Phosphorylation site mapping and mutational studies of the ACR4 intracellular domain

Arabidopsis CRINKLY4 (ACR4) is a receptor‐like kinase involved in the global development of the plant. The intracellular domain (ICD) of the receptor contains a kinase domain that is a member of the RD class of kinases in which phosphorylation within the activation loop is necessary for full activity of the kinase. In order to understand the role of phosphorylation in the ACR4 ICD, we have completed a comprehensive mapping of the autophosphorylation sites within the ICD via phosphopeptide enrichment followed by tandem mass spectrometry. A total of 16 phosphorylation sites were found encompassing all three subdomains of the ICD. Mutational alanine scanning of specific residues in the activation loop and the C‐terminal domain (CTD) of the ICD highlighted the critical role of some residues in the regulation of kinase activity. Thus, of the five potential phosphorylation sites within the activation loop, three are required for full activity. In one instance, a phosphomimetic mutation of a serine residue to aspartate in the activation loop was able to restore full kinase activity, suggesting that this site is phosphorylated upon kinase activation. Furthermore, mutations in the activation loop and the CTD were demonstrated to have an effect on the efficiency of peptide substrate phosphorylation in an in vitro phosphorylation assay.