The Calcineurin B-Like Calcium Sensors CBL1 and CBL9 Together with Their Interacting Protein Kinase CIPK26 Regulate the Arabidopsis NADPH Oxidase RBOHF

Abstract

Stimulus-specific accumulation of second messengers like reactive oxygen species (ROS) and Ca(2+) are central to many signaling and regulation processes in plants. However, mechanisms that govern the reciprocal interrelation of Ca(2+) and ROS signaling are only beginning to emerge. NADPH oxidases of the respiratory burst oxidase homolog (RBOH) family are critical components contributing to the generation of ROS while Calcineurin B-like (CBL) Ca(2+) sensor proteins together with their interacting kinases (CIPKs) have been shown to function in many Ca(2+)- signaling processes. In this study, we identify direct functional interactions between both signaling systems. We report that the CBL-interacting protein kinase CIPK26 specifically interacts with the N-terminal domain of RBOHF in yeast two-hybrid analyses and with the full-length RBOHF protein in plant cells. In addition, CIPK26 phosphorylates RBOHF in vitro and co-expression of either CBL1 or CBL9 with CIPK26 strongly enhances ROS production by RBOHF in HEK293T cells. Together, these findings identify a direct interconnection between CBL-CIPK-mediated Ca(2+) signaling and ROS signaling in plants and provide evidence for a synergistic activation of the NADPH oxidase RBOHF by direct Ca(2+)-binding to its EF-hands and Ca(2+)-induced phosphorylation by CBL1/9-CIPK26 complexes.

Two Distinct Families of Protein Kinases Are Required for Plant Growth under High External Mg2+ Concentrations in Arabidopsis

“…In this study, we revealed a novel role of subclass III SnRK2s in plant growth under high external Mg 2+ concentrations. Previous studies on CIPK26/3/9/23 have characterized the diverse and distinct functions of each of these CIPK genes (Kim et al, 2003;Li et al, 2006;Xu et al, 2006;Cheong et al, 2007;Pandey et al, 2007;Drerup et al, 2013;Kimura et al, 2013;Lyzenga et al, 2013). Other than the individual functions of CIPK26, CIPK3, CIPK9, and CIPK23, it seems that these CIPK genes also have some overlapping functions in planta, because CIPK26/3/9/23 formed a monophyletic group in the phylogenetic analysis (Supplemental Fig.…”

“…S4B). Based on a previous report that CBL1/CBL9 work together with CIPK26 in the activation of RBOHF (Drerup et al, 2013), we also considered whether the addition of CBL1/CBL9-GST to CIPK26-GST would affect the kinase activity of SRK2D-MBP (Fig. 3D).…”

“…This result provides evidence that SRK2D forms a protein complex with CIPK26 under a high external Mg 2+ concentration, where subclass III SnRK2s and CIPK26/3/9/23 play an important role in modulating Mg 2+ susceptibility. Recently, it was reported that both SRK2E/OST1 and CIPK26 are able to phosphorylate the NADPH oxidase RBOHF (Sirichandra et al, 2009;Drerup et al, 2013;Kimura et al, 2013). The effect of the phosphorylation of RBOHF by SRK2E/OST1 is still unknown (Sirichandra et al, 2009), but it was reported that CIPK26 enhances the activity of RBOHF through its phosphorylation together with CBL1 and CBL9 in human embryonic kidney 293T cells (Drerup et al, 2013).…”

“…This result indicated that the SRK2D RD could be phosphorylated by CIPK26. Based on a previous report that CIPK26 can activate the activity of the NADPH oxidase RESPIRATORY BURST OXIDASE PROTEIN F (RBOHF) only when together with the calcineurin B-like (CBL) calcium sensors CBL1 or CBL9 (Drerup et al, 2013), it is possible that these CBLs affect the phosphorylation of SRK2D by CIPK26. We tested the effects of coincubation of CBL1-GST or CBL9-GST with CIPK26-GST on the phosphorylation level of SRK2D K52N -MBP.…”

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus

“…The measurement of ROS production in HEK293T cells was performed essentially as described previously (11,61). See SI Experimental Procedures for details.…”