Phosphorylation of 35kDa protein in humanpremyelocytic HL-60 cells undergoing apoptosis by tumor necrosis factor

Abstract

Relationship between the state of protein phosphorylation and tumor necrosis factor $\alpha$(TNF) induced apoptosis was studied in HL-60 cells. Incubation of cells with TNF resulted in progressive increases in DNA fragmentation and the phosphorylation of a specific protein with 35 kDa of molecular weight. Serine residues in 35 kDa protein were specifically phosphorylated during apoptosis mediated by TNF. Calmidazolium, an inhibitor of $Ca^{2+}$/calmodulin-dependent protein kinase, inhibited the phosphorylation of 35 kDa protein as well as DNA fragmentation in cells treated with TNF. However, okadaic acid, known as a protein phosphatase inhibitor did not change the phosphorylation of 35 kDa protein but rather induced DNA fragmentation. These data provided support that phosphorylation status of 35 kDa protein is a critical requirement in the activation of programmed cell death in HL-60 cells. The involvement of phosphorylation of this specific protein in the apoptotic process was further supported by the finding that calmidazolium also suppressed the phosphorylation of 35 kDa protein and DNA fragmentation in the cells undergoing apoptosis mediated by other apoptosis inducers such as calcium ionophore, colchicine and cycloheximide. It was also showed that the increased intracellular $Ca^{2+}$ concentration is a necessary requirement of the phosphorylation of 35 kDa protein by $Ca^{2+}$/calmodulin-dependent protein kinase in TNF induced apoptosis. A $Ca^{2+}$/$Mg^{2+}$-dependent endonuclease which induces DNA fragemetation activated by TNF treatment and inhibited by calmodulin antagonist.