Wnt signals control cell fate decisions and orchestrate cell behavior in metazoan animals. In the fruit fly Drosophila, embryos defective in signaling mediated by the Wnt protein Wingless (Wg) exhibit severe segmentation defects. The Drosophila segment polarity gene naked cuticle (nkd) encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. Nkd antagonizes Wg via a direct interaction with the Wnt signaling component Dishevelled (Dsh). Here we describe two mouse and human proteins, Nkd1 and Nkd2, related to fly Nkd. The most conserved region among the fly and vertebrate proteins, the EFX domain, includes the putative EF hand and flanking sequences. EFX corresponds to a minimal domain required for fly or vertebrate Nkd to interact with the basic/PDZ domains of fly Dsh or vertebrate Dvl proteins in the yeast two-hybrid assay. During mouse development, nkd1 and nkd2 are expressed in multiple tissues in partially overlapping, gradient-like patterns, some of which correlate with known patterns of Wnt activity. Mouse Nkd1 can block Wnt1-mediated, but not beta-catenin-mediated, activation of a Wnt-dependent reporter construct in mammalian cell culture. Misexpression of mouse nkd1 in Drosophila antagonizes Wg function. The data suggest that the vertebrate Nkd-related proteins, similar to their fly counterpart, may act as inducible antagonists of Wnt signals.

The overall size of an animal is determined by the number of cell divisions, the rate of destruction of cells, and the average size of cells (Conlon and Raff 1999;Edgar 2006). With some species, size is a relatively rigid outcome of the genetically controlled program of development. Other species, such as many fish and trees, can continue to grow through much of their lives. In both cases growth is influenced by continuing assessment of the energy state of the growing organism and the availability of nutrients, interpreted in the context of the organism's genetic program. The cell-intrinsic machinery that specifies whether or not a cell should divide and how large a cell should grow has been unveiled in elegant molecular detail (Conlon and Raff 1999;Edgar 2006). Much remains to be learned about the cell-extrinsic mechanisms that coordinate the growth behaviors of individual cells. Global cell-extrinsic controls are needed to ensure that a properly proportioned animal is produced, with a size suited to its environment and genetic program.Whole-animal growth control can be envisioned as having three components that operate in concert: (1) sensory and homeostatic inputs, (2) processing within the CNS, and (3) instructive outputs to the periphery. Sensory inputs to the CNS provide information regarding the energy status of the organism and the available nutrient status of the environment. The CNS assesses this information in light of the genetic program and instincts about future energy requirements; e.g., for growth, reproduction, migration, and hibernation. The CNS converts the processed information into output signals that alter feeding behavior and spread through the body to coordinate growth. The transmitted output signals instruct cells in peripheral tissues to grow, to cease growth, or to die, by influencing cell-intrinsic programs. Recent studies have shed light on the input and output signals involved in growth control (1 and 3). Much less is known about the central processing mechanisms (2).A great deal of elegant work has identified signaling mechanisms by which energy states are sensed and the information is relayed to the CNS (Morton et al. 2006;Melcher et al. 2007). In mammals, dietary free fatty acids can act on the anterior pituitary gland to inhibit growth hormone secretion (Dieguez and Casanueva 1995). In

The ability of protein kinase C (PKC) to regulate the responsiveness of adenylyl cyclase to different activators was assessed. Membranes prepared from Sf9 cells infected with recombinant baculoviruses encoding either type II or IV adenylyl cyclase were incubated with recombinant PKCalpha (purified from Sf9 cells), and the effects on adenylyl cyclase activity were measured after reconstitution with Gsalpha, Gbetagamma, or forskolin. PKCalpha treatment of type II adenylyl cyclase leads to increases in basal, forskolin-stimulated, and betagamma-stimulated activities and greater sensitivity to stimulation by Gsalpha. Paradoxically, most of the betagamma potentiation of Gsalpha-stimulated activity is eliminated by pretreatment with PKCalpha. By contrast, treatment of type IV adenylyl cyclase with PKCalpha has little effect on the basal, forskolin-stimulated, or betagamma-stimulated activities but markedly reduces the Gsalpha-stimulated and betagamma-potentiated activity of this isoform. These studies demonstrate that protein kinases can alter both the activity of adenylyl cyclase isoforms and their responsiveness to G protein regulation, thereby altering the ability of adenylyl cyclases to integrate signals derived from multiple hormonal inputs.

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The recent determination of the crystal structure of adenylyl cyclase has elucidated many structural features that determine the regulatory properties of the enzyme. In addition, the characterization of adenylyl cyclase by mutagenic techniques and the identification of the binding site for P-site inhibitors have led to modeling studies that describe the ATP-binding site. Despite these advances, the catalytic mechanism of adenylyl cyclase remains uncertain, especially with respect to the role that magnesium ions may play in this process. We have identified four mutant mammalian adenylyl cyclases defective in their metal dependence, allowing us to further characterize the function of metal ions in the catalytic mechanism of this enzyme. The wild-type adenylyl cyclase shows a biphasic Mg 2؉ dose-response curve in which the high-affinity component displays cooperativity (Hill coefficient of 1.4). Two mutations (C441R and Y442H) reduce the affinity of the adenylyl cyclase for Mg 2؉ dramatically without affecting the binding of MgATP, suggesting that there is a metal requirement in addition to the ATP-bound Mg 2؉ . The results of this study thus demonstrate multiple metal requirements of adenylyl cyclase and support the existence of a Mg 2؉ ion essential for catalysis and distinct from the ATP-bound ion. We propose that adenylyl cyclase employs a catalytic mechanism analogous to that of DNA polymerase, in which two key magnesium ions facilitate the nucleophilic attack of the 3-hydroxyl group and the subsequent elimination of pyrophosphate.Intracellular levels of cAMP are primarily regulated at the level of its synthesis by adenylyl cyclase. cAMP, in turn, regulates a wide variety of cellular processes such as protein phosphorylation levels, gene expression, and ion channel conductance. Currently, nine isoforms of mammalian adenylyl cyclase have been identified by molecular cloning techniques (reviewed in Refs. 1-3), and they display a common deduced topology composed of a short cytoplasmic amino terminus followed by a region of six transmembrane domains (M1) and a large cytoplasmic loop (C1). This motif is then repeated with a second transmembrane region (M2) and a large cytoplasmic carboxyl terminus (C2). Sequence comparison has revealed that each cytoplasmic loop contains subdomains (denoted C1a and C2a) that are highly conserved among all adenylyl cyclase isoforms and that also display great homology to each other. The C1a and C2a domains can be expressed separately, and catalytic activity is reconstituted when they are mixed in vitro, although each domain by itself shows no activity (4, 5).Many of the structural motifs of adenylyl cyclase responsible for catalytic activity and for the recognition of regulatory molecules are presently being uncovered. A region in the C2 domain of type II adenylyl cyclase (residues 956 -982), for example, has been implicated in the binding of the G protein 1 ␤␥ subunits (6), whereas another region in the C1 domain of type I adenylyl cyclase (residues 495-522) appears to be involved ...