Substrate-Induced Degradation of the Α/β-Fold Hydrolase KARRIKIN INSENSITIVE2 Requires a Functional Catalytic Triad but is Independent of MAX2.

Derived from burnt vegetation, karrikins (KAR) are butenolide chemicals that stimulate seed germination and enhance seedling responses to light. Genetic and biochemical studies have identified KARRIKIN INSENSITIVE2 (KAI2) as a putative karrikin receptor protein (reviewed by Waters et al., 2014Waters M.T. Scaffidi A. Sun Y.K. Flematti G.R. Smith S.M. The karrikin response system of Arabidopsis.Plant J. 2014; 79: 623-631Crossref PubMed Scopus (91) Google Scholar). KAI2 is an α/β-fold hydrolase and a paralog of DWARF14 (D14; AtD14 in Arabidopsis), and both possess the conserved catalytic triad of Ser, His, and Asp residues typical of this class of hydrolytic enzymes. D14 proteins are likely receptors for strigolactones (SL), a group of butenolide plant hormones involved in the regulation of shoot architecture, and an intact catalytic triad is essential for D14-mediated SL responses (Hamiaux et al., 2012Hamiaux C. Drummond R.S. Janssen B.J. Ledger S.E. Cooney J.M. Newcomb R.D. Snowden K.C. DAD2 is an α/β hydrolase likely to be involved in the perception of the plant branching hormone, strigolactone.Curr. Biol. 2012; 22: 2032-2036Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). It has been proposed that the catalytic serine of KAI2 and AtD14 initiates nucleophilic attack on the butenolide moiety of KAR and SLs, respectively (Scaffidi et al., 2012Scaffidi A. Waters M.T. Bond C.S. Dixon K.W. Smith S.M. Ghisalberti E.L. Flematti G.R. Exploring the molecular mechanism of karrikins and strigolactones.Bioorg. Med. Chem. Lett. 2012; 22: 3743-3746Crossref PubMed Scopus (66) Google Scholar, Zhao et al., 2013Zhao L.-H. Zhou X.E. Wu Z.S. Yi W. Xu Y. Li S. Xu T.H. Liu Y. Chen R.Z. Kovach A. et al.Crystal structures of two phytohormone signal-transducing α/β hydrolases: karrikin-signaling KAI2 and strigolactone-signaling DWARF14.Cell Res. 2013; 23: 436-439Crossref PubMed Scopus (181) Google Scholar), and that these proteins have both enzyme-like and receptor-like qualities (Waters et al., 2014Waters M.T. Scaffidi A. Sun Y.K. Flematti G.R. Smith S.M. The karrikin response system of Arabidopsis.Plant J. 2014; 79: 623-631Crossref PubMed Scopus (91) Google Scholar). Recently, it was reported that AtD14 was degraded as a result of the SL perception process (Chevalier et al., 2014Chevalier F. Nieminen K. Sánchez-Ferrero J.C. Rodríguez M.L. Chagoyen M. Hardtke C.S. Cubas P. Strigolactone promotes degradation of DWARF14, an α/β hydrolase essential for strigolactone signaling in Arabidopsis.Plant Cell. 2014; 26: 1134-1150Crossref PubMed Scopus (154) Google Scholar). Crucially, this event was dependent on the F-box protein MORE AXILLARY GROWTH2 (MAX2), which is required for seedling responses to both SL and KAR in Arabidopsis (Nelson et al., 2011Nelson D.C. Scaffidi A. Dun E.A. Waters M.T. Flematti G.R. Dixon K.W. Beveridge C.A. Ghisalberti E.L. Smith S.M. F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana.Proc. Natl. Acad. Sci. USA. 2011; 108: 8897-8902Crossref PubMed Scopus (313) Google Scholar). Since the proteasome inhibitor MG132 also blocked degradation of AtD14, it was concluded that AtD14 was likely ubiquitinated in a MAX2-dependent process and thus targeted for degradation by the ubiquitin–proteasome system (Chevalier et al., 2014Chevalier F. Nieminen K. Sánchez-Ferrero J.C. Rodríguez M.L. Chagoyen M. Hardtke C.S. Cubas P. Strigolactone promotes degradation of DWARF14, an α/β hydrolase essential for strigolactone signaling in Arabidopsis.Plant Cell. 2014; 26: 1134-1150Crossref PubMed Scopus (154) Google Scholar). However, it was not clear if this degradation event was a requirement for or a consequence