Alteration of substrate specificity of a protein kinase: The variable N-terminal domain of Ca²⁺-dependent protein kinase is important for the recognition of the substrate, transcription factor RSG

Abstract

Protein kinases are major signaling molecules that are involved in a variety of cellular processes. However, molecular mechanisms of how protein kinases discriminate specific substrates are still largely unknown. Ca²⁺-dependent protein kinases (CDPKs) play central roles in Ca²⁺ signaling in plants. Previously, we found that a tobacco (Nicotiana tabacum) CDPK negatively regulates transcription factor REPRESSION OF SHOOT GROWTH (RSG) that is involved in gibberellin (GA) feedback regulation. Here, we found that the variable N-terminal domain of NtCDPK1 is necessary for the recognition of RSG. A mutation (R10A) in the variable N-terminal domain of NtCDPK1 reduced both RSG-binding and RSG phosphorylation while leaving kinase activity intact. Furthermore, R10A mutation suppressed in vivo function of NtCDPK1. The substitution of variable N-terminal domain of an Arabidopsis CDPK AtCPK9 with that of NtCDPK1 conferred RSG kinase activities. Our results open the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways.