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Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living
Brent R Martin1, Thomas J Deerinck, Mark H Ellisman
1Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Biarsenical labeling of fused green fluorescent protein (GFP) creates self-aggregates. This technique reveals that protein kinase A (PKA) holoenzyme dissociation is substrate-dependent for type I and relies on autophosphorylation for type II.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- The tetracysteine sequence fused to green fluorescent protein (GFP) enables self-aggregation upon biarsenical labeling.
- This aggregation phenomenon can be modulated by temperature and photobleaching.
- Protein Kinase A (PKA) is a crucial enzyme involved in various cellular signaling pathways.
Purpose of the Study:
- To investigate the self-aggregation properties of biarsenical labeling fused to GFP.
- To explore the regulation and dissociation mechanisms of PKA holoenzyme using this novel labeling technique.
- To compare the dissociation dynamics of Type I and Type II PKA under various cellular conditions.
Main Methods:
- Fusion of a tetracysteine-tagged GFP to regulatory (R) and catalytic (C) subunits of PKA.
- Biarsenical labeling to induce self-aggregation and visualize PKA localization in living cells and in vitro.
- Manipulation of cAMP levels, coexpression of inhibitors (PKI), substrates, and DAKAP1alpha to study PKA dissociation.
Main Results:
- Biarsenical labeling of fused GFP induces dye-triggered, temperature-dependent aggregates that can be dispersed by photobleaching.
- PKA holoenzyme (RIalpha-Calpha and RIIalpha-Calpha) is trapped in fluorescent puncta upon labeling.
- Type I PKA dissociation requires coexpression of PKI or substrate, contrary to classical models.
- Type II PKA dissociation is induced by elevated cAMP alone, dependent on RIIalpha autophosphorylation.
- DAKAP1alpha overexpression promotes outer mitochondrial colocalization of R and C subunits with similar regulation.
Conclusions:
- The biarsenical labeling of fused GFP provides a powerful tool to study protein complex dynamics in real-time.
- Effective separation of Type I PKA holoenzyme is dependent on substrate availability.
- Type II PKA dissociation is regulated by autophosphorylation of the RIIalpha subunit, independent of substrate.
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