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Molecular biology --- Osmotic Pressure

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Deoxycytidine kinase (dCK) is an enzyme involved in the deoxynucleoside salvage pathway; is phosphorylates deoxycytidine, but also deoxyaadenosine and deoxyguanosine. In addition, dCK phosphorylates and activates several nucleoside analogues used as chemotherapeutic agents. Previous studies have shown that dCK is a phosphoprotein and that its activity is modulated by phosphorylation of its Ser-74 residue. However, the signaling pathways regulating this phosphorylation are still not very well documented. In order to define them better, we studied the effects of hyperosmotic stress, the only condition known to decrease dCK activity. The first results showed that hyperosmotic stress, triggered by addition of 500 mM sorbitol, induced a bimodal effect on dCK activity in the leukemic cells CCRF-CEM : a decrease in dCK activity was observed after 30 min, followed by a return to the initial activity and finally by a second, steeper, decrease after 240 min. Given that the effect of hyperosmotic stress was higher at this time, we started by investigating its action after 240 min. We observed that the decrease in dCK activity at this point was not only due to its dephosphorylation on Ser-74, but also to its partial degradation. Use of protease inhibitors revealed that this degradation was partly due to its cleavage by caspases. Furthermore, sorbitol induced the catabolism of adenylic nucleotides and the degradation of Mcl-1, an anti-apoptotic protein, indicating its cytotoxicity in leukemic cells. Since dCK degradation complicated the analysis of the signaling pathways involved in the regulation of its activity, we continued our work by studying dCK regulation by sorbitol after 30 min in CCRF-CEM cells, when dCK is not yet degraded. In order to determine whether dCK dephosphorylation on Ser-74 induced by osmotic stress was due to the inhibition of a protein kinase or the stimulation of a protein phosphatase (PP), we used the different protein kinase and phosphatase effectors. The results indicated that neither the p70S6 kinase nor the p38 kinase, whose activities are modified by osmotic stress, would be involved in dCK inactivation by sorbitol. Finally, we showed that neither PP2A nor PP1 would play a major role in dCK regulation by osmotic stress induced by sorbitol.

Deoxycytidine Kinase --- Neoplasms --- Osmotic Pressure --- Apoptosis

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PHY Physiology & Biochemistry --- cell physiology --- osmotic pressure --- plant cell

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Phaseolus polyanthus --- Phaseolus polyanthus --- Phaseolus vulgaris --- Phaseolus vulgaris --- Embryonic development --- Embryonic development --- Osmotic pressure --- Osmotic pressure --- Hybridization --- Hybridization

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Ultrafiltration --- Ultrafiltration --- membranes --- membranes --- Inorganic compounds --- Inorganic compounds --- organic compounds --- organic compounds --- Permeability --- Permeability --- Viscosity --- Viscosity --- Polymers --- Polymers --- Osmotic pressure --- Osmotic pressure --- processing --- processing