The Molecular Mechanism Of Bluelight-dependent Phosphorylation And Degradation Of Arabidopsis Cryptochrome2

Cryptochromes are blue light receptors playing important roles in growth and development of plants. Arabidopsis CRY1 and CRY2 can be phosphorylated under blue light, subsequently interact with other proteins in a blue light dependent manner to transduce blue light signal. This kind of blue light-dependent phosphorylation follows up the initiation of blue light transduction and galvanizes the activity of CRY. However, the mechanism under CRYphosphorylation is not clear yet.By using CRY2 as bait to screen the Arabidopsis c DNA library in yeast, 4 kinases were picked out due to the interaction with CRY2 in a blue light-dependent manner. These kinases were named PPK1, PPK2, PPK3 and PPK4, respectively. PPK is the abbreviation for Photoregulatory Protein Kinase. Undoubtedly, the discovery of these 4 PPKs supports us a new way to study the mechanism of blue light-dependent phosphorylation of CRY2.The blue light-dependent interaction between CRY2 and PPKs(PPK1-PPK4) is verified by liquid assay of yeast two-hybrid and mass spectrometry analyses for plant CRY2 interacting complex. This means PPKs may be the kinases of CRY2 whichwe are looking for. Co-expression of CRY2 with any of the four PPKs in HEK293 T cells resulted in the generation of a phosphatase-sensitive slower-migrating form of CRY2, indicating that PPKs phosphorylate CRY2. Moreover, other protein kinases(CK1.3, CK1.4 or MAPK12) can not phosphorylate CRY2 under the same conditions, indicating the specificity of CRY2 phosphorylation by PPKs. Consistent with the CRY2 phosphorylation in plants, the PPK-catalyzed CRY2 phosphorylation in HEK293 T cells was dependent on the total fluence, fluence rate or photon density.In the next step mass spectrometry analyses, phosphorylation was detected on at lease 18 serine residues and 6 threonine residues of CRY2. Among these phosphosites, 7 phosphoserines(S506,S523,S525,S526,S598,S599 and S605) were reproducibly detected in all the three independent experiments. We did the same mass spectrometry study for CRY2 phosphorylation sites analyses in HEK293 T cells co-transfected to express CRY2 and PPKs. Results of our MS analyses demonstrate that all the 7 major phosphosites of CRY2 were similarly detected in CRY2 proteins purified from plants or from the HEK293 T cells. In contrast, little phosphorylation was detected in the CRY2 protein purified from HEK293 T cells co-expressing CK1.3, CK1.4 or catalytically inactive PPK1D267 Nmutant.Subsequently,how PPKs affect the biochemistry activity and function of CRY2 was investigated in Arabidopsis. Because the PPK genes are known to function redundantly, we analyzed the ppk123 and ppk124 triple mutants that impaired multiple PPK genes and the ami R4 k transgenic lines that express artificial micro RNAs to knockdown multiple PPK genes. Similar to the cry2 mutant, the ppk123 and ppk124 triple mutants and ami R4 k lineexhibited delayed flowering in long-day photoperiods. As expected, m RNA expression of the FT genewas significantly reduced in the ppk123 and ppk124 triple mutants. Importantly, blue light-dependent CRY2 phosphorylation is clearly impaired in the ppk123 mutant, the ppk124 mutant, and the ami R4 k transgenic line. The blue light-dependent degradation of CRY2 was also suppressed in all three mutant genotypes. Based on results of this study, we propose that in plants PPKs phosphorylate CRY2 under blue light activating its function and degradation.Arabidopsis cryptochrome CRY2 undergoes blue light-specific phosphorylation-dependent ubiquitination and is eventually degraded through proteasome pathway. Ubiquitination-dependent degradation is the main way for cryptochromes desensitizing their signal transduction. However,the specific ubiquitin E3 ligase for CRY2 ubiquitination has not been discovered and the mechanism of CRY2 degradation is not clear yet.In our study, the stability of CRY2 protein is analyzed by genetic method to figure out the molecular mechanism regulating blue light-dependent proteolysis of CRY2. We found that the F-box proteins ZTL and LKP2, which mediate blue-light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1/SPA1 protein complex in the blue light-dependent CRY2 degradation is more likely attributable to its CUL4-based E3 ubiquitin ligase activity than its activity as the cryptochrome-signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4-1mutant, the cop1-5 null mutant, and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele(axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that the photoexcited CRY2 undergoes the ubiquitination catalyzed by the CUL4-based and CUL1-based E3 ubiquitin ligases further clarifying the molecular mechanism under CRY2 ubiquitination and degradation.