Identification and characterization of cAMP-dependent protein kinase and its possible direct interactions with protein phosphatase-1 in marine dinoflagellates.

We have examined the nature of signal transduction involving reversible protein phosphorylation in marine Prorocentrale species. Of particular interest is the marine dinoflagellate Prorocentrum lima in which the tumour promoter okadaic acid is produced and may interfere with signal transduction. We have identified cAMP-dependent protein kinase (PKA) activity in P. lima, P. micans, and P. minimum. The P. lima enzyme was characterized biochemically and appears to consist of two different isoforms in the R2C2 configuration. Whole cell extracts of P. micans and P. minimum treated with the specific PKA inhibitor peptide PKI (5-24) or cAMP demonstrated altered intensities of phosphopeptide 32P labeling, most likely involving regulation of a protein phosphatase via PKA activity. A primary candidate for PKA regulation is protein phosphatase-1 (PP-1), which in P. lima possesses a classical PKA consensus phosphorylation site. We demonstrate that a peptide fragment of PP-1 from P. lima corresponding to this PKA phosphorylation site can be effectively phosphorylated by PKA and dephosphorylated by calcineurin. We speculate that PP-1 activity among several lower eukaryotes may be mediated directly by reversible phosphorylation. Higher eukaryotes may have developed inhibitor proteins to provide more complex regulation of protein phosphatase activity.