Daily changes in the phosphoproteome of the dinoflagellate Lingulodinium polyedra

Dinoflagellates are unicellular and generally marine protists and have been used as a model for the study of the biochemical bases of daily rhythms. Rhythms can result from changes in gene expression, and in the dinoflagellate Lingulodinium polyedra this occurs at a translational rather than a transcriptional level. However, the implication of other post-transcriptional mechanisms has not been previously examined. We report here that the abundance of phosphorylated proteins can also be rhythmic over the course of a diurnal cycle in L. polyedra. LC-MS/MS analyses of enriched phosphopeptide preparations identified 2485 unique peptides of which 348 showed significant rhythms in abundance over a 24-h light-dark cycle as judged by JTK_Cycle. The abundance of these rhythmic peptides typically showed peaks at either midday and midnight, and intriguingly, some peptides showed two peaks of abundance. Many of the phosphopeptides were found to be derived from proteins with functions related to RNA/DNA binding, metabolism, and phosphatase/kinase activities. The sequence motif surrounding the phosphosite was used to predict the kinase involved, and the kinases CK2, CDK9, and CDK4 appeared responsible for phosphorylation of most of the rhythmic phosphorylated peptides.