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Identification and characterization of cAMP-dependent protein kinase and its possible direct interactions with
J F Dawson1, K H Wang, C F Holmes
1Department of Biochemistry, University of Alberta, Edmonton, Canada. john_dawson@darwin.biochem.ualberta.ca
Abstract:
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.
Insights
Marine dinoflagellates like Prorocentrum lima utilize cAMP-dependent protein kinase (PKA) for signal transduction. This study reveals PKA may regulate protein phosphatase-1 (PP-1) activity through reversible phosphorylation in these species.
Area of Science:
- Marine biology
- Molecular biology
- Biochemistry
Background:
- Signal transduction pathways involving reversible protein phosphorylation are crucial in cellular processes.
- Marine dinoflagellates, such as Prorocentrum lima, produce compounds like okadaic acid that can interfere with cellular signaling.
- Understanding these pathways in lower eukaryotes provides insights into evolutionary conservation and divergence.
Purpose of the Study:
- To investigate signal transduction mechanisms, specifically cAMP-dependent protein kinase (PKA) activity, in marine Prorocentrum species.
- To characterize PKA activity in Prorocentrum lima and its potential role in regulating protein phosphatases.
- To explore the involvement of reversible protein phosphorylation in the regulation of protein phosphatase-1 (PP-1) in lower eukaryotes.
Main Methods:
- Identification and biochemical characterization of cAMP-dependent protein kinase (PKA) activity in Prorocentrum lima, P. micans, and P. minimum.
- Analysis of phosphopeptide labeling in response to PKA activators and inhibitors.
- Investigation of protein phosphatase-1 (PP-1) phosphorylation sites and their regulation by PKA and calcineurin using peptide fragments.
Main Results:
- cAMP-dependent protein kinase (PKA) activity was detected in P. lima, P. micans, and P. minimum.
- Biochemical characterization of P. lima PKA revealed two isoforms (R2C2 configuration).
- PKA activity influences phosphopeptide labeling in P. micans and P. minimum, suggesting regulation of protein phosphatases. P. lima's PP-1 contains a PKA phosphorylation site, which is phosphorylated by PKA and dephosphorylated by calcineurin.
Conclusions:
- PKA plays a role in signal transduction in marine dinoflagellates.
- Protein phosphatase-1 (PP-1) activity in Prorocentrum lima is likely regulated by PKA-mediated reversible phosphorylation.
- This phosphorylation-based regulation of PP-1 may be a conserved mechanism in lower eukaryotes, contrasting with more complex regulation in higher eukaryotes.