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bIn response to stress, the heart undergoes a pathological remodeling process associated with hypertrophy and the reexpression of a fetal gene program that ultimately causes cardiac dysfunction and heart failure. In this study, we show that A-kinase-anchoring protein (AKAP)-Lbc and the inhibitor of NF-B kinase subunit ␤ (IKK␤) form a transduction complex in cardiomyocytes that controls the production of proinflammatory cytokines mediating cardiomyocyte hypertrophy. In particular, we can show that activation of IKK␤ within the AKAP-Lbc complex promotes NF-B-dependent production of interleukin-6 (IL-6), which in turn enhances fetal gene expression and cardiomyocyte growth. These findings provide a new mechanistic hypothesis explaining how hypertrophic signals are coordinated and conveyed to interleukin-mediated transcriptional reprogramming events in cardiomyocytes.V entricular myocyte hypertrophy is the primary response whereby the heart responds to stress due to hemodynamic overload, myocardial infarction, or neurohumoral activation (1). It is associated with a nonmitotic growth of cardiomyocytes, increased organization of myofibers, and the reexpression of an embryonic gene program. These events alter cardiac contractility, calcium handling, and myocardial energetics and lead to maladaptive changes that, in the long term, reduce cardiac output and cause heart failure (2, 3).Most stimuli known to promote cardiomyocyte hypertrophy activate G protein-coupled receptors (GPCRs), including ␣1-and ␤-adrenergic receptor (ARs), type I angiotensin II receptors (AT1-Rs), and endothelin I receptors (ET1-Rs) (4-8). It is now clear that the multiple signaling pathways activated by these receptors converge at a limited number of nuclear transcription factors that ultimately regulate the expression of genes associated with hypertrophy (9). In this context, targeting the signaling complexes coordinating the activity of such transcriptional regulators emerges as a primary strategy for new therapeutic approaches aimed at preventing myocardial dysfunction.The transcription factor NF-B has recently been recognized as a mediator of the growth responses induced by a variety of prohypertrophic agonists (10, 11). Under resting conditions, NF-B is retained in the cytosol through an interaction with an inhibitor called IB (inhibitor of NF-B) (12). Upon stimulation, phosphorylation of IB by the inhibitor of IB kinase (IKK) complex, which includes IKK␣, IKK␤, and a regulatory protein termed IKK␥ (12, 13), targets IB for polyubiquitination and subsequently for its degradation by the 26S proteasome (14). This permits the translocation of NF-B to the nucleus, where it can activate the transcription of target genes.While it is now clear that inhibition of NF-B signaling in cardiomyocytes strongly reduces the hypertrophic responses activated by neurohumoral and biomechanical stimuli, including adrenergic agonists, angiotensin-II (Ang-II), proinflammatory cytokines, and aortic banding (15)(16)(17)(18)(19)(20)(21)(22), it is currently poorly unde...

Spatial regulation of tyrosine phosphorylation is important for many aspects of cell biology. However, phosphotyrosine accounts for less than 1% of all phosphorylated substrates, and it is typically a very transient event in vivo. These factors complicate the identification of key tyrosine kinase substrates, especially in the context of their extraordinary spatial organization. Here, we describe an approach to identify tyrosine kinase substrates based on their subcellular distribution from within cells. This method uses an unnatural amino acid-modified Src homology 2 (SH2) domain that is expressed within cells and can covalently trap phosphotyrosine proteins on exposure to light. This SH2 domain-based photoprobe was targeted to cellular structures, such as the actin cytoskeleton, mitochondria, and cellular membranes, to capture tyrosine kinase substrates unique to each cellular region. We demonstrate that RhoA, one of the proteins associated with actin, can be phosphorylated on two tyrosine residues within the switch regions, suggesting that phosphorylation of these residues might modulate RhoA signaling to the actin cytoskeleton. We conclude that expression of SH2 domains within cellular compartments that are capable of covalent phototrapping can reveal the spatial organization of tyrosine kinase substrates that are likely to be important for the regulation of subcellular structures.protein phosphorylation | proteomics | signal transduction

In response to stress or injury the heart undergoes a pathological remodeling process, associated with hypertrophy, cardiomyocyte death and fibrosis, that ultimately causes cardiac dysfunction and heart failure. It has become increasingly clear that signaling events associated with these pathological cardiac remodeling events are regulated by scaffolding and anchoring proteins, which allow coordination of pathological signals in space and time. A-kinase anchoring proteins (AKAPs) constitute a family of functionally related proteins that organize multiprotein signaling complexes that tether the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to ensure integration and processing of multiple signaling pathways. This review will discuss the role of AKAPs in the cardiac response to stress. Particular emphasis will be given to the adaptative process associated with cardiac hypoxia as well as the remodeling events linked to cardiac hypertrophy and heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.