Role of tyrosine phosphorylation in radiation-induced cell cycle-arrest of leukemic B-cell precursors at the G2-M transition checkpoint.

Here we provide experimental evidence that ionizing radiation induces inhibitory tyrosine phosphorylation of the p34(cdc2) kinase in human leukemic B-cell precursors. Herbimycin A markedly reduced tyrosine phosphorylation of p34(cdc2) in irradiated leukemic B-cell precursors, thereby preventing radiation-induced cell cycle arrest at the G2-M transition checkpoint. Thus, tyrosine phosphorylation is directly responsible for the inactivation of P34(cdc2) in irradiated human leukemic B-cell precursors and activation of protein tyrosine kinases is a proximal and mandatory step in radiation-induced G2-arrest arrest at the G(2)-M checkpoint. Human WEE1 kinase isolated from unirradiated or irradiated leukemic B-cell precursors had minimal tyrosine kinase activity towards p34(cdc2). We detected no increase of human WEE1 kinase activity after radiation of leukemic B-cell precursors, as measured by (a) autophosphorylation, (b) tyrosine phosphorylation of a synthetic peptide derived from the p34(cdc2) amino-terminal region or (c) recombinant human p34(cdc2)-cyclin B complex. Thus the signaling pathway leading to inhibitory tyrosine phosphorylation of p34(cdc2) and G2-arrest in irradiated human leukemic B-cell precursors functions independent of p49(WEE1Hu) and enzymes which augment the tyrosine kinase activity of p49(WEE1Hu).