In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K–PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

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.

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.