Bistratene A, a polyether toxin isolated from the colonial ascidian Lissoclinum bistratum, causes incomplete differentiation of human
leukemia (HL-60) cells apparently through a mechanism not involving
protein kinase C. In view of the importance of phosphorylation/dephosphorylation in cellular growth and differentiation we have investigated
protein phosphorylation in these cells following exposure to
bistratene A, using two-dimensional
polyacrylamide gel electrophoresis. Marked increases in the phosphorylation of a
protein of 20 kDa, pl 6.7, and a basic
protein of 25 kDa were observed after incubation with
bistratene A. A comparison was made with cells treated with 12-O-tetradecanoylphorbol 13-acetate and
bryostatin 5. While changes in phosphorylation patterns were observed with these two compounds, the 20 kDa and 25 kDa
proteins did not undergo phosphorylation changes. The 20 kDa
protein was induced rapidly by very low concentrations of
bistratene A reaching near maximal levels with 10 nM at 15 min exposure. This
protein was found to be localised to the cytoplasm. Phosphoaminoacid analysis demonstrated that the majority of 32P was present in
serine and
tyrosine residues. The increased phosphorylation of the 20 kDa
protein appeared to be due to hyperphosphorylation of existing
protein although there was some increase in the amount of the
protein. These results suggest that
bistratene A will be a useful tool with which to investigate cellular differentiation mechanisms.