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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America. >Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches
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Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches

机译：Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches

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Protein phosphorylation by cyclic AMP-dependent protein kinase (PKA) underlies key cellular processes, including sympathetic stimulation of heart cells, and potentiation of synaptic strength in neurons. Unrestrained PKA activity is pathological, and an enduring challenge is to understand how the activity of PKA catalytic subunits is directed in cells. We developed a light-activated cross-linking approach to monitor PKA subunit interactions with temporal precision in living cells. This enabled us to refute the recently proposed theory that PKA catalytic subunits remain tethered to regulatory subunits during cAMP elevation. Instead, we have identified other features of PKA signaling for reducing catalytic subunit diffusion and increasing recapture rate. Comprehensive quantitative immunoblotting of protein extracts from human embryonic kidney cells and rat organs reveals that regulatory subunits are always in large molar excess of catalytic subunits (average similar to 17-fold). In the majority of organs tested, type II regulatory (RII) subunits were found to be the predominant PKA subunit. We also examined the architecture of PKA complexes containing RII subunits using cross-linking coupled tomass spectrometry. Quantitative comparison of cross-linking within a complex of RII beta and C beta, with or without the prototypical anchoring protein AKAP18 alpha, revealed that the dimerization and docking domain of RII beta is between its second cAMP binding domains. This architecture is compatible with anchored RII subunits directing the myristylated N terminus of catalytic subunits toward the membrane for release and recapture within the plane of the membrane.

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