of SL perception. In addition, it is not known if KAI2 undergoes a similar degradation process as part of karrikin signaling. We investigated KAI2 protein levels in Arabidopsis seedlings exposed to KAI2 substrates. First, we assessed the relative effect of KAR1 and KAR2 upon KAI2 levels in Landsberg erecta (Ler) seedlings grown in liquid culture. We found that both karrikin analogs could trigger the destabilization of KAI2, but surprisingly KAR2 was at least 100-fold more potent than KAR1 (Figure 1A). Notably, this difference is more accentuated than that observed in morphological responses: KAR1 is about 10-fold less potent than KAR2 in terms of inhibiting hypocotyl elongation (Waters et al., 2012Waters M.T. Nelson D.C. Scaffidi A. Flematti G.R. Sun Y.K. Dixon K.W. Smith S.M. Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis.Development. 2012; 139: 1285-1295Crossref PubMed Scopus (348) Google Scholar), suggesting that molecular activity and the degree of developmental outputs are not linearly related. Substantially reduced levels of KAI2 could be detected within 2 h of continuous exposure to 5 μM KAR2, reaching a plateau within 4–8 h (Figure 1B), potentially reflecting a steady-state level in which the rates of protein degradation and biosynthesis are similar. Besides KAR, KAI2 can also mediate seed and seedling responses to the synthetic SL analog GR24 (Waters et al., 2012Waters M.T. Nelson D.C. Scaffidi A. Flematti G.R. Sun Y.K. Dixon K.W. Smith S.M. Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis.Development. 2012; 139: 1285-1295Crossref PubMed Scopus (348) Google Scholar). GR24 as used routinely is a racemic mixture (rac-GR24) of two enantiomers, which have distinct biological activity in Arabidopsis (Scaffidi et al., 2014Scaffidi A. Waters M.T. Sun Y.K. Skelton B.W. Dixon K.W. Ghisalberti E.L. Flematti G.R. Smith S.M. Strigolactone hormones and their stereoisomers signal through two related receptor proteins to induce different physiological responses in Arabidopsis.Plant Physiol. 2014; 165: 1221-1232Crossref PubMed Scopus (191) Google Scholar). To examine whether other butenolides that signal via KAI2 might also affect KAI2 stability, we tested these two enantiomers of GR24. We found that, while less potent than KAR2, GR24ent-5DS stimulated a reduction in KAI2 levels, but GR245DS did not (Figure 1C). This differential effect of the two enantiomers is consistent with previously observed KAI2-dependent responses in Arabidopsis in which only GR24ent-5DS is effective (Scaffidi et al., 2014Scaffidi A. Waters M.T. Sun Y.K. Skelton B.W. Dixon K.W. Ghisalberti E.L. Flematti G.R. Smith S.M. Strigolactone hormones and their stereoisomers signal through two related receptor proteins to induce different physiological responses in Arabidopsis.Plant Physiol. 2014; 165: 1221-1232Crossref PubMed Scopus (191) Google Scholar). We considered that apparent KAR2-dependent reduction in KAI2 protein levels might reflect reduced transcription, or a change in the epitope recognized by the KAI2 antibody. However, a GFP–KAI2 fusion protein expressed from the constitutive 35S promoter and detected with an anti-GFP antibody showed similar KAR2-dependent destabilization, suggesting that changes in KAI2 levels do not result from a change in KAI2 transcripts or from obfuscation of the native KAI2 epitope (Figure 1D). Thus KAR2, and to a lesser extent KAR1 and GR24ent-5DS, trigger a reduction in KAI2 protein levels by a post-translational mechanism. To establish whether MAX2 is necessary for the response of KAI2 to KAR2, we examined KAI2 responses in three different max2 mutants. Surprisingly, patterns of KAI2 destabilization were indistinguishable between all max2 alleles and their respective wild-types, although the loss of KAI2 was much more