An AKAP-Lbc-RhoA interaction inhibitor promotes the translocation of aquaporin-2 to the plasma membrane of renal collecting duct principal cells

Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking

Pharmacological interference with protein-protein interactions of A-kinase anchoring proteins as a strategy for the treatment of disease

A-kinase anchoring proteins (AKAPs) control the localization of cAMP-dependent protein kinase A (PKA) by tethering PKA to distinct cellular compartments. Through additional direct protein-protein interactions with PKA substrates and other signaling molecules they form multi-protein complexes. Thereby, AKAPs regulate the access of PKA to its substrates in a temporal and spatial manner as well as the local crosstalk of cAMP/PKA with other signaling pathways. Due to the increasing information on their molecular functioning and three-dimensional structures, and their emerging roles in the development of diseases, AKAPs move into the focus as potential drug targets. In particular, targeting AKAP-dependent protein-protein interactions for interference with local signal processing inside cells potentially allows for the development of therapeutics with high selectivity and fewer side effects

Pharmacological targeting of AKAP-directed compartmentalized cAMP signalling

The second messenger cyclic adenosine monophosphate (cAMP) can bind and activate protein kinase A (PKA). The cAMP/PKA system is ubiquitous and involved in a wide array of biological processes and therefore requires tight spatial and temporal regulation. Important components of the safeguard system are the A-kinase anchoring proteins (AKAPs), a heterogeneous family of scaffolding proteins defined by its ability to directly bind PKA. AKAPs tether PKA to specific subcellular compartments, and they bind further interaction partners to create local signalling hubs. The recent discovery of new AKAPs and advances in the field that shed light on the relevance of these hubs for human disease highlight unique opportunities for pharmacological modulation. This review exemplifies how interference with signalling, particularly cAMP signalling, at such hubs can reshape signalling responses and discusses how this could lead to novel pharmacological concepts for the treatment of disease with an unmet medical need such as cardiovascular disease and cancer

Small-Molecule Protein-Protein Interaction Inhibitor of Oncogenic Rho Signaling

Uncontrolled activation of Rho signaling by RhoGEFs, in particular AKAP13 (Lbc) and its close homologs, is implicated in a number of human tumors with poor prognosis and resistance to therapy. Structure predictions and alanine scanning mutagenesis of Lbc identified a circumscribed hot region for RhoA recognition and activation. Virtual screening targeting that region led to the discovery of an inhibitor of Lbc-RhoA interaction inside cells. By interacting with the DH domain, the compound inhibits the catalytic activity of Lbc, halts cellular responses to activation of oncogenic Lbc pathways, and reverses a number of prostate cancer cell phenotypes such as proliferation, migration, and invasiveness. This study provides insights into the structural determinants of Lbc-RhoA recognition. This is a successful example of structure-based discovery of a small protein-protein interaction inhibitor able to halt oncogenic Rho signaling in cancer cells with therapeutic implications

Roles of A-kinase anchoring proteins and phosphodiesterases in the cardiovascular system

A-kinase anchoring proteins (AKAPs) and cyclic nucleotide phosphodiesterases (PDEs) are essential enzymes in the cyclic adenosine 3'-5' monophosphate (cAMP) signaling cascade. They establish local cAMP pools by controlling the intensity, duration and compartmentalization of cyclic nucleotide-dependent signaling. Various members of the AKAP and PDE families are expressed in the cardiovascular system and direct important processes maintaining homeostatic functioning of the heart and vasculature, e.g., the endothelial barrier function and excitation-contraction coupling. Dysregulation of AKAP and PDE function is associated with pathophysiological conditions in the cardiovascular system including heart failure, hypertension and atherosclerosis. A number of diseases, including autosomal dominant hypertension with brachydactyly (HTNB) and type I long-QT syndrome (LQT1), result from mutations in genes encoding for distinct members of the two classes of enzymes. This review provides an overview over the AKAPs and PDEs relevant for cAMP compartmentalization in the heart and vasculature and discusses their pathophysiological role as well as highlights the potential benefits of targeting these proteins and their protein-protein interactions for the treatment of cardiovascular diseases

A Novel Approach For The Identification of Cytoskeletal and Adhesion A-Kinase Anchoring Proteins

A-kinase anchoring proteins (AKAPs) are signaling scaffolds which provide spatial and temporal organization of signaling pathways in discrete subcellular compartments. Through tethering the cyclic-AMP dependent protein kinase A (PKA), AKAPs target PKA activity to distinct regions in the cell, bringing PKA in close proximity to its target proteins. This provides a high level of specificity and regulation of PKA and its role in mediating a number of biological processes, one of which is cell migration. Cell migration is a highly dynamic and fundamental process, when misregulated can lead to a number of pathologies. The process of cell migration requires integration and coordination of actin cytoskeletal dynamics, adhesion turnover, and contractility. The important role of PKA in regulating the cellular processes involved in cell migration has been extensively studied. Our lab has shown that PKA activity and spatial distribution through AKAPs are localized to the leading edge of migrating cells and are required for effective cell migration, yet the specific AKAPs responsible remain unknown. Traditional methods for identifying AKAPs suffer from a number of limitations. Therefore the objective of the enclosed work is to establish and characterize a novel approach for the identification of cytoskeletal and adhesion-associated AKAPs. We show for the first time, an in vitro approach to identify cytoskeletal AKAPs which may be responsible for localizing PKA to the leading edge of migrating cells

