Martin Brent R, Deerinck Thomas J, Ellisman Mark H, Taylor Susan S, Tsien Roger Y
Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Chem Biol. 2007 Sep;14(9):1031-42. doi: 10.1016/j.chembiol.2007.07.017.
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.
与绿色荧光蛋白（GFP）N端融合的四半胱氨酸序列YRECCPGCCMWR在活细胞或体外经双砷标记后会发生自我聚集。这种染料触发的聚集体形成温度依赖性形态，并可通过光漂白分散。将双砷聚集的GFP与蛋白激酶A（PKA）的调节（R）亚基或催化（C）亚基融合，经双砷标记后，完整的全酶会被困在紧密的荧光斑点中。与PKA激活的经典模型相反，除非共表达假底物抑制剂PKI或局部浓缩的底物，升高的环磷酸腺苷（cAMP）不会使RIα和Cα扩散得很远。然而，单独升高cAMP时，RIIα会释放Cα，这依赖于RIIα抑制域的自磷酸化。DAKAP1α的过表达诱导R和C在线粒体外共定位，并表现出类似的调节。总体而言，I型PKA的有效分离依赖于底物，而II型PKA的解离则依赖于自磷酸化。
