Involvement of protein phosphorylation in the early steps of transduction of the oligogalacturonide signal in tobacco cells

Two structurally unrelated protein kinase inhibitors, staurosporine and 6-dimethylaminopurine, a substituted purine identified as an inhibitor of a variety of animal protein kinases were able to inhibit the oligogalacturonide-induced stimulation of the phenylpropanoid pathway in Nicotiana tabacum cells. Extracellular alkalinization and oxidative burst, which occur in a few minutes after elicitor addition, were highly susceptible to the inhibitors. When cells were incubated in the presence of 500 mu M 6-dimethylaminopurine, elicitor-induced oxidative burst was completely prevented, although a reduced extracellular alkalinization was still induced by oligogalacturonides. The possible significance of this result is discussed in terms of potential involvement of different protein kinases in the pH and oxidative responses. Two protein phosphatase inhibitors, cantharidin and calyculin A, were able to induce extracellular alkalinization and oxidative burst in tobacco cells, confirming the involvement of protein phosphorylation in the two membrane responses. In the presence of 6-dimethylaminopurine, pH and oxidative responses induced by phosphatase inhibitors, as well as the oligogalacturonide-dependent responses, were greatly reduced, showing that the purine very likely acts as an inhibitor of plant protein kinases. In vitro phosphorylation studies indicate that most protein kinases active in a purified plasma membrane fraction are highly susceptible to staurosporine. On the contrary, only a few phosphorylated bands appear affected by 6-dimethylaminopurine in the same conditions, suggesting a more specific action of this inhibitor. The phosphorylation of two polypeptides, 95 kDa and 20 kDa, which was increased by oligogalacturonides, was susceptible to staurosporine and 6-dimethylaminopurine. The substituted purine appears as a tool to characterize plasma membrane protein kinases highly susceptible to the inhibitor, which appeared involved in the oxidative burst.