Indicative of the importance of protein phosphorylation in the core circadian clock mechanism, chronically applied inhibitors of both protein kinases and phosphoprotein phosphatases have significant effects on the period, phase, and light-dependent regulation of circadian rhythms in the dinoflagellate Lingulodinium polyedrum. This study was aimed at identifying the presence of the affected phosphatase(s). Dephosphorylation of a PP1/PP2A-specific substrate by L. polyedrum extracts was inhibited by okadaic acid only at concentrations greater than 100 nM, as in vivo, by mammalian inhibitor-2 (I-2), and by an endogenous inhibitor with properties similar to I-2, indicating that a type-1 protein phosphatase (PP1) was predominant. A cDNA encoding a highly conserved PP1 was isolated, the 1st such signaling molecule identified in dinoflagellates. Anti-sera specific for this type of phosphatase recognized a 34 kDa protein in L. polyedrum extract, this being the same size as the PP1 encoded by the isolated cDNA. These findings are consistent with the suggestion that the L. polyedrum PP1 may be a part of the clock mechanism in this species.
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