The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that PDE4 exists in two conformations that bind the inhibitor rolipram with affinities differing by more than 100-fold. Here we report that these two conformations are the consequence of PDE4 binding to its metal cofactor such as Mg(2+). Using a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay, we identified that L-791,760, a fluorescent inhibitor, binds to the apoenzyme (free enzyme) and the holoenzyme (enzyme bound to Mg(2+)) with comparable affinities (K(d) approximately 30 nM). By measuring the displacement of the bound L-791,760, we have also identified that other inhibitors bind differentially with the apoenzyme and the holoenzyme depending upon their structure. CDP-840, SB-207499, and RP-73401 bind preferentially to the holoenzyme. The conformational-sensitive inhibitor (R)-rolipram binds to the holoenzyme and apoenzyme with affinities (K(d)) of 5 and 300 nM, respectively. In contrast to its high affinity (K(d) approximately 2 microM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K(d) approximately 170 microM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.

Chx10 and VSX1 are transcription factors that share a homeodomain (HD), 1 and a CVC motif, named after CHX10, VSX1, and ceh-10 (1-3). CHX10 is expressed early in development in dividing retinal progenitor cells (RPCs) and is also expressed in mature bipolar neurons and a subset of Mü ller glia (2, 4). Null mutations results in microphthalmia in humans (5) and mice (6), and antisense Chx10 RNA injected into zebrafish embryos impairs retinal development (7). Recessive mutations identified in patients with microphthalmia that modified residues in helix III of the CHX10 HD (R200P and R200Q) ablated DNA binding in vitro (5). The ocular retardation (or J ) mouse carries a mutation that generates a premature stop codon in helix

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.The mammalian retina consists of three nuclear layers. The outer nuclear layer houses the cell bodies of photoreceptors (rods and cones), whereas those of horizontal, bipolar, and amacrine interneurons and Müller glia reside in the inner nuclear layer. The innermost layer is the ganglion cell layer, which consists of a mixture of ganglion and amacrine neurons. These three cellular areas are separated by outer and inner plexiform layers that house synaptic connections. This intricate laminated structure develops through the amplification of multipotent progenitor cells, generation of more restricted post-mitotic transition cells, and maturation of these cells into terminally differentiated neurons and glia (1). In rodents, ganglion, horizontal, cone, and amacrine transition cells are born in the prenatal period, bipolar and Müller cells are born post-natally, and rods are born throughout retinal development (2). Transcription factors play critical roles in each of the stages of retinal development, but many gaps remain in our understanding of the specific target genes involved. Here, we focus on one of these factors, the homeobox gene CHX10, and its role in sculpting the characteristics of bipolar interneurons.Homeodomain (HD) 4 proteins regulate retinal development from the earliest stages of optic vesicle formation to the final stages of maturation in the adult (reviewed in Refs. 3 and 4)). Previous molecular analysis linked a naturally occurring mutation in the homeobox of Chx10 to the ocular retardation phenotype in mice (or J ) (5). These mice display a dramatic decrease in retinal progenitor cell (RPC) proliferation and lack bipolar cells, phenotypes that reflect the expression of Chx10 in both of these cell types 5 (5, 6). Molecular and genetic studies have begun to reveal some aspects of the mechanism by which Chx10 regulates retinal development...

A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5 end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor and SLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709-713, 1996). The remaining four slt mutations are new alleles of previously identified splicing genes: slt15, previously identified as prp17 (slt15/prp17-100), slt16/smd3-1, slt17/slu7-100, and slt21/prp8-21. slt11-1 and slt22-1 are synthetically lethal with mutations in the 3 end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however, slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, including slt15/prp17-100, slt17/slu7-100, and prp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179-5205, 1992), as well as with slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.Precursor-mRNA (pre-mRNA) splicing takes place in the spliceosome through a two-step transesterification reaction. At least 40 splicing factors have been identified by genetic means in the yeast Saccharomyces cerevisiae. Most have been implicated in specific steps of the splicing pathway (31,41). During the process of spliceosome assembly, small nuclear RNAs (snRNAs) and the pre-mRNA substrate, in association with protein factors, undergo extensive conformational changes which establish RNA-RNA interactions that are important for both splicing reactions. Among these factors are the DExD or DExH proteins: RNA-dependent ATPases (possibly RNA helicases), which include Prp2p, Prp5p, Prp16p, Prp22p, Prp28p (26, 31), Prp43p (2), and Slt22p (also called Brr2p) (22,34,53). Their functions are essential for the formation and maintenance of RNA-RNA interactions in the spliceosome.With few exceptions (28,29,36), Watson-Crick base pairing is important for most RNA-RNA interactions in the formation of the spliceosome (26). In particular, intermolecular basepairing interactions that occur between U2 and U6 snRNAs, forming helices I and II (Fig. 1A), are likely...

Type 4 phosphodiesterases (PDE4s) are metallohydrolases that catalyze the hydrolysis of cAMP to AMP. At the bottom of its active site lie two divalent metal ions in a binuclear motif which are involved in both cAMP binding and catalysis [(2000) Science 288, 1822-1825; (2000) Biochemistry 39, 6449-6458]. Using a SPA-based equilibrium [(3)H]rolipram binding assay, we have determined that Mg(2+), Mn(2+), and Co(2+) all mediated a high-affinity (K(d) between 3 and 8 nM) and near stoichiometric (R)-rolipram binding to PDE4. In their absence, (R)-rolipram binds stoichiometrically to the metal ion-free apoenzyme with a K(d) of approximately 150 nM. The divalent cation dose responses in mediating the high-affinity rolipram/PDE4 interaction mirror their efficacy in catalysis, suggesting that both metal ions of the holoenzyme are involved in mediating the high-affinity (R)-rolipram/PDE4 interaction. The specific rolipram binding to the apo- and holoenzyme is differentially displaced by cAMP, AMP, and other inhibitors, providing a robust tool to dissect the components of metal ion-dependent and independent PDE4/ligand interactions. cAMP binds to the holoenzyme with a K(s) of 1.9 microM and nonproductively to the apoenzyme with a K(d) of 179 microM. In comparison, AMP binds to the holo- and apoenzyme with K(d) values of 7 and 11 mM, respectively. The diminished Mg(2+)-dependent component of AMP binding to PDE4 suggests that most of the Mg(2+)/phosphate interaction in the cAMP/PDE4 complex is disrupted upon the hydrolysis of the cyclic phosphoester bond, leading to the rapid release of AMP.

The PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg 2+ doser esponse (EC 50 of V V0.15 and s 10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKAcatalytic subunit increases the enzyme's sensitivity to Mg 2+ , leading to 4-fold increased cAMP hydrolysis without affecting its K m . The phosphorylation also increases the potencies of (R)-and (S)-rolipram without affecting CDP-840 and SB-207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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