pronounced in Ler background than in Col-0 (Figure 1E). Next, to test whether the loss of KAI2 requires a functional catalytic serine, we mutated this to alanine (S95A). We expressed both wild-type and mutant proteins under the control of the native KAI2 promoter in the null kai2-2 background. Whereas KAI2 levels responded in the wild-type (KAI2:KAI2) control similarly to Ler, KAI2 S95A levels showed little to no reduction over the same time period in two independent transgenic lines (Figure 1F). This result suggests that substrate-induced KAI2 instability is a consequence of ligand attack mediated by the catalytic serine. When purified KAI2 and KAI2 S95A proteins were incubated with KAR2 in vitro, no change in protein abundance or size was observed (Supplemental Figure 1A). In addition, KAI2 levels did not change substantially in response to KAR2 in cell-free lysates extracted from Ler seedlings (Supplemental Figure 1B). Together, these findings imply that KAR2 does not affect KAI2 stability directly but that KAI2 breakdown requires cellular integrity. To establish whether KAR2 triggers degradation of KAI2 via the 26S proteasome, we tested the effect of the proteasomal inhibitor MG132. Surprisingly, MG132 could not prevent the loss of KAI2 in the presence of KAR2, but did lead to an increase in abundance of poly-ubiquitinated proteins, suggesting that proteasome activity was indeed blocked (Figure 1G). Furthermore, we could find no evidence that KAI2 protein is ubiquitinated in planta (Figure 1H). We also tested the broad-spectrum serine protease inhibitor AEBSF and the cysteine protease inhibitors ALLN and E-64d. None of these compounds could prevent KAI2 destabilization by KAR2 (Figure 1I and 1J; Supplemental Figure 1C). Overall, these data imply that KAI2-dependent butenolide signaling induces the degradation or loss of KAI2, via a mechanism that follows enzymatic attack upon the substrate. This mechanism does not depend on MAX2 or the 26S proteasome, but does require other unidentified cellular components. Changes in tertiary structure of DAD2/D14 following interaction with GR24 in vitro have been inferred based on a shift in protein melting temperature, and the corresponding serine mutant does not show such a response (Hamiaux et al., 2012Hamiaux C. Drummond R.S. Janssen B.J. Ledger S.E. Cooney J.M. Newcomb R.D. Snowden K.C. DAD2 is an α/β hydrolase likely to be involved in the perception of the plant branching hormone, strigolactone.Curr. Biol. 2012; 22: 2032-2036Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). Assuming these changes are also true for KAI2 in planta, our results are consistent with a model in which serine-mediated hydrolytic attack upon the ligand triggers a conformational change in protein structure, which in turn leads to protein destabilization. Recent crystallographic data indicating that the pyran ring of KAR1 interacts with phenylalanine residues lining the KAI2 active-site pocket, distal from the catalytic triad itself (Guo et al., 2013Guo Y. Zheng Z. La Clair J.J. Chory J. Noel J.P. Smoke-derived karrikin perception by the α/β-hydrolase KAI2 from Arabidopsis.Proc. Natl. Acad. Sci. USA. 2013; 110: 8284-8289Crossref PubMed Scopus (121) Google Scholar), are at odds with the functional requirement for the catalytic serine. It is formally possible that the S95A mutation prevents the reported conformational changes in KAI2 induced by KAR1 binding, without KAR1 actually interacting with the catalytic triad. However, we do not favor this explanation for two reasons. First, crystallography of rice D14 has shown the hydrolyzed butenolide moiety (D ring) from rac-GR24 covalently attached to the active-site serine (Zhao et al., 2013Zhao L.