Calcium-dependent protein phosphorylation may mediate the gibberellic acid response in barley aleurone.
Sian Ritchie and Simon Gilroy
Department of Biology, 208 Mueller Lab, The Pennsylvania State University, University Park, PA 16802
Abstract.
Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts in order to inhibit, and therefore identify, events in the transduction of the gibberellic acid (GA) and abscisic acid (ABA) signals regulated by protein kinase activities. Syntide-2, a substrate designed for Ca2+- and calmodulin- (CaM-) dependent kinases, selectively inhibited the GA response, leaving constitutive and ABA-regulated events unaffected. Microinjection of syntide did not inhibit the GA-induced increase in cytosolic [Ca2+], suggesting it inhibited GA action downstream of the Ca2+ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of 30 and 54 kD. A 54 kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by 10 µM Ca2+, was calmodulin (CaM) -independent, but was inhibited by the CaM antagonist N-(6-Aminohexyl)-5-chloro-1-napthalene-sulfonamide (W7; 250 mM), suggesting it was a CaM-domain protein kinase (CDPK) -like activity . Thus syntide-2 may inhibit the GA response of the aleurone via an interaction with this kinase, implicating the 54 kD kinase as a Ca2+-dependent regulator of the GA response in these cells.
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