PHOSPHORYLATION STUDIES ON RAT P53 USING THE BACULOVIRUS EXPRESSION SYSTEM - MANIPULATION OF THE PHOSPHORYLATION STATE WITH OKADAIC ACID AND INFLUENCE ON DNA-BINDING

To elucidate the role of phosphorylation of p53 we used the baculoviru s expression system to obtain high yields of protein eventually in dis tinct phosphorylation states. Initially, we obtained only marginal pho sphorylation, despite high levels of expression. Two-dimensional phosp hopeptide maps exhibited the same pattern as known from rat cells alth ough some sites were underrepresented. Coexpression of simian virus 40 (SV40) large T antigen or cyclin-dependent kinases, cdc2 or cdk2, had only marginal effects on the phosphorylation state of p53. However, w hen we employed the phosphatase inhibitor okadaic acid, overall phosph orylation of p53 was drastically enhanced in a dose-dependent manner a nd resembled that of p53 from SV40-transformed rat cells. This hyperph osphorylation resulted in enhanced binding of a consensus oligonucleot ide as revealed by electrophoretic mobility shift assays. To assess th e role of individual phosphorylation sites, we generated a set of muta nts at putative or identified sites. All mutants retained the ability to bind wild-type conformation-specific antibody Pab1620, to complex w ith SV40 large T antigen, and to bind to the consensus oligonucleotide . Moreover, most mutants exhibited enhanced DNA binding upon okadaic a cid treatment, except for a mutant at the cdk site which failed to do so. These data show that: (a) insect cells contain all the protein kin ases necessary for phosphorylation of a mammalian protein, p53; (b) in insect cells the ratio of kinase/phosphatase activities differs from that in mammalian cells so that underphosphorylation of recombinant pr oteins in this system may result from high phosphatase activities rath er than saturation of kinases with recombinant substrate; (c) the syst em can be manipulated to obtain subpopulations of recombinant protein in a desired phosphorylation state, and (d) phosphorylation may regula te the DNA-binding activity of p53.