On the mechanism of MgATP-dependent gating of CFTR Cl- channels

CFTR, the product of the gene mutated in cystic fibrosis, is an ATPase that functions as a Cl- channel in which bursts of openings separate relatively long interburst closed times (Tib). Channel gating is controlled by phosphorylation and MgATP, but the underlying molecular mechanisms remain controversial. To investigate them, we expressed CFTR channels in Xenopus oocytes and examined, in excised patches, how gating kinetics of phosphorylated channels were affected by changes in [MgATP], by alterations in the chemical structure of the activating nucleotide, and by mutations expected to impair nucleotide hydrolysis and/or diminish nucleotide binding affinity. The rate of opening to a burst (1/tauib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR's nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Burst duration of neither wild-type nor mutant channels was much influenced by [MgATP]. Poorly hydrolyzable nucleotide analogs, MgAMPPNP, MgAMPPCP, and MgATPyS, could open CFTR channels, but only to a maximal rate of opening similar to20-fold lower than attained by MgATP acting on the same channels. NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Corresponding NBD1 mutations did not affect timing of burst termination in normal, hydrolytic conditions. However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. In light of recent biochemical and structural data, the results suggest that: nucleotide binding to both NBDs precedes channel opening; at saturating nucleotide concentrations the rate of opening to a burst is influenced by the structure of the phosphate chain of the activating nucleotide; normal, rapid exit from bursts occurs after hydrolysis of the nucleotide at NBD2, without requiring a further nucleotide binding step; if hydrolysis at NBD2 is prevented, exit from bursts occurs through a slower pathway, the rate of which is modulated by the structure of the NBD1 catalytic site and its bound nucleotide. Based on these and other results, we propose a mechanism linking hydrolytic and gating cycles via ATP-driven dimerization of CFTR's NBDs

Direct Interaction of PP2A Phosphatase with GABAB Receptors Alters Functional Signaling

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABABR2 subunit, promotes internalization of the GABAB receptor (GABABR) and leads to smaller GABABR-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABABR1 subunit, and that GABABRs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABABR interaction can be regulated by intracellular Ca2+ Finally, a peptide that potentially reduces recruitment of PP2A to GABABRs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABABRs may be a useful strategy to increase receptor signaling for treating diseases.SIGNIFICANCE STATEMENT Dysregulation of GABAB receptors (GABABRs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABABRs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABABR1 subunit, and that this interaction is sensitive to intracellular Ca2+ We demonstrate that a short peptide corresponding to the PP2A interaction site on GABABR1 competes for PP2A binding, enhances phosphorylation GABABR2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABABRs may provide a specific strategy to modulate GABABR signaling in disease conditions

Opposing changes in phosphorylation of specific sites in synapsin I during Ca2+-dependent glutamate release in isolated nerve terminals

Synapsins are major neuronal phosphoproteins involved in regulation of neurotransmitter release. Synapsins are well established targets for multiple protein kinases within the nerve terminal, yet little is known about dephosphorylation processes involved in regulation of synapsin function. Here, we observed a reciprocal relationship in the phosphorylation-dephosphorylation of the established phosphorylation sites on synapsin I. We demonstrate that, in vitro, phosphorylation sites 1, 2, and 3 of synapsin I (P-site 1 phosphorylated by cAMP-dependent protein kinase; P-sites 2 and 3 phosphorylated by Ca2+ calmodulin-dependent protein kinase II) were excellent substrates for protein phosphatase 2A, whereas P-sites 4, 5, and 6 (phosphorylated by mitogen-activated protein kinase) were efficiently dephosphorylated only by Ca2+-calmodulin-dependent protein phosphatase 2B-calcineurin. In isolated nerve terminals, rapid changes in synapsin I phosphorylation were observed after Ca2+ entry, namely, a Ca2+-dependent phosphorylation of P-sites 1, 2, and 3 and a Ca2+-dependent dephosphorylation of P-sites 4, 5, and 6. Inhibition of calcineurin activity by cyclosporin A resulted in a complete block of Ca2+-dependent dephosphorylation of P-sites 4, 5, and 6 and correlated with a prominent increase in ionomycin-evoked glutamate release. These two opposing, rapid, Ca2+-dependent processes may play a crucial role in the modulation of synaptic vesicle trafficking within the presynaptic terminal

Dopamine-dependent tuning of striatal inhibitory synaptogenesis.

