Activation of deoxycytidine kinase in lymphocytes is calcium dependent and involves a conformational change detectable by native immunostaining

Gergely Keszler, T. Spasokoukotskaja, Z. Csapó, Iannis Talianidis, Staffan Eriksson, M. Staub, M. Sasvári

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)947-955
Number of pages9
JournalBiochemical Pharmacology
Volume67
Issue number5
DOIs
Publication statusPublished - Mar 1 2004

Fingerprint

Deoxycytidine Kinase
Lymphocytes
Chemical activation
Calcium
Enzymes
Western Blotting
Cladribine
Aphidicolin
Salvaging
Phosphorylation
Enzyme Activation
Thapsigargin
Antibodies
Enzyme activity
Post Translational Protein Processing
Nucleosides
Epitopes
Proteins
Cells
Maintenance

Keywords

  • 2-Chlorodeoxyadenosine (Cladribine)
  • BAPTA-AM
  • Calcium
  • Conformational change
  • Deoxycytidine kinase
  • Native immunoblot

ASJC Scopus subject areas

  • Pharmacology

Cite this

Activation of deoxycytidine kinase in lymphocytes is calcium dependent and involves a conformational change detectable by native immunostaining. / Keszler, Gergely; Spasokoukotskaja, T.; Csapó, Z.; Talianidis, Iannis; Eriksson, Staffan; Staub, M.; Sasvári, M.

In: Biochemical Pharmacology, Vol. 67, No. 5, 01.03.2004, p. 947-955.

Research output: Contribution to journalArticle

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