No abstract

Since the discovery in 1957 that cyclic AMP acts as a second messenger for the hormone adrenaline, interest in this molecule and its companion, cyclic GMP, has grown. Over a period of nearly 50 years, research into second messengers has provided a framework for understanding transmembrane signal transduction, receptor-effector coupling, protein-kinase cascades and downregulation of drug responsiveness. The breadth and impact of this work is reflected by five different Nobel prizes.

There is a large body of functional data that supports the existence of subcellular compartmentation of the components of cyclic AMP action in the heart. Data from isolated perfused hearts and from purified ventricular myocytes imply a fixed and hormone-specific spatial relationship amongst components of cyclic AMP synthesis, response, and degradation. Available data demonstrate that within a cardiac myocyte, not all cyclic AMP gains access to all cyclic AMP-dependent protein kinase (PKA), that not all PKA interacts with all possible cellular substrates of PKA, and that only a subset of the myocyte's phosphodiesterases (PDEs) may degrade cyclic AMP after a given synthetic stimulus. Molecular mechanisms contributing to compartmentation are being discovered: localization of receptors, G proteins, and adenylyl cyclases in caveolar versus noncaveolar regions of the sarcolemma; localization of PKA by A-kinase anchoring proteins; localization of PKA substrates, PDE isoforms, and phosphoprotein phosphatases in discrete subcellular regions; and differential regulation of multiple isoforms of adenylyl cyclase, phosphoprotein phosphatase, and PDE in distinct subcellular compartments.

The ␤-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating ␤-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of ␤ 1 -adrenergic receptor, and manipulating the affinity of G s ␣ for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6؋ increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the ␤-adrenergic signaling network may be understood within the context of integrative cellular physiology.In the cardiac myocyte, the ␤-adrenergic signaling network responds to the catecholamines norepinephrine and epinephrine, providing coordinated control of contractility, metabolism, and gene regulation (1, 2). These actions are initiated by ␤-adrenergic receptor coupling with G s and subsequent stimulation of adenylyl cyclase, which synthesizes the classical second messenger cyclic AMP. Cyclic AMP promotes dissociation of the protein kinase A (PKA) 1 holoenzyme, whose catalytic subunits phosphorylate a wide spectrum of target proteins. The ␤-adrenergic control of cardiac contractility is believed to be dominated by PKA phosphorylation of the L-type calcium channel and phospholamban (3), important players in the regulation of calcium dynamics and transport. Although this network mediates the contractile response to catecholamines in the healthy myocyte, altered ␤-adrenergic signaling may also play an incompletely understood role in the progression of heart failure (1, 3).Modules in this network have received considerable attention as possible therapeutic targets in congestive heart failure, including ␤-receptors, adenylyl cyclase (4), and phospholamban (5). Recent studies (6 -8) suggest an important role of compartmentation in signaling specificity. Wherea...

The increased expression of hsp27 and alphaB-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of alphaB-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.

The major limitation to our understanding of the clinical importance of enterotoxigenic Escherichia coli in diarrheal illness has been the lack of a simple rapid assay for the enterotoxin produced by certain E. coli. On the basis of the activation of adenylate cyclase by heat-labile enterotoxin of E. coli (LT) and by cholera toxin (CT) in intestinal and other tissues, cultured Chinese hamster ovary (CHO) cells with known morphological responses to dibutyryl cyclic adenosine 5'-monophosphate (AMP) were exposed to these enterotoxins. Crude culture filtrates of LT-producing E. coli and CT stimulated cyclic AMP accumulation and cell elongation in CHO cells. The similarity of time course, concentration dependence, and potentiation by phosphodiesterase inhibitors suggested cyclic AMP mediation of the morphological change. Heat inactivated CT and LT in this system. Choleragenoid inhibited CT; antiserum against CT inhibited both enterotoxin effects. In contrast to culture filtrates of 16 strains of E. coli known to produce LT, culture filtrates from 13 E. coli that do not produce LT did not alter CHO cell morphology. The morphological change is a simple, specific assay for these enterotoxins and detect 3 x 10-17 mol of CT or a 1: 250 dilution of crude culture filtrate of LT-producing E. coli 334.

Endogenous protein kinase inhibitors are essential for a wide range of physiological functions. These endogenous inhibitors may mimic peptide substrates as in the case of the heat-stable protein kinase inhibitor (PKI), or they may mimic nucleotide triphosphates. Natural product inhibitors, endogenous to the unique organisms producing them, can be potent exogenous inhibitors against foreign protein kinases. Balanol is a natural product inhibitor exhibiting low nanomolar K i values against serine and threonine specific kinases, while being ineffective against protein tyrosine kinases. To elucidate balanol's specific inhibitory effects and provide a basis for understanding inhibition-regulated biological processes, a 2.1 Å resolution crystal structure of balanol in complex with cAMP-dependent protein kinase (cAPK) was determined. The structure reveals conserved binding regions and displays extensive complementary interactions between balanol and conserved cAPK residues. This report describes the structure of a protein kinase crystallized with a natural ATP mimetic in the absence of metal ions and peptide inhibitor.Protein kinases, of which the human genome is predicted to encode several thousand, reversibly phosphorylate diverse molecular targets and are critical enzymes in the initiation, regulation, and abrogation of both normal and abnormal cellular functions (reviewed in refs 1-6). These often exquisitely specific kinases play essential roles in apoptosis, cell proliferation, gene expression, glycogen metabolism, immune response, neurotransmission, oncogenesis, and secondary messenger signal transduction (7-18). This biological pervasiveness underscores the importance of more explicitly characterizing kinase activation and inhibition. Additionally, there is significant therapeutic value in achieving selective pharmacological control of members of this important class of enzymes, since unregulated or otherwise defective protein kinase activities to date have been implicated in asthma, cancer, cardiovascular disorders, central nervous system (CNS) 1 diseases, diabetes, human immunodeficiency virus (HIV) infections, inflammation, psoriasis, and rheumatoid arthritis (19)(20)(21)(22)(23)(24).To more explicitly characterize protein kinase activation and inhibition, cAMP-dependent protein kinase, the most completely characterized and mechanistically simplest protein kinase (25, reviewed in refs 26-31), is a logical singular choice for biochemical and structural research focusing on cellular phosphorylation events at the molecular level. The inactive cAPK holoenzyme consists of two regulatory (R) and two catalytic (C) subunits that dissociate in response to elevated levels of intracellular cAMP. The triply phosphorylated, active C subunit then phosphorylates serine or threonine residues of peptide substrates containing the consensus sequence Arg-Arg-X-Ser[Thr]-Hyd, where X is variable and Hyd is any hydrophobic residue (3,14,32,33). The C subunit of cAPK shares with other kinase family members a conserved cat...

A number of balanol analogs have been designed and synthesized that, unlike balanol itself, exhibit dramatic selectivity between PKA and PKC. Thus, despite the substantial homology between the catalytic domains of PKA and PKC, there is enough difference to allow for the development of potent and selective inhibitors acting in this region. These inhibitors should be useful tools for analyzing signal transduction pathways and may also aid in the development of drugs with significant therapeutic potential.