Raf-like protein kinase heterocomplexes directly regulate the plant plasma membrane H⁺-ATPase

The plasma membrane proton pump [PM H⁺-adenosine triphosphatase (PM H⁺-ATPase)] is essential in plants. C-terminal phosphorylation events regulate proton pump activity, such as Thr⁸⁸¹ phosphorylation in Arabidopsis AHA1. We discovered a sequential protein phosphorylation pathway in which two distinct types of Raf-like protein kinases, C5-Raf and C7-Raf, form a heterocomplex that phosphorylates Thr⁸⁸¹ to activate PM H⁺-ATPases. This regulatory system is highly conserved across lineages from liverworts to angiosperms. In Arabidopsis, a C5-Raf Raf36 regulates plant growth through the phosphorylation of multiple Arabidopsis H⁺-ATPases (AHAs). Additionally, another C5-Raf HT1 functions with C7-Rafs CBC1/2 to phosphorylate AHA1^T881, thereby generating a driving force for light-induced stomatal opening. Our findings provide a framework for understanding PM H⁺-ATPase activation in various physiological processes, particularly in elucidating the complete mechanistic understanding of light-induced stomatal opening.