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.
The ether phospholipid platelet-activating factor and certain similar phospholipids, including lysophosphatidylcholine, are known to stimulate both H' transport and protein phosphorylation in plant microsomal membranes. In the present work, several polypeptides in highly purified tonoplast membranes from zucchini (Cucurbita pepo L.) showed platelet-activating factor-dependent phosphorylation. Comparison of protein phosphorylation in different membrane fractions separated by sucrose step density gradient centrifugation indicated that some of the phosphoproteins were contaminants or were common to several membrane fractions, but platelet-activating factor-dependent phosphorylation of peptides at 30, 53, and perhaps 100 kilodaltons was tonoplast specific. The phosphoprotein of 53 kilodaltons was shown by three different approaches (one-and two-dimensional polyacrylamide gel electrophoresis, western blots, and immunoprecipitation) to cross-react with antibody raised against the B subunit of the tonoplast ATPase from red beet (Beta vulgaris L.).Although the vacuolar H+-ATPases of plants, animals, and fungi have been receiving increasing attention, much of the current work is focused on their structure and evolution (9,14,18) and relatively little is known of their regulation. Increased activity of the enzyme has been observed as an adaptation to salt stress (19) and during adaptation to CAM (27), and regulation is also assumed to occur in the turgor responses of guard cells and other systems.In vitro, proton transport and ATPase activity in a light microsomal fraction from zucchini (Cucurbita pepo L.) were shown to be increased in the presence of the ether phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine (12,20,24 enhanced influx of calcium ions into platelets and the tumover of phosphorylated phosphatidylinositols (5). In plants, PAF presumably mimicks the action of PAF-like plant phospholipids (12, 24) such as lysophosphatidylcholine. A protein kinase stimulated by PAF, lysophospholipids, and Ca2" has been demonstrated in higher plants (12,21,23,24). We proposed that the PAF/lysophospholipid-stimulated protein kinase could be involved in a signal transduction chain participating in cell regulation in higher plants (20)(21)(22)(23)(24).Membrane fractionation studies showed that H+ transport in zucchini microsomes was attributable mostly to the tonoplast (12). The present work, therefore, examines in more detail the phosphorylation of tonoplast proteins and its relationship to the activation of tonoplast H+ transport by PAF. MATERIALS AND METHODS Plant MaterialZucchini seeds (Cucurbita pepo L.) were surface-sterilized with sodium hypochlorite and grown for 4 d in the dark at 280C on vermiculite. Hypocotyl hooks of about 1 cm were used for membrane preparations. Membrane PreparationsThe preparation of light microsomes from zucchini has been described (20). Chopped hypocotyls were degassed in homogenization buffer (1 g:1 mL) containing 4% (v/v) ethanolamine, 0.4 M sodium ,B-glycerophosphate, 20 mm...
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