A type-1 phosphoprotein phosphatase from a dinoflagellate as a possible component of the circadian mechanism

Area of Science:

• * Molecular Biology
• * Chronobiology
• * Marine Biology

Background:

• * Protein phosphorylation is crucial for circadian clock mechanisms.
• * Circadian rhythms in the dinoflagellate Lingulodinium polyedrum are affected by kinase and phosphatase inhibitors.
• * The specific phosphatases involved in L. polyedrum's circadian clock were previously unidentified.

Purpose of the Study:

• * To identify the protein phosphatase(s) involved in the circadian clock of Lingulodinium polyedrum.
• * To investigate the presence and characteristics of phosphatases in L. polyedrum extracts.
• * To isolate and characterize the gene encoding the identified phosphatase.

Main Methods:

• * Enzyme assays using a PP1/PP2A-specific substrate with L. polyedrum extracts.
• * Inhibition studies using okadaic acid and mammalian inhibitor-2 (I-2).
• * Isolation of a cDNA encoding a conserved protein phosphatase type 1 (PP1).
• * Western blot analysis using antisera specific to the identified PP1.

Main Results:

• * L. polyedrum extracts showed predominant type-1 protein phosphatase (PP1) activity, indicated by inhibition patterns similar to mammalian I-2.
• * Okadaic acid only inhibited phosphatase activity at high concentrations (>100 nM).
• * A cDNA encoding a highly conserved PP1 was isolated from L. polyedrum.
• * Western blot confirmed a 34 kDa protein in extracts, matching the size of the encoded PP1.

Conclusions:

• * A type-1 protein phosphatase (PP1) is the predominant phosphatase in Lingulodinium polyedrum.
• * The identified L. polyedrum PP1 is highly conserved and likely plays a role in the dinoflagellate's circadian clock mechanism.
• * This is the first signaling molecule of its kind identified in dinoflagellates.