Phosphorylation Studies on Rat p53 Using the Baculovirus Expression System

Abstract

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.

Interaction partners of Dlk/ZIP kinase: co-expression of Dlk/ZIP kinase and Par-4 results in cytoplasmic retention and apoptosis

“…Recombinant baculoviruses encoding histidine-tagged wildtype or mutant forms of Dlk were generated and propagated as described (Fuchs et al, 1995). His-tagged Dlk expressed in the baculovirus system was puri®ed by a nity chromatography on Ni-NTA agarose (Qiagen, Hilden, Germany) as described (KoÈ gel et al, 1998).…”

Cloning and characterization of Dlk, a novel serine/threonine kinase that is tightly associated with chromatin and phosphorylates core histones

“…Therefore, we made use of the baculovirus expression system which has been shown to exert posttranslational modifications of recombinant proteins similar or identical to vertebrate cells (Fuchs et al, 1995). Dlk was recloned into a baculovirus transfer vector and thereby provided with a 6xHis tag at its amino terminus to facilitate puri®cation (see Materials and methods).…”

“…Spodoptera frugiperda cells (Sf9) were grown as monolayer cultures in TC100 medium supplemented with 10% fetal calf serum or in serum-free medium (Life Technologies, Gibco BRL) at 278C. The origin of wildtype baculovirus Autographa californica (Ac) and the generation of recombinant baculovirus have been described (Fuchs et al, 1995).…”

“…Dlk was recloned into a baculovirus transfer vector and thereby provided with a 6xHis tag at its amino terminus to facilitate puri®cation (see Materials and methods). Recombinant virus was generated as described (Fuchs et al, 1995). Sf9 cells were infected with His-Dlk-baculovirus and harvested 2 ± 3 days postinfection.…”

Restoration of the tumor suppressor p53 by downregulating cyclin B1 in human papillomavirus 16/18-infected cancer cells

“…Of importance, Aurora A phosphorylates Ser315 of p53 and overexpression of Aurora A induces MDM2-mediated p53 ubiquitination and degradation (Katayama et al, 2004). Interestingly, it has been revealed for almost 20 years that Cdk1/cyclin B phosphorylates Ser315 (Bischoff et al, 1990), but only the in vitro function of this phosphorylation has been reported (Fuchs et al, 1995;Wang and Prives, 1995). The in vivo function of this phosphorylation by Cdk1/cyclin B1 remains undefined.…”