The transmission of information from the plasma membrane to the actin cytoskeleton is essential to control a variety of dynamic cellular processes including cell shape, motility, and adherence (1). Critical mediators of these events are the Rho family small molecular weight GTPases, Rho, Rac, and Cdc42, which regulate distinct actin remodeling events. Rho is primarily responsible for the assembly of actin stress fibers and focal adhesions, and Rac controls the formation of lamellipodia, while Cdc42 induces filopodial formation (1, 2). In addition to these effects, Rho has also been implicated in a variety of other critical cellular functions including gene transcription (3,4) and progression through the cell cycle (5).Lysophosphatidic acid (LPA) and thrombin are the extracellular ligands that induce Rho signaling events (2). Binding of either ligand to distinct classes of cell surface receptors triggers a series of events that mobilize the pertussis toxin-insensitive heterotrimeric G protein subunits G␣ 12 and G␣ 13 (6, 7). The intracellular targets for either G␣ subunit are a growing family of guanine nucleotide exchange factors (GEFs) (8). These exchange factors dock with activated G␣ 12 and G␣ 13 and facilitate GTP loading of Rho. Individual cells express several Rho-GEFs, which activate distinct Rho signaling pathways (9). For example, p115 Rho-GEF interacts with G␣ 13 through a regulator of G protein signaling (RGS) domain located in the amino-terminal region of the exchange factor (10, 11). Likewise, a related module, the Lsc homology domain, governs docking of G␣ 13 to exchange factors such as PDZ Rho-GEF, KIAA0380,.A universal hallmark of exchange factors that activate GTPases of the Rho family is a conserved region of ϳ250 residues that contains a Dbl homology (DH) domain followed by a pleckstrin homology domain (9). The DH domain contains the nucleotide exchange activity, whereas the pleckstrin homology domain is thought to be involved in the subcellular localization of GEFs (2). GTPase selectivity is governed by determinants located within the DH domain that discriminate Rho-specific from Rac or Cdc42-specific exchange factors. Several families of Rho-specific exchange factors have been recognized. Members of the Lbc family were originally identified in a screen for transforming genes from human myeloid leukemias (15). OncoLbc is a 424-residue oncogenic protein with unregulated exchange factor activity that transforms NIH-3T3 cells in a Rhodependent manner (16). Subsequently, a proto-oncogenic form has been isolated with a COOH-terminal region that attenuates its transforming potential (17). More recently, a splice variant called Brx has been identified that is specifically expressed in testis and estrogen-sensitive tissues (18). Interestingly, Lbc does not possess RGS-like domains, suggesting that different mechanisms might be involved in its activation in response to extracellular signals.In this study, we demonstrate that a novel Lbc splice variant, AKAP-Lbc, is also an A-kinase-anchoring protein (...

Summary Elevated catecholamines in the heart evoke transcriptional activation of the Myocyte Enhancer Factor (MEF) pathway to induce a cellular response known as pathological myocardial hypertrophy. We have discovered that the A-Kinase Anchoring Protein AKAP-Lbc is up-regulated in hypertrophic cardiomyocytes. It coordinates activation and movement of signaling proteins that initiate MEF2-mediated transcriptional reprogramming events. Live-cell imaging, fluorescent kinase activity reporters and RNA interference techniques show that AKAP-Lbc couples activation of protein kinase D (PKD) with the phosphorylation-dependent nuclear export of the class II histone deacetylase HDAC5. These studies uncover a role for AKAP-Lbc in which increased expression of the anchoring protein selectively amplifies a signaling pathway that drives cardiac myocytes towards a pathophysiological outcome.

In response to various pathological stresses, the heart undergoes a pathological remodeling process that is associated with cardiomyocyte hypertrophy. Because cardiac hypertrophy can progress to heart failure, a major cause of lethality worldwide, the intracellular signaling pathways that control cardiomyocyte growth have been the subject of intensive investigation. It has been known for more than a decade that the small molecular weight GTPase RhoA is involved in the signaling pathways leading to cardiomyocyte hypertrophy. Although some of the hypertrophic pathways activated by RhoA have now been identified, the identity of the exchange factors that modulate its activity in cardiomyocytes is currently unknown. In this study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critical for activating RhoA and transducing hypertrophic signals downstream of ␣1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbcspecific short hairpin RNAs strongly reduces both ␣1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, ␣1-ARs promote AKAP-Lbc activation via a pathway that requires the ␣ subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor (GEF) involved in the signaling pathways leading to cardiomyocytes hypertrophy.cardiac hypertrophy ͉ Rho GTPase ͉ G protein-coupled receptor

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.

The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network. Scaffolds that bind protein kinase A are called “A-kinase anchoring proteins” (AKAPs). In this review, we discuss recent advances in our understanding of how PKA is compartmentalized within the cardiac myocyte by AKAPs and how AKAP complexes modulate cardiac function in both health and disease.

Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division [1,2] and have long been recognized as major anchoring sites for cAMP-dependent protein kinase (PKA) [3,4]. Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs) [5,6]. AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Here, we show that pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. We demonstrate that both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo.

