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. 2022:2462:45-57.
doi: 10.1007/978-1-0716-2156-1_4.

Affinity Purification of Ubiquitinated Proteins Using p62-Agarose to Assess Ubiquitination of Clade A PP2Cs

Jose Julian  1   2 , Alberto Coego  3 , Abdulwahed F Alrefaei  4 , Pedro L Rodriguez  3
Affiliations

Affinity Purification of Ubiquitinated Proteins Using p62-Agarose to Assess Ubiquitination of Clade A PP2Cs

Jose Julian et al. Methods Mol Biol. 2022.

Abstract

Certain E3 ubiquitin ligases play a key role in the abscisic acid (ABA) pathway by targeting clade A type 2C protein phosphatases (PP2Cs) for degradation. At early stages of ABA signaling, degradation of PP2Cs is a complementary step to PP2Cs inhibition by ABA receptors. At later steps, protein levels of PP2Cs are increased as a negative feedback mechanism. Subsequently, E3 ligases targeting PP2Cs are critical to recover the basal PP2C levels and reset the ABA signaling. BTB/POZ AND MATH DOMAIN proteins (BPMs) are substrate adaptors of a multimeric cullin3-RING based E3 ligase and target for degradation clade A PP2Cs. In this chapter, we provide a detailed protocol to assess the ubiquitination of PP2CA, a clade A PP2C, mediated by BPMs using agarose-immobilised p62-derived ubiquitin-associated (UBA) domain, which efficiently binds ubiquitinated proteins.

Keywords: ABA; Affinity purification; BPM; E3 ubiquitin ligase; PP2C; Pulldown; p62.

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References

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