Myocyte hypertrophy is the major compensatory response of the heart to chronic stress. It is induced by the activation of a network of interdependent, intracellular signaling pathways.1 An important pathway activated during the hypertrophic response is the calcineurin Abeta-NFATc transcription factor pathway.2 Our laboratory has recently discovered that calcineurin Abeta and NFATc transcription factors can associate with the scaffold protein mAKAPbeta.3 mAKAPbeta is a scaffold protein that forms a multimolecular signalosome located to the nuclear envelope of cardiac myocytes. Preliminary data demonstrate that calcineurin Abeta binds to a specific site on mAKAPbeta that lacks any of the consensus calcineurin binding sequences previously described. In this report, it is shown that a peptide, which contains the mAKAPbeta -calcineurin Abeta binding domain, associates with calcineurin Abeta in a calcium/calmodulin dependent manner. In addition, the binding of this mAKAPbeta peptide to calcineurin Abeta has no effect on calcineurin?s phosphatase activity. In fact, calcineurin Abeta bound to this mAKAPbeta peptide is catalytically active and capable of dephosphorylating NFAT. This is novel since other scaffold proteins that associate with calcineurin Abeta have been reported to inhibit its phosphatase activity. Furthermore, in our laboratory it has been shown that mAKAPbeta is required for both the nuclear translocation of NFATc and the induction of myocyte hypertrophy in vitro.4 In this report it is demonstrated that inhibition of calcineurin Abeta association to mAKAPbeta affects NFATc phosphorylation state and attenuates the norepinephrine induced hypertrophic response in primary neonatal cardiac myocytes. This study supports the hypothesis that the formation of multimolecular signaling complexes, like the mAKAPbeta signalosome, is necessary for the integration and fidelity of signal transduction involved in physiological processes like hypertrophy. Although hypertrophy is an adaptive response; it is often accompanied by maladaptive remodeling of the heart that can result in heart failure, a leading cause of death in the United States. Research in the signaling complexes involved in myocyte hypertrophy, like the mAKAPbeta signalosome, may lead to the development of novel treatments for pathologic hypertrophy and heart failure.

<p dir="ltr">Membrane-dependent enzymes play crucial roles in cellular signaling by transducing extracellular signals into intracellular responses. Phospholipase Cepsilon (PLCe) and diacylglycerol kinase alpha (DGKa) are membrane-associated enzymes regulated by G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), controlling signaling pathways essential for numerous cellular processes. PLCe catalyzes the hydrolysis of phosphatidylinositol phosphates into inositol phosphates (IPX) and diacylglycerol (DAG), triggering calcium release from intracellular stores and activating protein kinase C (PKC)-dependent pathways. While PLCe is crucial for normal cardiovascular function, hyperactivation or sustained activation can lead to hypertrophy. Due to structural heterogeneity, previous studies focused on isolated regulatory domains or the catalytic core. In this work, I present the first cryo-EM reconstruction of the largest PLCe fragment to date in complex with an antigen-binding fragment (Fab). This structure reveals the domain architecture of the N-terminal regions of the lipase and defines an extended membrane-binding surface critical for maximal basal and G protein-dependent activity. These findings lay the groundwork for high-resolution structures of the full-length enzyme and its complexes with the small GTPase Rap1A. Additionally, I explored the role of mAKAP in the Rap1A–PLCe pathway, alongside the guanine nucleotide exchange factor (GEF) function of PLCe toward Rap1A. In parallel, cryo-EM studies of DGKa bound to a covalent inhibitor were initiated. DGKa reduces DAG, thereby limiting PKC activity, and its inhibition is emerging as a promising cancer immunotherapy target. We have established a protocol for structural studies of full-length DGKa, which will elucidate its structures in basal and inhibited states.</p>