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Abstract
The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif. This led to the hypothesis that Fyn could affect proteins in complex with PKA. To test this, we employed a quantitative mass spectrometry approach to identify Fyn-dependent binding partners in complex with PKA-C. We found Fyn enhanced the binding of PKA-C to several cytoskeletal regulators that localize to the centrosome and Golgi apparatus. Three of these Fyn-induced PKA interactors, AKAP9, PDE4DIP, and CDK5RAP2, were validated biochemically and were shown to exist in complex with Fyn and PKA in a glioblastoma cell line. Intriguingly, the complexes formed between PKA-C and these known AKAPs were dependent upon Fyn catalytic activity and expression levels. In addition, we identified Fyn-regulated phosphorylation sites on proteins in complex with PKA-C. We also identified and biochemically validated a novel PKA-C interactor, LARP4, which complexed with PKA in the absence of Fyn. These results demonstrate the ability of Fyn to influence the docking of PKA to specific cellular scaffolds and suggest that Fyn may affect the downstream substrates targeted by PKA.
Highlights
The cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is a serine/threonine kinase that plays many important roles in fundamental cellular processes, including cell proliferation, differentiation, and migration [1]
We recently identified a novel interaction between the tyrosine kinase Fyn and PKA, in which Fyn co-immunoprecipitates with and phosphorylates Y69 of the α isoform of the catalytic PKA subunit (PKA–Cα) [21]
AKAP9, PDE4DIP, and CDK5RAP2 are known interactors of the PKA holoenzyme, we identified two novel PKA–Cα interactors common to both SILAC experiments, LARP4 and
Summary
The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase that plays many important roles in fundamental cellular processes, including cell proliferation, differentiation, and migration [1]. Many PKA interactors have evolved to sequester PKA within distinct subcellular locations for spatiotemporal regulation of its activity [2,3,4,5,6,7]. The C subunits remain inactive when bound to a pseudo-substrate consensus sequence within the R subunits, and are activated and canonically released upon the binding of cyclic adenosine monophosphate (cAMP). The subcellular localization of PKA is mediated by A-kinase anchoring proteins (AKAPs), a class of scaffolding proteins that interacts with PKA-R subunits.
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