REGULATION OF PHOSPHOPROTEIN P18 IN LEUKEMIC-CELLS - CELL-CYCLE-REGULATED PHOSPHORYLATION BY P34(CDC2) KINASE

p18 is a phosphoprotein that is expressed at very high levels in leuke mic cells, at moderately high levels in proliferating normal lymphocyt es, and at low levels in quiescent lymphocytes. Induction of terminal differentiation of leukemic cells in culture results in a decrease in cellular proliferation. These phenotypic changes are associated with r apid phosphorylation of p18, followed by a more gradual decrease in th e level of its mRNA expression. More than 12 different phosphorylation products of p18 have been identified in different cells by high resol ution two-dimensional polyacrylamide gel electrophoresis. Previous stu dies have suggested that p18 may be a substrate for protein kinase C i n some cellular processes and protein kinase A in others. In this repo rt, we show that the phosphorylation of p18 increases as cells progres s toward the G2-M phases of the cell cycle in proliferating leukemic c ells. We have examined the hypothesis that the putative role of p18 in cellular proliferation may be mediated by its involvement in the p34( cdc2) kinase signal transduction pathway. We have produced recombinant p18 in bacterial cells and shown that it can be phosphorylated in vit ro by purified p34(cdc2) kinase with a stoichiometry of 0.86 mol of PO 4/mol of substrate. We have used site-directed mutagenesis to demonstr ate that the site of p34(cdc2) phosphorylation is the serine at positi on 38. This same site has previously been shown to be phosphorylated i n vivo in bovine brain along with another serine at position 25. The o bservation that p18 gets phosphorylated in the G2-M phases of the cell cycle and the demonstration that p18 is phosphorylated efficiently by p34(cdc2) kinase in vitro at a residue that is also phosphorylated in vivo support the hypothesis that p18 may be a physiologic substrate f or p34(cdc2) kinase in vivo. We have also examined the effect of inhib iting the expression of p18 on cell cycle progression. These experimen ts demonstrated that antisense inhibition of the expression of p18 in K562 erythroleukemia cells is associated with a decrease in cellular p roliferation and accumulation of cells in the G2-M phases of the cycle . The implications of these findings to the proposed role of p18 in th e regulation of cellular proliferation are discussed.