Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice

Abstract

The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery.

Transcriptome Analysis Highlights Defense and Signaling Pathways Mediated by Rice pi21 Gene with Partial Resistance to Magnaporthe oryzae

“…Calcium-dependent protein kinases (CDPKs/CPKs) and calcium-related proteins play an important role in plant defense. The OsCPK10 (Fu et al, 2013) and OsCPK18 (Xie et al, 2014) were described as positive and negative regulators of M. oryzae resistance, respectively. Overexpression of OsCPK4 gene enhances resistance to rice blast disease and the constitutive accumulation of OsCPK4 protein prepares rice plants for a rapid and potentiated defense response (Bundó and Coca, 2016).…”

The plant cell wall integrity maintenance mechanism – A case study of a cell wall plasma membrane signaling network

“…MPKs are normally activated by double phosphorylation of the TXY motive, which is mediated by a MKK. Recent results from research in rice (Oryza sativa) showed that the calcium dependent kinase 18 (CPK18) activate MPK5 through phosphorylation in two threonine residues (Thr-14 and -32) in a domain different from the one normally required for MAPkinase activation (Xie et al, 2014). Taken together these selected examples illustrate how combinations of MAPkinases and different signaling molecules could translate and integrate signals to mediate CWI maintenance.…”

A rapid and cost-effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation

“…In a typical kinase reaction, however, 2–4 μg of substrate was used in one reaction [25]. We analyzed the linear range of FDIT method in a higher concentration (2–8 μg) with casein, OVA and BSA, representing high, low or no phosphorylation standards (MAN0002351, Molecular Probes, Inc).…”