Catecholamines as well as phorbol esters can induce the phosphorylation and desensitization of the ␣ 1B -adrenergic receptor (␣ 1B AR). In this study, phosphoamino acid analysis of the phosphorylated ␣ 1B AR revealed that both epinephrine-and phorbol ester-induced phosphorylation predominantly occurs at serine residues of the receptor. The findings obtained with receptor mutants in which portions of the C-tail were truncated or deleted indicated that a region of 21 amino acids (393-413) of the carboxyl terminus including seven serines contains the main phosphorylation sites involved in agonist-as well as phorbol ester-induced phosphorylation and desensitization of the ␣ 1B AR. To identify the serines invoved in agonist-versus phorbol ester-dependent regulation of the receptor, two different strategies were adopted, the seven serines were either substituted with alanine or reintroduced into a mutant lacking all of them. Desensitization is a general regulatory phenomenon of G protein-coupled receptors resulting in the attenuation of the receptor-mediated response. Two major patterns of desensitization referred to as homologous and heterologous desensitization can be distinguished. Homologous desensitization is defined as a rapid loss of responsiveness for a receptor repeatedly exposed to its specific agonist, whereas in heterologous desensitization stimulation of a receptor by an agonist can attenuate the response mediated by other receptors eliciting similar cellular effects (1).In the G protein-coupled receptor family (2), receptor desensitization has been extensively characterized for rhodopsin mediating phototransduction in retinal rod cells and for the ␤ 2 -adrenergic receptor (␤ 2 AR) 1 which mediates catecholamineinduced stimulation of adenylyl cyclase. The second messengerdependent cAMP-dependent protein kinase can phosphorylate and desensitize the ␤ 2 AR both in response to its agonist as well as to other agents increasing the cellular content of cAMP. On the other hand, a prominent role in homologous desensitization of rhodopsin and ␤ 2 AR is played by the second messengerindependent rhodopsin kinase (3) and ␤-adrenergic receptor kinase (␤ARK) (4), respectively. Once the receptor is occupied by the agonist, it is recognized by the kinase and becomes phosphorylated. The subsequent uncoupling of the receptor and G protein is then mediated by arrestin proteins, which specifically bind to the phosphorylated receptor (5, 6). Rhodopsin kinase and ␤ARK are members of the newly discovered family of G protein-coupled receptor kinases (GRK) (7). These protein kinases have the unique ability to recognize and phosphorylate their G protein-coupled receptor substrates predominantly in their active (i.e. agonist-occupied) conformations. Recently, we have provided evidence that the ␣ 1B AR coupled to Gq-mediated activation of phospholipase C can be phosphorylated by at least two types of protein kinases, a protein kinase C (PKC) upon its stimulation by phorbol esters (8) and protein kinases belonging to the GRK family o...

Using the yeast two-hybrid system, we identified the 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the ␣ 1b -adrenergic receptor (AR). Direct association between the ␣ 1b -AR and 2 was demonstrated using a solid phase overlay assay. The ␣ 1b -AR/ 2 interaction occurred inside the cells, as shown by the finding that the transfected ␣ 1b -AR and the endogenous 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the ␣ 1b -AR revealed that the binding site for 2 does not involve canonical YXX⌽ or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The ␣ 1b -AR specifically interacted with 2 , but not with the 1 , 3 , or 4 subunits belonging to other AP complexes. The deletion of the 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.Desensitization to the effects of hormones and neurotransmitters is a fundamental regulatory mechanism of G proteincoupled receptor (GPCR) 1 function defined by a specific loss of responsiveness for those receptors that have been repeatedly stimulated by the agonist. Receptor desensitization results from the combination of multiple biochemical events occurring at different time frames: receptor-G protein uncoupling in response to receptor phosphorylation (seconds to minutes), internalization or endocytosis of cell surface receptors to intracellular compartments (minutes), and down-regulation of the total pool of receptors due to their decreased synthesis and/or increased degradation (hours) (1). A prominent role in homologous desensitization of GPCRs is played by G protein-coupled receptor kinases. Once the receptor is occupied by the agonist, it is recognized by the G protein-coupled receptor kinases and becomes phosphorylated (2). The subsequent uncoupling of the receptor from the G protein is then mediated by arrestin proteins, which preferentially bind to the agonist-occupied phosphorylated receptor (3). However, during the last decade, ␤-arrestins have emerged as key regulatory molecules controlling various steps of receptor desensitization. Beyond their role in physical uncoupling of GPCRs from the G proteins (3), it has been demonstrated that ␤-arrestins target GPCRs to the endocytic machinery (4). In fact, it is believed that for those GPCRs that internalize in a clathrin-dependent manner, like the ␤ 2 -adrenergic receptor (AR), targeting of the receptor-␤-arrestin complexes to clathrin-coated vesicles is mediated by a dual interaction of ␤-arrestin with both clathrin heavy chain and the ␤ 2 subunit of the heterotetrameric ...