Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins*

Abstract

Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr²⁰⁹, Ser²⁴⁷, Ser²⁷⁰, and Ser³⁰³ as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr²⁰⁹ and Ser²⁴⁷ on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr²⁰⁹ peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response.