Exposure to toxic concentrations of aluminum activates a MAPK-like protein in cell suspension cultures of Coffea arabica.

Addition of a toxic concentration of aluminum (Al) to cell suspension cultures of Coffea arabica L. induced the rapid and transient activation of a protein kinase that phosphorylates myelin basic protein (MBP), as revealed by in-gel kinase assays. This enzyme with an apparent molecular mass of 58 kDa was activated shortly after cells were exposed to 50 microM AlCl(3), a concentration previously shown to produce toxicity in plant cells in vitro. The activity of this kinase dropped to basal levels after 20 min of Al addition; this activity is specific for MBP as it could not be detected when casein or histone H1 were used as substrates. Analysis of the same cell extracts with antibodies that specifically recognize bis-phosphorylated (active) mitogen-activated protein kinases (MAP kinases), revealed the presence of a phosphoprotein with an apparent molecular mass of 58 kDa, which showed the same response to Al as the protein kinase revealed by the in-gel kinase assays. Furthermore, immunoprecipitation with an antibody directed against mammalian MAP kinases depleted both the enzymatic activity and the phosphoprotein from the cell extracts, suggesting that the 58 kDa kinase and the 58 kDa phosphoprotein from C. arabica cells are the same protein, and that it can be actually a member of the MAP kinase family of protein kinases. Since its activity is enhanced dramatically after addition of AlCl(3) to the medium, we can speculate that Al toxicity in plants could be perceived through the MAP kinase signal transduction pathway.

MAP kinase-like activity in transformed Catharanthus roseus hairy roots varies with culture conditions such as temperature and hypo-osmotic shock.

Mitogen activated protein (MAP) kinase-like activity was determined in extracts obtained from transformed Catharanthus roseus hairy roots by the ability to phosphorylate myelin basic protein (MBP). Both in solution and in gel kinase assays showed variation in activity, depending on root developmental stage. In gel kinase assays, using the extract soluble fraction, revealed a 56 kDa polypeptide with phosphorylation activity on MBP. In addition, another 75 kDa polypeptide was observed in the particulate fraction. Immunodetection with monoclonal antibodies against ERK-1, a mammalian MAP kinase, and with anti-phosphotyrosine antibodies cross-reacted with the 56 kDa polypeptide, named SMK56, from the soluble fraction, suggesting that this polypeptide could be related with members of the MAP kinase family. Antibodies against the dually phosphorylated threonine-tyrosine motif, characteristic of active forms of MAP kinases, also cross-reacted with this 56 kDa polypeptide. Changes in the levels of SMK56 were detected within the first 30 min of root exposure to low temperatures or hypo-osmotic shock, suggesting that this protein may be involved in the perception of environmental changes.