Feedback inhibition of AMT1 NH(4)(+)-transporters mediated by CIPK15 kinase

BACKGROUND: Ammonium (NH(4)(+)), a key nitrogen form, becomes toxic when it accumulates to high levels. Ammonium transporters (AMTs) are the key transporters responsible for NH(4)(+) uptake. AMT activity is under allosteric feedback control, mediated by phosphorylation of a threonine in the cytosolic C-terminus (CCT). However, the kinases responsible for the NH(4)(+)-triggered phosphorylation remain unknown. RESULTS: In this study, a functional screen identified protein kinase CBL-Interacting Protein Kinase15 (CIPK15) as a negative regulator of AMT1;1 activity. CIPK15 was able to interact with several AMT1 paralogs at the plasma membrane. Analysis of AmTryoshka, an NH(4)(+) transporter activity sensor for AMT1;3 in yeast, and a two-electrode-voltage-clamp (TEVC) of AMT1;1 in Xenopus oocytes showed that CIPK15 inhibits AMT activity. CIPK15 transcript levels increased when seedlings were exposed to elevated NH(4)(+) levels. Notably, cipk15 knockout mutants showed higher (15)NH(4)(+) uptake and accumulated higher amounts of NH(4)(+) compared to the wild-type. Consistently, cipk15 was hypersensitive to both NH(4)(+) and methylammonium but not nitrate (NO(3)(−)). CONCLUSION: Taken together, our data indicate that feedback inhibition of AMT1 activity is mediated by the protein kinase CIPK15 via phosphorylation of residues in the CCT to reduce NH(4)(+)-accumulation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-020-00934-w.