Auxin Perception Leads to SCF E3 Ligase Mediated Degradation of AuxIAA Proteins

The identification of Aux/IAAs as repressors of auxin responsive gene expression (Ulmasov et al. 1997) and the observation that they are very instable, especially upon auxin stimulation (Abel et al. 1994; Gray et al. 2001), placed the proteolysis machinery at a central position in auxin signaling. Moreover, the isolation and characterization of several Arabidopsis auxin-resistant mutants revealed that a ubiquitination-mediated degradation pathway is likely to be involved. Ubiquitin is a regulatory protein that is first activated by a ubiquitin-activating enzyme E1, which transfers the ubiquitin component to a ubiquitin-conjugating enzyme, E2. E2 subsequently acts in concert with an E3 ubiquitin-ligase, to link the ubiquitin to lysine residues in the target protein (Fig. 1). A final poly-ubiquitylation step is generally thought to label these target proteins for degradation by the 26S proteasome (Moon et al. 2004).

Arabidopsis mutants revealed that the E3 ligase that participates in auxin signaling is the SKP1/Cullin/F-box (SCF) complex comprising the Arabidopsis SKP1-like protein ASK1, CULLIN1 (CUL1), the F-Box protein Transport Inhibitor Response 1 (TIR1), and the E2-interacting RING domain protein RBX1 (SCFTIR1; Fig. 1) (Hellmann et al. 2003; Gray et al. 1999, 2002; Rueg-

Fig. 1 Auxin signaling in Arabidopsis thaliana is regulated by proteolysis of Aux/IAA proteins mediated by the SCFTIR1 E3 ubiquitin ligase. Aux/IAAs, which inhibit auxin responsive gene expression (AuxRE: Auxin Responsive Element) through binding ARFs (through their Protein-Protein Interaction Domain—PPID), are likely labelled for proteolysis by ubiquitination. This process is mediated by the ubiquitin activating enzyme E1, the ubiquitin conjugating enzyme E2 and the ubiquitin ligase E3. E1 transfers the ubiquitin component to E2 that in turn binds to and acts in concert with E3 to ubiq-uitinate the substrate protein. Once targets are ubiquitinated, they are degraded by the 26S proteasome. SCFTIR1, the E3 ligase complex that participates in auxin signaling, consists of CULLIN1 (AtCULl), the Arabidopsis SKP1 homolog ASK1, and the F-box protein TIR1. Auxin binding to TIR1 enhances its interaction with the Aux/IAAs which leads to enhanced degradation of these proteins. Regulatory subunits of SCFTIR1 include RCE1, which modifies AtCULl by adding RUB1 that is previously activated by the AXR1-ECR1 sub-complex. The COP9 signalosome (CSN) removes RUB1 from AtCULl, leading to subsequent dissociation of the SCFTIR1 complex

Fig. 1 Auxin signaling in Arabidopsis thaliana is regulated by proteolysis of Aux/IAA proteins mediated by the SCFTIR1 E3 ubiquitin ligase. Aux/IAAs, which inhibit auxin responsive gene expression (AuxRE: Auxin Responsive Element) through binding ARFs (through their Protein-Protein Interaction Domain—PPID), are likely labelled for proteolysis by ubiquitination. This process is mediated by the ubiquitin activating enzyme E1, the ubiquitin conjugating enzyme E2 and the ubiquitin ligase E3. E1 transfers the ubiquitin component to E2 that in turn binds to and acts in concert with E3 to ubiq-uitinate the substrate protein. Once targets are ubiquitinated, they are degraded by the 26S proteasome. SCFTIR1, the E3 ligase complex that participates in auxin signaling, consists of CULLIN1 (AtCULl), the Arabidopsis SKP1 homolog ASK1, and the F-box protein TIR1. Auxin binding to TIR1 enhances its interaction with the Aux/IAAs which leads to enhanced degradation of these proteins. Regulatory subunits of SCFTIR1 include RCE1, which modifies AtCULl by adding RUB1 that is previously activated by the AXR1-ECR1 sub-complex. The COP9 signalosome (CSN) removes RUB1 from AtCULl, leading to subsequent dissociation of the SCFTIR1 complex ger et al. 1998). Aux/IAA proteins were shown to specifically interact with the F-box protein TIR1 (Gray et al. 2001), and the recent finding that auxin-binding to TIR1 enhances this interaction and thus leads to enhanced degra dation of Aux/IAAs (Dharmasiri et al. 2003a) uncovered TIR1, together with the closely related proteins AFB1, 2 and 3, as the long-sought auxin receptors (Fig. 1) (Kepinski and Leyser 2005; Dharmasiri et al. 2005a,b). In view of these findings, it is likely that auxin-promoted poly-ubiquitination by the SCFTIR1 E3 ligase marks Aux/IAA proteins for degradation. Remarkably, however, auxin-induced poly-ubiquitination has not yet been demonstrated for Aux/IAA proteins.

Several regulatory components of SCF E3 ligases have been identified. For example, it has been found that the CUL1 subunit of the SCF complex is modified by the addition of the ubiquitin-like protein RUB1/NEDD8 (del Pozo and Estelle 1999). RUB1/NEDD8 is activated by the E1 complex AXR1-ECR1, which catalyzes the transfer of RUB1 to the RUB conjugating enzyme RCE1 that acts together with RBX1 in the RUB modification of CUL1 (Fig. 1) (Gray et al. 2002; Dharmasiri et al. 2003b). Knock-out mutations in most of these regulatory components lead to auxin-resistant phenotypes, and the double mutant axr1/rce1 causes embryonic defects similar to mp, leading to the hypothesis that RUB modification positively regulates SCF activity (Lincoln et al. 1990; Bostick et al. 2004; Larsen and Cancel 2004; Dharmasiri et al. 2003b). The RUB-conjugated state of the SCF complex is regulated by the COP9 Signalosome (CSN), a protein complex that shares reasonable similarity to the lid of the 26S proteasome (Fu et al. 2001). CSN action has been demonstrated to be necessary for both auxin response and RUB1 removal from CUL1 (Schwechheimer et al. 2001), which probably destabilizes the SCF complex after its function so that new complexes can be formed (Fig. 1) (Cope and Deshaies 2003; Schwechheimer 2004). The CSN is also known to interact with other types of E3 ligases, such as the photomorphogenesis related COP1, and to be required for the nuclear import of this RING finger protein (Chamovitz et al. 1996; von Arnim et al. 1997; Seo et al. 2003). COP1 and the CSN have been shown to promote degradation of HY5 (Osterlund et al. 2000a,b), a transcription factor that positively regulates photomorpho-genesis, explaining why loss-of-function mutations in both COP1 and the CSN-subunit encoding genes cause constitutive photomorphogenesis (cop) rather than auxin-related phenotypes (Schwechheimer and Deng 2000).

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