The caspase-3 cleavage product of the plasma membrane Ca2+-ATPase 4b is activated and appropriately targeted.
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.
Pászty
Katalin
K
Membrane Research Group of the Hungarian Academy of Sciences, Nádor u.7., H-1051, Budapest, Hungary.
Antalffy
Géza
G
Penheiter
Alan R
AR
Homolya
László
L
Padányi
Rita
R
Iliás
Attila
A
Filoteo
Adelaida G
AG
Penniston
John T
JT
Enyedi
Agnes
A
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
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