An endogenous factor from soybean (Glycine max L.) cell cultures activates phosphorylation of a protein which is dephosphorylated in vivo in elicitor-challenged cells

The existence of specific binding sites for a -glucan elicitor of phytoalexin synthesis derived from the fungus Phytophthora megasperma f.sp. glycinea at the plasma membrane of soybean (Glycine max L.) tissues (W.E. Schmidt, J. Ebel (1987) Proc. Natl. Acad. Sci. USA 84, 4117–4121) might imply that stimulation of phytoalexin formation by the elicitor is a membrane-mediated process. Addition of the -glucan elicitor to soybean cellsuspension cultures, which has previously been shown to induce phytoalexin accumulation, also results in rapid changes in the phosphate turnover of several phosphoproteins. The effect of the elicitor on protein phosphorylation was tested after labeling of the cells with ³²P]orthophosphate. As shown by analysis using one-and two-dimensional gel electrophoresis, decreases as well as increases in the labeling of several phosphoroteins occurred rapidly, being detectable within 5 min after elicitor application, and persisted for at least 15 min. As judged by their relative molecular masses (M_r) and isoelectric points (pI), a number of proteins which were radioactively labeled in vivo were also phosphorylated in vitro by endogenous protein-kinase activity in the presence of Ca²⁺. The most pronounced effect was observed with a protein substrate with M_r=69000 and pI=5.7 (pp69) whose phosphate labeling markedly decreased in response to elicitor treatment in vivo. Phosphorylation of pp69 in vitro in the presence of -³²P]ATP was strongly enhanced by a phosphorylation-stimulating factor (effector) derived from soybean cell cultures and occurred predominantly at serine residues. The effector possessed a low apparent M_r (1000), was negatively charged at pH 7.3, and was relatively heat stable. The effector was inactivated by treatment with alkaline phosphatase from calf intestine. Phosphorylation of pp69 was only slightly stimulated by Ca²⁺, and was insensitive to cAMP, cGMP, calmodulin, a lipid mixture, a ganglioside mixture, or spermine under the assay conditions used. A 10 mM concentration of 3-phosphoglycerate increased pp69 phosphorylation to the extent of about 50% of that induced by the soybean effector. There was no evidence, however, that such concentrations of 3-phosphoglycerate occurred in effector preparations. The results are discussed in relation to hypothetical signal transduction during elicitor action on soybean cells.Abbreviations M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TPCK L-1-tosylamide-2-phenylethyl chloromethyl ketone