Proteomic analysis of acute promyelocytic leukemia: PML-RARapha promotes mitotic exit by increased expression and decreased phosphorylation of OP18 at serine 63.

We applied mass spectrometry based approach to explore the proteins differentially regulated by PML-RARalpha a translocation characteristic of acute promyelocytic leukemia (APL). Differentialy expressed proteins, a number of which are related to cell cycle function, including OP18, HSP70, GRP75 and Pin1 were identified by mass spectrometry. Further analysis of the OP18 pathway indicated that mRNA expression of OP18 was higher in APL patients and the increased OP18 protein expression upon PML-RAR induction was overcome by retinoic acid treatment. PML-RARalpha induced cell cycle progression and led to mitotic exit. RNA interference experiments revealed that siRNA against OP18 overcomes PML-RARalpha effects on cell cycle progression. In addition to increased OP18 expression by PML-RARalpha, 2D gel electrophoresis revealed an isomer of OP18, subsequently confirmed as Ser63 phosphomer to be downregulated by PML-RARalpha. Based on these findings, point mutation experiments indicated that decreased phosphorylation of Ser63 in OP18 is important for PML-RARalpha mediated cell cycle and mitotic index effects since constitutive phosphorylated mutant (Ser63-asp) of OP18 overcame the PML-RARalpha effects in U937-PR cells, NB4 and APL patients. In summary, our results demonstrate that the effect of PML-RAR on cell cycle progression and mitotic exit is via two mechanisms: increasing the expression of OP18 and decreasing the phosphorylation of OP18 at Ser63