RNAi screens have, to date, identified many genes required for mitotic divisions of Drosophila tissue culture cells. However, the inventory of such genes remains incomplete. We have combined the powers of bioinformatics and RNAi technology to detect novel mitotic genes. We found that Drosophila genes involved in mitosis tend to be transcriptionally co-expressed. We thus constructed a co-expression–based list of 1,000 genes that are highly enriched in mitotic functions, and we performed RNAi for each of these genes. By limiting the number of genes to be examined, we were able to perform a very detailed phenotypic analysis of RNAi cells. We examined dsRNA-treated cells for possible abnormalities in both chromosome structure and spindle organization. This analysis allowed the identification of 142 mitotic genes, which were subdivided into 18 phenoclusters. Seventy of these genes have not previously been associated with mitotic defects; 30 of them are required for spindle assembly and/or chromosome segregation, and 40 are required to prevent spontaneous chromosome breakage. We note that the latter type of genes has never been detected in previous RNAi screens in any system. Finally, we found that RNAi against genes encoding kinetochore components or highly conserved splicing factors results in identical defects in chromosome segregation, highlighting an unanticipated role of splicing factors in centromere function. These findings indicate that our co-expression–based method for the detection of mitotic functions works remarkably well. We can foresee that elaboration of co-expression lists using genes in the same phenocluster will provide many candidate genes for small-scale RNAi screens aimed at completing the inventory of mitotic proteins.

AbstractcAMP/PKA activation represents a key signaling mechanism for neurohormonal stimulation of diversified physiological processes. Using real-time, FRET-based imaging of PKA activity in neonatal cardiac myocytes, we report that sustained activation of PKA induced by β adrenoceptor (βAR) dictates signaling propagation for substrate phosphorylation and myocyte contraction. Activation of βARs in wild-type myocytes induces strong and sustained PKA activities, which are rapidly attenuated upon washing away agonist or adding antagonist to the cells. The sustained PKA activities promote signaling propagation to the sarcoplasmic reticulum for phosphorylation of phospholamban and increases in myocyte contraction. Addition of antagonist after βAR stimulation significantly attenuates PKA phosphorylation of phospholamban, and rapidly reduces contraction rate increases. Moreover, Stimulation of β 1 AR subtype induces PKA activities similar to those in wild-type cells. In contrast, stimulation of β 2 AR subtype induces strong initial activation of PKA similar to those induced by β 1 AR; however the activities are rapidly decreased to baseline levels. The transient PKA activities are sufficient for phosphorylation of the overexpressed β 2 ARs under agonist stimulation, but not phospholamban. Further analysis reveals that phosphodiesterase 4 is the major family that shapes PKA activities under βAR stimulation. Inhibition of phosphodiesterase 4 extendsβ 2 AR-induced PKA activities, promotes PKA phosphorylation of phospholamban, and ultimately enhances myocyte contraction responses. Together, our data has revealed insights into kinetics of PKA activities in signaling propagation under neurohormonal stimulation.

Progressive decrease in neuronal function is an established feature of Alzheimer's disease (AD). Previous studies have shown that amyloid beta (Abeta) peptide induces acute increase in spontaneous synaptic activity accompanied by neurotoxicity, and Abeta induces excitotoxic neuronal death by increasing calcium influx mediated by hyperactive alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors. An in vivo study has revealed subpopulations of hyperactive neurons near Abeta plaques in mutant amyloid precursor protein (APP)-transgenic animal model of Alzheimer's disease (AD) that can be normalized by an AMPA receptor antagonist. In the present study, we aim to determine whether soluble Abeta acutely induces hyperactivity of AMPA receptors by a mechanism involving beta(2) adrenergic receptor (beta(2)AR). We found that the soluble Abeta binds to beta(2)AR, and the extracellular N terminus of beta(2)AR is critical for the binding. The binding is required to induce G-protein/cAMP/protein kinase A (PKA) signaling, which controls PKA-dependent phosphorylation of GluR1 and beta(2)AR, and AMPA receptor-mediated excitatory postsynaptic currents (EPSCs). beta(2)AR and GluR1 also form a complex comprising postsynaptic density protein 95 (PSD95), PKA and its anchor AKAP150, and protein phosphotase 2A (PP2A). Both the third intracellular (i3) loop and C terminus of beta(2)AR are required for the beta(2)AR/AMPA receptor complex. Abeta acutely induces PKA phosphorylation of GluR1 in the complex without affecting the association between two receptors. The present study reveals that non-neurotransmitter Abeta has a binding capacity to beta(2)AR and induces PKA-dependent hyperactivity in AMPA receptors.

