Interaction between acylphosphatase and SERCA in SH-SY5Y cells.
Ca2+ transport by sarco/endoplasmic reticulum, tightly coupled with the enzymatic activity of Ca2+ -dependent ATPase, controls the cell cycle through the regulation of genes operating in the critical G, to S checkpoint. Experimental studies demonstrated that acylphosphatase actively hydrolyses the phosphorylated intermediate of sarco/endoplasmic reticulum calcium ATPase (SERCA) and therefore enhances the activity of Ca2+ pump. In this study we found that SH-SY5Y neuroblastoma cell division was blocked by entry into a quiescent G0-like state by thapsigargin, a high specific SERCA inhibitor, highlighting the regulatory role of SERCA in cell cycle progression. Addition of physiological amounts of acylphosphatase to SY5Y membranes resulted in a significant increase in the rate of ATP hydrolysis of SERCA. In synchronized cells a concomitant variation of the level of acylphosphatase isoenzymes opposite to that of intracellular free calcium during the G1 and S phases occurs. Particularly, during G1 phase progression the isoenzymes content declined steadily and hit the lowest level after 6 h from G0 to G1 transition with a concomitant significant increase of calcium levels. No changes in free calcium and acylphosphatase levels upon thapsigargin inhibition were observed. Moreover, a specific binding between acylphosphatase and SERCA was demonstrated. No significant change in SERCA-2 expression was found. These findings suggest that the hydrolytic activity of acylphosphatase increase the turnover of the phosphoenzyme intermediate with the consequences of an enhanced efficiency of calcium transport across endoplasmic reticulum and a subsequent decrease in cytoplasmic calcium levels. A hypothesis about the modulation of SERCA activity by acylphosphatase during cell cycle in SY5Y cells in discussed.
Cecchi
C
C
Department of Biochemical Sciences, University of Florence, Italy.
Liguri
G
G
Pieri
A
A
Degl'Innocenti
D
D
Nediani
C
C
Fiorillo
C
C
Nassi
P
P
Ramponi
G
G
D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D009447:Neuroblastoma; D011233:Precipitin Tests; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D002118:Calcium; D000252:Calcium-Transporting ATPases; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019284:Thapsigargin; D000097024:Acylphosphatase