To elucidate the role of phosphorylation of p53 we used the baculovims expression system to obtain high yields of protein eventually in distinct phosphorylation states. Initially, we obtained only marginal phosphorylation, despite high levels of expression. Two-dimensional phosphopeptide maps exhibited the same pattern as known from rat cells although 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, when we employed the phosphatase inhibitor okadaic acid, overall phosphorylation of p53 was drastically enhanced in a dose-dependent manner and resembled that of p53 from SV40-transformed rat cells. This hyperphosphorylation resulted in enhanced binding of a consensus oligonucleotide as revealed by electrophoretic mobility shift assays. To assess the role of individual phosphorylation sites, we generated a set of mutants at putative or identified sites. All mutants retained the ability to bind wild-type conformation-specific antibody Pab1620, to complex with SV40 large T antigen, and to bind to the consensus oligonucleotide. Moreover, most mutants exhibited enhanced DNA binding upon okadaic acid 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 kinases 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 proteins in this system may result from high phosphatase activities rather than saturation of kinases with recombinant substrate ; (c) the system can be manipulated to obtain subpopulations of recombinant protein in a desired phosphorylation state, and (d) phosphorylation may regulate the DNA-binding activity of p53.

To elucidate the role of phosphorylation of p53 we used the baculovims expression system to obtain high yields of protein eventually in distinct phosphorylation states. Initially, we obtained only marginal phosphorylation, despite high levels of expression. Two-dimensional phosphopeptide maps exhibited the same pattern as known from rat cells although 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, when we employed the phosphatase inhibitor okadaic acid, overall phosphorylation of p53 was drastically enhanced in a dose-dependent manner and resembled that of p53 from SV40-transformed rat cells. This hyperphosphorylation resulted in enhanced binding of a consensus oligonucleotide as revealed by electrophoretic mobility shift assays. To assess the role of individual phosphorylation sites, we generated a set of mutants at putative or identified sites. All mutants retained the ability to bind wild-type conformation-specific antibody Pab1620, to complex with SV40 large T antigen, and to bind to the consensus oligonucleotide. Moreover, most mutants exhibited enhanced DNA binding upon okadaic acid 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 kinases 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 proteins in this system may result from high phosphatase activities rather than saturation of kinases with recombinant substrate ; (c) the system can be manipulated to obtain subpopulations of recombinant protein in a desired phosphorylation state, and (d) phosphorylation may regulate the DNA-binding activity of p53.

Single antibody-forming cells (AFC) specific for alpha(1-3) dextran (Dex) from i.p.-immunized BALB/c mice were enumerated in soft agar cultures by blotting on antigen-precoated membranes and subsequent staining via enzyme-coupled anti-IgM antibodies. Short cultures (2 h) revealed AFC as harvested ex vivo, while in long-term cultures (4 days), in the presence of lipopolysaccharide (LPS) as B cell mitogen, cells or colonies developed by differentiation in vitro. Whereas the spleen contained most AFC ex vivo in a sharp-peak response at 4 and 5 days after i.p. injection of Dex in aqueous solution, peritoneal exudate cells (PEC) contained only very few AFC. However, the same PEC population developed Dex-specific cells or colonies after 4 days of culture. The isotype of antibodies was IgM. The frequency of these Dex-specific LPS-inducible precursor cells rose exponentially in the course of the immune response to a broad plateau and was still, 11 weeks after Dex injection, approximately 40-fold higher than in non-immunized mice. Since these cells increased in frequency after antigen injection, and since they could not be detected as AFC during 2 h ex vivo, they were regarded as memory cells. They seemed to be arrested in vivo, but could be induced to differentiation and/or proliferation in vitro. Although these cells had the functional characteristics of memory cells as defined above, they produced anti-Dex antibodies of IgM isotype. Their population might be critical for the protection of the peritoneal cavity against microbial invasion from the intestines, and it may be significant in this context that we could evoke a peritoneal memory cell response only when antigen was injected intraperitoneally, but not intravenously. In athymic BALB/c-nu/nu mice only few of these Dex-specific memory cells were found. It is possible that T cells exert a regulatory influence on this pathway of differentiation.

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