CULL-ING CURIOSITY: EXPLORING REGULATION AND NOVEL TARGETS OF CULLIN-BASED E3 LIGASES

Christina Marie Choi

Rapid turnover of proteins is an essential molecular process for plant development and stress response. The ubiquitin proteasome pathway (UPP) is a conserved mechanism used to regulate protein activities. The pathway uses a three-step enzymatic cascade that marks substrate proteins for degradation via the 26S proteasome. E3 ligases serve as the key step in facilitating substrate recognition and its subsequent ubiquitination. While the basic steps of ubiquitination are well understood, mechanisms that regulate recognition and degradation of target proteins are only poorly resolved. Additionally, one can expect that most substrates of the 26S proteasome have not been identified yet. RELATED TO APETALA2.4 (RAP2.4) is a transcription factor and known substrate of a CULLIN3-based E3 ligase utilizing BTB/POZ/MATH (BPM) proteins (CRL3BPM). To understand how RAP2.4 is regulated by the UPP, we investigated recognized of RAP2.4 by the CRLBPM complex and what signals degradation of RAP2.4. Speckled type POZ Protein (SPOP) is a human homolog of BPMs and RAP2.4 contains an SPOP binding consensus (SBC), a short sequence critical for interaction between SPOP and its targets. Additionally, RAP2.4 has a predicted PEST motif, a region enriched in four amino acids and associated with protein degradation. Loss of the PEST motif significantly impacted the rate of RAP2.4 degradation while loss of the SBC motif only had mild impacts. Change in degradation correlated with reduced interaction with BPMs. RAP2.4 is phosphorylated by ARABIDOPSIS KINASE10 (AKIN10). We hypothesized that phosphorylation contributes to protein stability. Mutagenesis of four identified sites neither influenced the rate of degradation nor interaction of RAP2.4 with BPMs. 14-3-3 proteins in conjunction with phosphorylated proteins participate in many cellular processes and can affect protein stability. For proof of principle, 14-3-3κ was selected and show interaction with RAP2.4 required an intact PEST motif but not phosphorylation. A mild stabilization of RAP2.4 is observed in the presence of 14-3-3κ providing insight of RAP2.4 regulation. Finally, the Salt Tolerance Zinc (STZ) family of six closely related zinc finger proteins were identified as novel targets of a 26S proteasome dependent degradation. While further work is needed, implications of these newly identified targets in plants are discussed.

CULL-ING CURIOSITY: EXPLORING REGULATION AND NOVEL TARGETS OF CULLIN-BASED E3 LIGASES

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