Dopaminergic projections to the striatum, crucial for the correct functioning of this brain region in adulthood, are known to be established early in development, but their role is currently uncharacterized. We demonstrate here that dopamine, by activating D(1)- and/or D(2)-dopamine receptors, decreases the number of functional GABAergic synapses formed between the embryonic precursors of the medium spiny neurons, the principal output neurons of the striatum, with associated changes in spontaneous synaptic activity. Activation of these receptors reduces the size of postsynaptic GABA(A) receptor clusters and their overall cell-surface expression, without affecting the total number of clusters or the size or number of GABAergic nerve terminals. These changes result from an increased internalization of GABA(A) receptors, and are mediated by distinct signaling pathways converging at the level of GABA(A) receptors to cause a transient PP2A/PP1-dependent dephosphorylation. Thus, tonic D(1)- and D(2)-receptor activity limits the extent of collateral inhibitory synaptogenesis between medium spiny neurons, revealing a novel role of dopamine in controlling the development of intrinsic striatal microcircuits

Antibody Labelling of Resilin in Energy Stores for Jumping in Plant Sucking Insects

The rubbery protein resilin appears to form an integral part of the energy storage structures that enable many insects to jump by using a catapult mechanism. In plant sucking bugs that jump (Hemiptera, Auchenorrhyncha), the energy generated by the slow contractions of huge thoracic jumping muscles is stored by bending composite bow-shaped parts of the internal thoracic skeleton. Sudden recoil of these bows powers the rapid and simultaneous movements of both hind legs that in turn propel a jump. Until now, identification of resilin at these storage sites has depended exclusively upon characteristics that may not be specific: its fluorescence when illuminated with specific wavelengths of ultraviolet (UV) light and extinction of that fluorescence at low pH. To consolidate identification we have labelled the cuticular structures involved with an antibody raised against a product of the Drosophila CG15920 gene. This encodes pro-resilin, the first exon of which was expressed in E. coli and used to raise the antibody. We show that in frozen sections from two species, the antibody labels precisely those parts of the metathoracic energy stores that fluoresce under UV illumination. The presence of resilin in these insects is thus now further supported by a molecular criterion that is immunohistochemically specific

Immunohistochemical assessment of protein phosphorylation state: the dream and the reality

The development of phosphorylation state-specific antibodies (PSSAs) in the 1980s, and their subsequent proliferation promised to enable in situ analysis of the activation states of complex intracellular signaling networks. The extent to which this promise has been fulfilled is the topic of this review. I review some applications of PSSAs primarily in the assessment of solid tumor signaling pathway activation status. PSSAs have received considerable attention for their potential to reveal cell type-specific activation status, provide added prognostic information, aid in the prediction of response to therapy, and most recently, demonstrate the efficacy of kinase-targeted chemotherapies. However, despite some successes, many studies have failed to demonstrate added value of PSSAs over general antibody immunohistochemistry. Moreover, there is still a large degree of uncertainty about the interpretation of complex and heterogeneous staining patterns in tissue samples and their relationship to the actual phosphorylation states in vivo. The next phase of translational research in applications of PSSAs will entail the hard work of antibody validation, gathering of detailed information about epitope-specific lability, and implementation of methods for standardization

Adenylyl Cyclases 1 and 8 Initiate a Presynaptic Homeostatic Response to Ethanol Treatment

BACKGROUND:Although ethanol exerts widespread action in the brain, only recently has progress been made in understanding the specific events occurring at the synapse during ethanol exposure. Mice deficient in the calcium-stimulated adenylyl cyclases, AC1 and AC8 (DKO), demonstrate increased sedation duration and impaired phosphorylation by protein kinase A (PKA) following acute ethanol treatment. While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition. METHODOLOGY/PRINCIPAL FINDINGS:Here, we have used phosphoproteomic techniques and identified several presynaptic proteins that are phosphorylated in the brains of wild type mice (WT) after ethanol exposure, including synapsin, a known PKA target. Phosphorylation of synapsins I and II, as well as phosphorylation of non-PKA targets, such as, eukaryotic elongation factor-2 (eEF-2) and dynamin is significantly impaired in the brains of DKO mice. This deficit is primarily driven by AC1, as AC1-deficient, but not AC8-deficient mice also demonstrate significant reductions in phosphorylation of synapsin and eEF-2 in cortical and hippocampal tissues. DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment. CONCLUSIONS/SIGNIFICANCE:These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade

