Evidence for cAMP-dependent protein kinase in the dinoflagellate, Amphidinium operculatum

Abstract

A cAMP dependent protein kinase (PKA) was identified in the dinoflagellate Amphidinium operculum. In vitro kinase activity towards kemptide, a PKA-specific substrate, was not detectable in crude lysates. However, fractionation of dinoflagellate extracts by gel filtration chromatography showed PKA-like activity toward kemptide at approximately 66 kDa. These findings suggest that possible low molecular mass inhibitors in crude lysates were removed by the gel filtration chromatography. Pre-incubation of extracts with cAMP prior to chromatography resulted in an apparent molecular mass shift in the in vitro kinase assay to 40 kDa. An in-gel kinase assay reflected activity of the free catalytic subunit at approximately 40 kDa. Furthermore, western blotting with an antibody to the human PKA catalytic subunit confirmed a catalytic subunit with a mass of approximately 40 kDa. Results from this study indicate that the PKA in A. operculatum has a catalytic subunit of similar size to that in higher eukaryotes, but with a holoenzyme of a size suggesting a dimeric, rather than tetrameric structure.