Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells.
The tetracysteine sequence YRECCPGCCMWR fused to the N terminus of green fluorescent protein (GFP) self-aggregates upon biarsenical labeling in living cells or in vitro. Such dye-triggered aggregates form temperature-dependent morphologies and are dispersed by photobleaching. Fusion of the biarsenical aggregating GFP to the regulatory (R) or catalytic (C) subunit of PKA traps intact holoenzyme in compact fluorescent puncta upon biarsenical labeling. Contrary to the classical model of PKA activation, elevated cAMP does not allow RIalpha and Calpha to diffuse far apart unless the pseudosubstrate inhibitor PKI or locally concentrated substrate is coexpressed. However, RIIalpha releases Calpha upon elevated cAMP alone, dependent on autophosphorylation of the RIIalpha inhibitory domain. DAKAP1alpha overexpression induced R and C outer mitochondrial colocalization and showed similar regulation. Overall, effective separation of type I PKA is substrate dependent, whereas type II PKA dissociation relies on autophosphorylation.
Martin
Brent R
BR
Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Deerinck
Thomas J
TJ
Ellisman
Mark H
MH
Taylor
Susan S
SS
Tsien
Roger Y
RY
D054758:A Kinase Anchor Proteins / Q000378:metabolism*; D004396:Coloring Agents / Q000494:pharmacology*; D000242:Cyclic AMP; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000378:metabolism; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019281:Dimerization; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes; D008027:Light; D051336:Mitochondrial Membranes / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D013696:Temperature
D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D004396:Coloring Agents; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D007527:Isoenzymes; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; C517757:PRKACA protein, human