.lastname: Iliás
authors.forename: Adelaida G
authors.initials: AG
authors.lastname: Filoteo
authors.forename: John T
authors.initials: JT
authors.lastname: Penniston
authors.forename: Agnes
authors.initials: A
authors.lastname: Enyedi
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004285:Dogs; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D053499:Plasma Membrane Calcium-Transporting ATPases; D021381:Protein Transport; D013379:Substrate Specificity
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C507517:ATP2B4 protein, human; D053499:Plasma Membrane Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium