Fyn Regulates Cyclic‐AMP Dependent Protein Kinase A Binding Interactions

Abstract

Cyclic‐AMP dependent protein kinase A (PKA) and Src family kinases (SFKs) are important for signal transduction in a multitude of cellular processes. While it is known that PKA positively regulates SFKs such as Src and Fyn through phosphorylation, we have discovered that Fyn can regulate PKA through a reciprocal phosphorylation event on the catatlytic subunit (PKA‐C). Here we report that PKA holoenzymes are physically associated with Fyn in HEK293 cells, suggesting the formation of a novel signaling complex. Interestingly, a Fyn mutant lacking the SH3 domain displayed markedly enhanced binding to the PKA catalytic subunit as compared to wild type (WT) Fyn. To determine whether Fyn regulates the interaction of PKA with intracellular binding partners, we employed stable isotopic labeling of amino acids in cell culture (SILAC) coupled with tandem mass spectrometry. Indeed, in cells overexpressing WT‐Fyn, PKA displayed enhanced binding to 40 intracellular proteins. The three most significantly enhanced binding partners (AKAP9, PDE4DIP, and CDK5RAP2) were A kinase anchoring proteins (AKAPs) known to function in microtubule nucleation and organization at the centrosome and Golgi apparatus. The ability of Fyn to modulate the physical interaction of PKA with these AKAPs was validated through coimmunoprecipitation and western blot analysis. Interestingly, this function of Fyn was independent of its kinase activity, as similar results were obtained in experiments using a kinase dead (KD) allele of Fyn. Taken together, our results suggest that Fyn complexes with PKA and directs its association with distinct AKAPs. Current studies aim to further characterize this novel molecular complex and identify downstream substrates of these kinases that may be targeted due to this interaction.Support or Funding InformationThis work was supported by the Arnold and Mabel Beckman foundation Beckman Scholars Program, a Scientist Development Grant from the AHA and an Endowed Professorship (to PBD) and the Vermont Genetics Network through U. S. National Institutes of Health Grant 8P20GM103449 from the INBRE program of the NIGMS.