Protein kinase activity in Cucumis sativus cotyledons: Effect of calcium and light

Abstract

Light signals received by phytochromes in plants may be transduced through protein phosphorylation. Ca 2+ as second messenger was involved in phytochrome-mediated cellular events. Our experiments with Cucumis sativus cotyledons, treated with red (R) and far-red (FR) light, showed a stimulatory effect on in vitro protein phosphorylation of histone, added as exogenous substrate to the cotyledon extracts, and also modified the phosphorylation of endogenous polypeptides. The effect of light treatments was mimicked by the addition of Ca 2+ to the phosphorylation buffer, indicating phytochrome- and Ca 2+-dependence on activity of some protein kinases (PKs). In-gel kinase assays were performed to characterize the PKs involved at the cotyledon stage of cucumber plants. Three proteins of about 75, 57 and 47 kDa with PK activity were detected between M r markers of 94 and 45 kDa. All three were able to phosphorylate histone and undergo autophosphorylation. However, only the 75 and 57 kDa proteins autophosphorylated and phosphorylated the substrate in a Ca 2+-dependent manner, and were inhibited when calmodulin (CaM) antagonists were added to the incubation buffer. Western-blot analysis with polyclonal antibodies directed against calcium-dependent protein kinase of rice (OsCDPK11) or Arabidopsis (AtCPK2) recognised 57 and 75 kDa polypeptides, respectively. These results indicate the presence in cucumber cotyledons of at least two proteins (ca. 75 and 57 kDa) with activity of PKs that could be calcium-dependent protein kinases (CDPKs). Both CDPKs could be modulated by phytochromes throughout FR-HIR and VLFR responses.