Molecular basis for blue light-dependent phosphorylation of Arabidopsis cryptochrome 2

Abstract

Plant cryptochromes undergo blue light-dependent phosphorylation to regulate their activity and abundance, but the protein kinases that phosphorylate plant cryptochromes have remained unclear. Here we show that photoexcited Arabidopsis cryptochrome 2 (CRY2) is phosphorylated in vivo on as many as 24 different residues, including 7 major phosphoserines. We demonstrate that four closely related Photoregulatory Protein Kinases (previously referred to as MUT9-like kinases) interact with and phosphorylate photoexcited CRY2. Analyses of the ppk123 and ppk124 triple mutants and amiR4k artificial microRNA-expressing lines demonstrate that PPKs catalyse blue light-dependent CRY2 phosphorylation to both activate and destabilize the photoreceptor. Phenotypic analyses of these mutant lines indicate that PPKs may have additional substrates, including those involved in the phytochrome signal transduction pathway. These results reveal a mechanism underlying the co-action of cryptochromes and phytochromes to coordinate plant growth and development in response to different wavelengths of solar radiation in nature.

Beyond the photocycle — how cryptochromes regulate photoresponses in plants?

“…This makes the correlative phenotypic analyses for the specific effect of PPKs on cryptochrome phosphorylation technically difficult if not impossible. Nevertheless, the ppk123 and ppk124 triple mutants and the amiR 4k transgenic lines expressing the artificial microRNAs targeting all four PPKs exhibited delayed flowering similar to that of the cry2 mutant [6,44 •• ], which is consistent with PPKs being positive regulators of CRY2 function. Mass spectrometry analyses of the CRY2 proteins phosphorylated by individual PPKs indicate that different PPKs catalyze phosphorylation of CRY2 at overlapping but not identical residues, suggesting the partial functional redundancy of the four PPKs.…”

“…Phosphorylation of CRY2 not only enhances its activity but also facilitates its ubiquitylation and degradation by the CUL4 COP1/SPAs and other E3 ubiquitin ligases [39,41–43]. The label-free quantitative mass spectrometry analyses of the Arabidopsis CRY2 proteins purified from plants identified at least two dozen phosphorylated residues of CRY2, including 18 serine and 6 threonine residues [44 •• ,45]. The level of phosphorylation in almost all those phosphorylated residues increases in response to blue light.…”

“…Although crypto-chromes may undergo autophosphorylation in vitro , their phosphorylation in vivo is primarily catalyzed by protein kinases. A recent mass spectrometry analysis of the CRY2 protein complex purified from plants identified four closely related CRY2-associated protein kinases referred to as photoregulatory protein kinases (PPK1 to PPK4) [44 •• ,47]. PPKs are plant-specific protein kinases evolutionarily derived from the ubiquitous Casein kinase I, which were previously called MUT9-like kinases [48].…”

“…PPKs are plant-specific protein kinases evolutionarily derived from the ubiquitous Casein kinase I, which were previously called MUT9-like kinases [48]. All four PPKs preferentially interact with photoexcited but unphosphorylated CRY2, and they catalyze blue light-dependent phosphorylation of CRY1 and CRY2 in human HEK293 cells co-expressing the respective CRY and PPKs [44 •• ] (Q. Liu, unpublished results). Unexpectedly, the previously reported CRY2 kinases CK1.3 and CK1.4 did not phosphorylate CRY2 in HEK293 cells in either the electrophoretic migration shift assay or the mass spectrometry analyses [44 •• ,49].…”

“…All four PPKs preferentially interact with photoexcited but unphosphorylated CRY2, and they catalyze blue light-dependent phosphorylation of CRY1 and CRY2 in human HEK293 cells co-expressing the respective CRY and PPKs [44 •• ] (Q. Liu, unpublished results). Unexpectedly, the previously reported CRY2 kinases CK1.3 and CK1.4 did not phosphorylate CRY2 in HEK293 cells in either the electrophoretic migration shift assay or the mass spectrometry analyses [44 •• ,49]. Therefore, the previously proposed function of plant Casein kinase I in CRY phosphorylation remains to be further investigated.…”