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  • Recently a non proteolytic role of COP in stabilizing

    2020-05-15

    Recently, a non-proteolytic role of COP1 in stabilizing PIF3 proteins in the dark has been revealed . Previous studies have shown that the endogenous levels of PIF3 protein in etiolated seedlings were nearly undetectable . Ling found that Angiotensin (1-7) the GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) plays important roles in COP1-regulated PIF3 protein turnover . Endogenous and recombinant PIF3 proteins were dramatically degraded in the gain-of-function mutant , while in the triple loss-of-function mutant PIF3 accumulated in abundance. The quadruple mutant recovered the PIF3 protein that was undetectable in to levels comparable with wild-type plants, suggesting that BIN2 with its homologs acts downstream of COP1 to stabilize PIF3. Furthermore, phosphorylation of PIF3 by BIN2 has been shown to be required for COP1-stabilizing PIF3 actions. Ling then explored the biochemical mechanism and deduced that the COP1–SPA complex interferes with the BIN2–PIF3 interaction, thus stabilizing PIF3 proteins. Yeast two-hybrid and pull-down assays showed that SPA1 and BIN2 interact with the same regions of PIF3, and COP1 interacts only with BIN2 and not with PIF3. and assays further reveal that COP1 and SPA1 inhibit the BIN2–PIF3 interaction as well as the BIN2-mediated phosphorylation of PIF3 (). Taken together, these data demonstrate that the COP1–SPA1 complex stabilizes PIF3 by blocking BIN2-mediated PIF3 phosphorylation and degradation, in which COP1 sequesters BIN2, while SPA competitively interferes with the interaction between PIF3 and BIN2. This study provides a new mechanism in that COP1 acts as a blocker to stabilize the negative photomorphogenesis regulator in a non-proteolytic manner. Although EIN3 and PIF3 act similarly in repressing photomorphogenesis, we previously proposed a different mode for COP1 stabilizing EIN3 . Genetic analysis showed that overexpression of EIN3 in the mutant rescued ’s defects in skotomorphogenesis (cotyledon opening and expansion) and seedling emergence, suggesting the Angiotensin (1-7) of to . Moreover, COP1 promotes EIN3 protein accumulation by regulating its protein stability but not its transcript levels. To investigate how COP1 stabilizes EIN3, extensive biochemical analysis has been performed with the following results. (i) COP1 physically interacts with EBF1 and EBF2 in the nucleus. (ii) COP1 directly ubiquitinates EBF1/2 . (iii) COP1 degrades EBF1/2 proteins through the 26S proteasome system. Therefore, COP1, EBF1/2, and EIN3 comprise a tandem E3 ubiquitin ligase module through which EIN3 protein levels are maintained at high levels in the dark and are gradually decreased with the seedling growing upward to the soil surface (). This elaborate tandem E3 ubiquitin ligase module ensures the efficient and precise regulation of target protein levels according to multiple signals. Concluding Remarks It has long been known that COP1 directly targets the photomorphogenesis positive regulators for protein ubiquitination and degradation. The two reports highlighted here propose novel modes of action for COP1 – ‘blocker mode’ and the ‘tandem E3 ubiquitin ligase module’ – to uncover the mechanisms underlying how COP1 stabilizes the photomorphogenesis negative regulators. To stabilize PIF3, COP1 blocks the interaction between PIF3 and its kinase BIN2. To stabilize EIN3, COP1 acts as the E3 ligase of EIN3’s E3 ligases EBF1/2 for degradation. Some open questions remain to be explored in future. (i) How does COP1 target different substrates for specific pathways? One clue is that the WD40 repeats have been shown to be responsible for the interactions between COP1 and the positive photomorphogenesis regulators, whereas COP1 interacts with EBF1/2 through the RING-finger and coiled-coil domains. New experimental strategies such as protein complex co-crystallization and structure determination will help us to better understand the principles of COP1’s multiple functions. (ii) What are the relationships of these two newly reported noncanonical modes for COP1? Two recent studies have reported that EBF1 and EBF2 can interact with PIF3 and mediate light-induced PIF3 protein degradation 11, 12. It is tempting to speculate whether etiolated seedlings also adopt the COP1–EBF1/2–PIF3 tandem E3 ligase cascade to stabilize PIF3 abundance. Thus, it will be interesting to investigate whether COP1 uses both pathways to stabilize the substrate or one specific mode under certain conditions. (iii) What are the roles of SPAs in COP1’s noncanonical actions? Regarding the tandem E3 ubiquitin ligase module, it remains unclear whether SPAs are involved. For the blocker mode, evidence from spaQ mutants suggests that SPA proteins mediate the phosphorylation of PIF3 by BIN2. Moreover, protein–protein interaction analysis shows that SPA1, but not full-length COP1, directly interacts with PIF3. However, in the in vitro phosphorylation assay COP1 alone could decrease BIN2-mediated PIF3 phosphorylation without SPA1. Therefore, further investigations are needed to clarify the role of SPAs.