-H. Zhou X.E. Wu Z.S. Yi W. Xu Y. Li S. Xu T.H. Liu Y. Chen R.Z. Kovach A. et al.Crystal structures of two phytohormone signal-transducing α/β hydrolases: karrikin-signaling KAI2 and strigolactone-signaling DWARF14.Cell Res. 2013; 23: 436-439Crossref PubMed Scopus (181) Google Scholar), making it more likely that a similar mechanism holds for the action of KAI2 on GR24ent-5DS and karrikins. Second, karrikin analogs with a saturated, non-aromatic pyran ring are still biologically active, albeit at reduced levels (Scaffidi et al., 2012Scaffidi A. Waters M.T. Bond C.S. Dixon K.W. Smith S.M. Ghisalberti E.L. Flematti G.R. Exploring the molecular mechanism of karrikins and strigolactones.Bioorg. Med. Chem. Lett. 2012; 22: 3743-3746Crossref PubMed Scopus (66) Google Scholar). Hence, pi-pi stacking of these saturated karrikin analogs with the phenylalanine residues is unlikely to occur. Instead, to account for the observed ligand–receptor structure (Guo et al., 2013Guo Y. Zheng Z. La Clair J.J. Chory J. Noel J.P. Smoke-derived karrikin perception by the α/β-hydrolase KAI2 from Arabidopsis.Proc. Natl. Acad. Sci. USA. 2013; 110: 8284-8289Crossref PubMed Scopus (121) Google Scholar), it is plausible that the karrikin ligand may be in a position that precedes subsequent relocation allowing catalysis, or has moved within the KAI2 pocket after interaction with the catalytic triad. Under the conditions of protein crystallization, KAR1 may remain in this position. The KAI2 degradation mechanism is evidently different from that proposed for rac-GR24-induced destabilization of AtD14 (Chevalier et al., 2014Chevalier F. Nieminen K. Sánchez-Ferrero J.C. Rodríguez M.L. Chagoyen M. Hardtke C.S. Cubas P. Strigolactone promotes degradation of DWARF14, an α/β hydrolase essential for strigolactone signaling in Arabidopsis.Plant Cell. 2014; 26: 1134-1150Crossref PubMed Scopus (154) Google Scholar). KAI2 degradation is independent of MAX2, is not proteasome mediated, and we could not detect ubiquitinated KAI2; pertinently, these three observations are mutually supportive because MAX2 is part of a complex that poly-ubiquitinates substrates such as D53 for proteasomal degradation (Jiang et al., 2013Jiang L. Liu X. Xiong G. Liu H. Chen F. Wang L. Meng X. Liu G. Yu H. Yuan Y. et al.DWARF 53 acts as a repressor of strigolactone signalling in rice.Nature. 2013; 504: 401-405Crossref PubMed Scopus (506) Google Scholar, Zhou et al., 2013Zhou F. Lin Q. Zhu L. Ren Y. Zhou K. Shabek N. Wu F. Mao H. Dong W. Gan L. et al.D14-SCFD3-dependent degradation of D53 regulates strigolactone signalling.Nature. 2013; 504: 406-410Crossref PubMed Scopus (528) Google Scholar). The mechanism for removal of KAI2 protein after signaling is therefore currently unclear. One possibility is lysosomal or vacuolar degradation, a process that would require cellular compartmentalization. Nevertheless, the process is highly specific, being triggered only by ligands that operate through KAI2. Complete absence of the protein (i.e. in kai2-2 mutants) imparts a phenotype that is opposite to that of karrikin signaling. Therefore, loss of KAI2 is likely to be a consequence rather than a cause of the signaling process. Furthermore, that an enzyme be destroyed as a result of its activity is unusual, suggesting that KAI2 does not simply modify a substrate into a further bioactive compound. Rather, KAI2 degradation may be an integral element of its function, allowing removal of the activated or "used" receptor in its post-signaling state. Screening KAI2 mutant proteins for stabilized but constitutively active variants might be instructive in establishing the biological significance of, and mechanistic basis for, KAI2 turnover. No conflict of interest declared. Download .pdf (1.29 MB) Help with pdf files Document S1. Supplemental Figure 1, Supplemental Methods, and Supplemental References