Protein phosphorylation and binding of a 14-3-3 protein in Vicia guard cells in response to ABA.
Under drought stress, ABA promotes stomatal closure to prevent water loss. Although protein phosphorylation plays an important role in ABA signaling, little is known about these processes at the biochemical level. In this study, we searched for substrates of protein kinases in ABA signaling through the binding of a 14-3-3 protein to phosphorylated proteins using Vicia guard cell protoplasts. ABA induced binding of a 14-3-3 protein to proteins with molecular masses of 61, 43 and 39 kDa, with the most remarkable signal for the 61 kDa protein. The ABA-induced binding to the 61 kDa protein occurred only in guard cells, and reached a maximum within 3 min at 1 microM ABA. The 61 kDa protein localized in the cytosol. ABA induced the binding of endogenous vf14-3-3a to the 61 kDa protein in guard cells. Autophosphorylation of ABA-activated protein kinase (AAPK), which mediates anion channel activation, and ABA-induced phosphorylation of the 61 kDa protein showed similar time courses and similar sensitivities to the protein kinase inhibitor K-252a. AAPK elicits the binding of the 14-3-3 protein to the 61 kDa protein in vitro when AAPK in guard cells was activated by ABA. The phosphorylation of the 61 kDa protein by ABA was not affected by the NADPH oxidase inhibitor, H(2)O(2), W-7 or EGTA. From these results, we conclude that the 61 kDa protein may be a substrate for AAPK and that the 61 kDa protein is located upstream of H(2)O(2) and Ca(2+), or on Ca(2+)-independent signaling pathways in guard cells.
Takahashi
Yohei
Y
Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka, 810-8560, Japan.
Kinoshita
Toshinori
T
Shimazaki
Ken-ichiro
K
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000040:Abscisic Acid / Q000494:pharmacology*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D008970:Molecular Weight; D010766:Phosphorylation / Q000187:drug effects; D019441:Plant Epidermis / Q000235:genetics / Q000378:metabolism; D010937:Plant Growth Regulators / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D031306:Vicia / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D010937:Plant Growth Regulators; D010940:Plant Proteins; D047428:Protein Kinase Inhibitors; D000040:Abscisic Acid; D011494:Protein Kinases