Post-Training Dephosphorylation of eEF-2 Promotes Protein Synthesis for Memory Consolidation

Memory consolidation, which converts acquired information into long-term storage, is new protein synthesis-dependent. As protein synthesis is a dynamic process that is under the control of multiple translational mechanisms, however, it is still elusive how these mechanisms are recruited in response to learning for memory consolidation. Here we found that eukaryotic elongation factor-2 (eEF-2) was dramatically dephosphorylated within 0.5–2 hr in the hippocampus and amygdala of mice following training in a fear-conditioning test, whereas genome-wide microarrays did not reveal any significant change in the expression level of the mRNAs for translational machineries or their related molecules. Moreover, blockade of NMDA receptors with MK-801 immediately following the training significantly impeded both the post-training eEF-2 dephosphorylation and memory retention. Notably, with an elegant sophisticated transgenic strategy, we demonstrated that hippocampus-specific overexpression of eEF-2 kinase, a kinase that specifically phosphorylates and hence inactivates eEF-2, significantly inhibited protein synthesis in the hippocampus, and this effects was more robust during an “ongoing” protein synthesis process. As a result, late phase long-term potentiation (L-LTP) in the hippocampus and long-term hippocampus-dependent memory in the mice were significantly impaired, whereas short-term memory and long-term hippocampus-independent memory remained intact. These results reveal a novel translational underpinning for protein synthesis pertinent to memory consolidation in the mammalian brain

HIV-1 Promotes Renal Tubular Epithelial Cell Protein Synthesis: Role of mTOR Pathway

Tubular cell HIV-infection has been reported to manifest in the form of cellular hypertrophy and apoptosis. In the present study, we evaluated the role of mammalian target of rapamycin (mTOR) pathway in the HIV induction of tubular cell protein synthesis. Mouse proximal tubular epithelial cells (MPTECs) were transduced with either gag/pol-deleted NL4-3 (HIV/MPTEC) or empty vector (Vector/MPTEC). HIV/MPTEC showed enhanced DNA synthesis when compared with Vector/MPTECs by BRDU labeling studies. HIV/MPTECs also showed enhanced production of β-laminin and fibronection in addition to increased protein content per cell. In in vivo studies, renal cortical sections from HIV transgenic mice and HIVAN patients showed enhanced tubular cell phosphorylation of mTOR. Analysis of mTOR revealed increased expression of phospho (p)-mTOR in HIV/MPTECs when compared to vector/MPTECs. Further downstream analysis of mTOR pathway revealed enhanced phosphorylation of p70S6 kinase and associated diminished phosphorylation of eEF2 (eukaryotic translation elongation factor 2) in HIV/MPTECs; moreover, HIV/MPTECs displayed enhanced phosphorylation of eIF4B (eukaryotic translation initiation factor 4B) and 4EBP-1 (eukaryotic 4E binding protein). To confirm our hypothesis, we evaluated the effect of rapamycin on HIV-induced tubular cell downstream signaling. Rapamycin not only attenuated phosphorylation of p70S6 kinase and associated down stream signaling in HIV/MPTECs but also inhibited HIV-1 induced tubular cell protein synthesis. These findings suggest that mTOR pathway is activated in HIV-induced enhanced tubular cell protein synthesis and contributes to tubular cell hypertrophy

Genetic Architecture of Hybrid Male Sterility in Drosophila: Analysis of Intraspecies Variation for Interspecies Isolation

Background: The genetic basis of postzygotic isolation is a central puzzle in evolutionary biology. Evolutionary forces causing hybrid sterility or inviability act on the responsible genes while they still are polymorphic, thus we have to study these traits as they arise, before isolation is complete. Methodology/Principal Findings: Isofemale strains of D. mojavensis vary significantly in their production of sterile F 1 sons when females are crossed to D. arizonae males. We took advantage of the intraspecific polymorphism, in a novel design, to perform quantitative trait locus (QTL) mapping analyses directly on F1 hybrid male sterility itself. We found that the genetic architecture of the polymorphism for hybrid male sterility (HMS) in the F1 is complex, involving multiple QTL, epistasis, and cytoplasmic effects. Conclusions/Significance: The role of extensive intraspecific polymorphism, multiple QTL, and epistatic interactions in HMS in this young species pair shows that HMS is arising as a complex trait in this system. Directional selection alone would be unlikely to maintain polymorphism at multiple loci, thus we hypothesize that directional selection is unlikely to be the only evolutionary force influencing postzygotic isolation