cAMP and protein kinase A (PKA) activation represents a key signaling mechanism upon ␤-adrenergic stimulation under stress. Both ␤ 1 -and ␤ 2 -adrenoreceptor (ARs) subtypes induce cAMP accumulation, yet play distinct roles in cardiac contraction and myocyte apoptosis. Differences in controlling cAMP/ PKA activities through the assembly of complexes between the receptors and cAMP-specific phosphodiesterases contribute to the distinct biological outcomes. Here, we demonstrate that ␤ 2 ARs form signaling complexes with a set of PDE4D isoforms expressed in cardiac myocytes. PDE4D9 and PDE4D8 bind to the ␤ 2 AR at resting conditions; however, agonist stimulation induces dissociation of PDE4D9 from the receptor but recruitment of PDE4D8 to the receptor. Agonist stimulation also induces recruitment of PDE4D5 to the ␤ 2 AR. Moreover, the receptor-associated PDE4D isoforms play distinct roles in controlling cAMP activities and regulating the PKA phosphorylation of the receptor and myocyte contraction rate responses. Knockdown of PDE4D9 with short hairpin RNA enhances the ␤ 2 AR-induced cAMP signaling, whereas knockdown of PDE4D8 only slightly prolongs the receptor-induced cAMP signaling in myocytes. Inhibition of PDE4D9 and PDE4D5 enhances the base-line levels of contraction rates, whereas inhibition of PDE4D9 and PDE4D8 enhances the maximal contraction rate increases upon activation of ␤ 2 AR. Our data underscore the complex regulation of intracellular cAMP by ␤ 2 ARassociated phosphodiesterase enzymes to enforce the specificity of the receptor signaling for physiological responses.cAMP/PKA 2 activation represents a key signaling mechanism upon stimulation of G protein-coupled receptors for cardiac contraction and energy metabolism under stress conditions. Activation of ␤ARs, a group of prototypical G protein-coupled receptors, is one of the major neurohormonal mechanisms controlling cAMP/PKA activities for physiological responses in animal hearts (1). ␤ 1 AR and ␤ 2 AR are highly homologous receptors expressed in animal heart for enhancing cardiac performance. ␤ 1 AR plays a dominant role in stimulating heart rate and strength of myocyte contraction, whereas ␤ 2 AR produces only modest chronotropic effects (1). One of the emerging mechanisms that safeguard the specificity of G protein-coupled receptor/cAMP signaling is the control of cAMP transients via degradation by cyclic nucleotide PDEs (2).PDEs include 11 families based on their amino acid sequence homology, substrate specificities, and pharmacological properties (2). Each of the 11 PDE families has one to four distinct genes. In addition, most PDE genes encode for multiple splicing variants through the usage of multiple promoters and alternative splicing. In animal hearts, PDE4 and PDE3 are two major families expressed, which account for more than 90% of PDE activities (3). These PDEs play a critical role for the subcellular specificity in cAMP signaling by preventing diffusion of cAMP from one microdomain to another (4, 5).Our previous studies have identified th...

We investigated the role of phospholipase D (PLD) and its product phosphatidic acid (PA) in myogenic differentiation of cultured L6 rat skeletal myoblasts. Arginine-vasopressin (AVP), a differentiation inducer, rapidly activated PLD in a Rho-dependent way, as shown by almost total suppression of activation by C3 exotoxin pretreatment. Addition of 1-butanol, which selectively inhibits PA production by PLD, markedly decreased AVP-induced myogenesis. Conversely, myogenesis was potentiated by PLD1b isoform overexpression but not by PLD2 overexpression, establishing that PLD1 is involved in this process. The expression of the PLD isoforms was differentially regulated during differentiation. AVP stimulation of myoblasts induced the rapid formation of stress fiber-like actin structures (SFLSs). 1-Butanol selectively inhibited this response, whereas PLD1b overexpression induced SFLS formation, showing that it was PLD dependent. Endogenous PLD1 was located at the level of SFLSs, and by means of an intracellularly expressed fluorescent probe, PA was shown to be accumulated along these structures in response to AVP. In addition, AVP induced a PLD-dependent neosynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), which also was accumulated along actin fibers. These data support the hypothesis that PLD participates in myogenesis through PA- and PIP2-dependent actin fiber formation.