Activation of deoxycytidine kinase in lymphocytes is calcium dependent and involves a conformational change detectable by native immunostaining.
Deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, plays a seminal role in the bioactivation of a wide array of cytotoxic nucleoside analogues. Recently, activation of dCK has been considered as a protective cellular response to a number of DNA-damaging agents in lymphocytes. Regarding the molecular mechanism of the enzyme activation, a post-translational modification by protein phosphorylation has been suggested. Here we provide evidence that both the activation process and the maintenance of the activated state require free cytosolic calcium. BAPTA-AM, a cell-permeable calcium chelator selectively inhibited the activation of dCK in a time- and concentration-dependent manner while extracellular calcium depletion had no effect. On the other hand, elevation of cytoplasmic calcium levels by thapsigargin did not potentiate the enzyme, referring to the permissive function of calcium in the activation process. Denaturing Western blots of extracts from lymphocytes incubated with 2-chlorodeoxyadenosine, aphidicolin and/or BAPTA-AM clearly demonstrated that dCK protein levels were unchanged during these treatments. However, a striking correlation was found between enzyme activity and the intensity of dCK-specific signals in native Western blots. Extracts from CdA-treated cells were much better recognized by the antibody raised against the C-terminal peptide of dCK than the BAPTA-AM-treated samples. These results indicate that the calcium-dependent activation of dCK is accompanied by a conformational change that renders the C-terminal epitope more accessible to the antibody.
Keszler
Gergely
G
Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, P.O. Box 260, H-1444 Budapest, Hungary. keszler@puskin.sote.hu
Spasokoukotskaja
Tatjana
T
Csapo
Zsolt
Z
Talianidis
Iannis
I
Eriksson
Staffan
S
Staub
Maria
M
Sasvari-Szekely
Maria
M
D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003842:Deoxycytidine Kinase / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D003842:Deoxycytidine Kinase; D002118:Calcium