Cyclic Nucleotide Signaling and the Cardiovascular System

The cyclic nucleotides 3',5'-adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) play important roles in the control of cardiovascular function under physiological and pathological conditions. In this book, which is a reprint of a Special Issue of the Journal of Cardiovascular Development and Disease entitled "Cyclic Nucleotide Signaling and the Cardiovascular System", internationally recognized experts give an overview of this vibrant scientific field. The first series of articles deal with the localization and function of membrane-bound and soluble adenylate cyclases, followed by articles on the roles of phosphodiesterase isoforms in the heart. Cyclic nucleotide signaling takes place in nanodomains and the A-kinase anchor proteins (AKAPS) are essential for the compartmentalized assembly of signaling proteins into functional complexes. Reviews on the role of AKAP proteins in the physiology and pathophysiology of the heart are also included in this book. Cyclic nucleotides act through effector proteins and articles on EPAC and POPDC proteins inform the reader of recent developments on these topics. A major advancement in our understanding of cyclic nucleotide signaling came through the use of genetically encoded cAMP sensor molecules, and a series of articles review the current insight that these reporter molecules have provided. The final set of articles in this book deals with the association of the cyclic nucleotide pathway and cardiovascular disease as well as the development of novel therapeutic approaches. Thomas Brand and Enno Klussmann Special Issue Editor

AKAP7γ Regulation of PKA Substrate Phosphorylation

In the cell, cAMP-protein kinase A (PKA) signaling is compartmentalized. Two different receptor types, both utilizing cAMP and PKA as second messenger and signal effector, are able to convey separate signals that result in phosphorylation of distinct substrates. Signal compartmentation is possible primarily because of A Kinase Anchoring Proteins (AKAPs) that bind both PKA and a target substrate, effectively co-localizing them. AKAPs are also capable of binding to other necessary signaling components like adenylate cyclase and phosphodiesterase, thus enabling AKAPs to coordinate a signaling microdomain containing many of necessary components. In this thesis I present multiple lines of evidence demonstrating how AKAP7g is able to regulate PKA phosphorylation. First, I show that AKAP7g is able to self-associate, forming dimers and possibly higher order oligomers. I predict via computational modeling that this behavior will increase both the speed and magnitude of PKA signaling. Next, I demonstrate that AKAP7g participates in a highly dynamic phosphorylation-state dependent interaction with phospholamban (PLB), and predict via computational modeling that this allows low concentrations of AKAP7g to regulate phosphorylation of much higher concentrations of substrate. Finally I demonstrate via computational modeling that contrary to the widely accepted hypothesis of AKAP signaling, the catalytic subunit of PKA is likely retained within the AKAP-PKA complex during signaling events. I further show that the structure of the complex is an important determinant of substrate phosphorylation. This work offers new insight into the function of AKAPs and offers an update to the AKAP signaling hypothesis

Mechanoregulation of leading edge PKA activity during ovarian cancer cell migration

Ovarian cancer is the deadliest of all the gynecologic cancers and is known for its clinically occult and asymptomatic dissemination. Most ovarian malignancies are diagnosed in the late stages of the disease and the high rate of morbidity is thought to be due, in part, to the highly metastatic nature of ovarian carcinomas. Cancer metastasis relies on the ability of cells to migrate away from primary tumors and invade into target tissues. Though the processes are distinct, cancer cell invasion relies on the underlying migration machinery to invade target tissues. Cell migration requires the coordinated effort of numerous spatially-regulated signaling pathways to extend protrusions, create new adhesion to the extracellular matrix (ECM), translocate the cell body, and retract the cell rear. Our lab established that the cyclic-AMP dependent protein kinase (PKA) subunits and enzymatic activity are localized to the leading edge of migrating cells and are required for cell movement. Despite the importance for localized PKA activity during migration, neither its role in regulating ovarian cancer cell migration and invasion nor the mechanism regulating leading edge PKA activity have been determined. Therefore, the objective of the enclosed work is to establish the importance of PKA for ovarian cancer cell migration and invasion and elucidate the molecular mechanism governing leading edge PKA. We demonstrate, for the first time, that PKA activity and spatial distribution through A-Kinase Anchoring Proteins (AKAPs) is required for efficient ovarian cancer cell migration and invasion. Additionally, we establish a link between leading edge PKA activity in migrating cells, ECM stiffness sensing, and the regulation of both PKA activity and ovarian cancer cell migration by the mechanical properties of the ECM. Finally, we delineate the hierarchy of cell signaling events that regulate leading edge PKA activity and, ultimately, the migration of ovarian cancer cells. Specifically, we elucidate a mechanism where leading edge protrusions elicit leading edge calcium currents through the stretch-activated calcium channel (SACC) of the transient receptor potential family melastatin 7 (TrpM7) to activate actomyosin contractility. ECM substrate stiffness is sensed by the actin cytoskeleton and actomyosin contractility, which, in turn, regulates the activity of leading edge PKA activity. These studies have provided important insights into the regulation of cell migration and have established the mechanistic details governing leading edge PKA activity during cell migration