The RING finger E3 ligases PIR1 and PIR2 mediate PP2CA degradation to enhance abscisic acid response in Arabidopsis

Abstract

Recent work has established a core ABA signaling pathway in which A-type PP2C protein phosphatases act as central negative modulators. Although ABA signaling inhibits PP2C activity through ABA-receptor complex, it remains unknown if other mechanisms exist to modulate the level of PP2Cs. Here, we identified a RING domain ubiquitin E3 ligase, PIR1 (PP2CA interacting RING finger protein 1), that interacted with PP2CA. Of the two splicing isoforms, PIR1.2 was isolated from leaf tissue. The PIR1.2 exhibited E3 ligase activity and determined PP2CA stability in the presence of ABA. Consistent with the conclusion that PIR1 promotes ABA signaling by removing PP2CA, a negative modulator, the pir1 knockout mutant displayed an ABA-hyposensitive phenotype. We further showed that PIR2, the closest homologue of PIR1.2, also interacted with PP2CA. Although the pir2 knockout mutant did not display altered ABA response, the pir1-1/pir2 double mutant became more insensitive to ABA than the wild-type or pir1-1 and pir2 single mutants. Using a cell-free degradation assay, ABA promoted degradation of PP2CA, however, such degradation was delayed when incubated with protein extract prepared from the pir1-1/pir2 double mutant. Our data suggest that PIR1 and PIR2 positively modulate ABA signaling by targeting PP2CA for degradation.

Keywords: 26S proteasome; E3 ligase; RING finger protein; abscisic acid; protein degradation; protein phosphatase.