Phosphorylation of the V-ATPase subunit C by protein kinase A

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Abstracts / Comparative Biochemistry and Physiology, Part A 150 (2008) S131-S138

most challenging topics in V-ATPase research. Recently a novel class of specific V-ATPase inhibitors – the archazolids, cytotoxic macrolactons produced by the myxobacterium Archangiumgephyra – has been discovered. Like the well known and highly potent V-ATPase inhibitors concanamycin and bafilomycin which belong to the family of the plecomacrolides, archazolids inhibit with an IC50 value in the nanomolar range. They also share at least part of the binding site which is located in the VO subunit c of the V-ATPase. In this study the binding site of archazolid is characterized and the structure–function relationship of the inhibitor is investigated in more detail in order to develop new selective inhibitors of the V-ATPase. We will explore, on the one hand, the amino acids binding the inhibitor by site directed mutagenesis of the genes encoding involved subunits. On the other hand, we will investigate derivatives of archazolid in order to analyze the crucial structural features of the inhibitor. Supported by the VolkswagenStiftung Funding Initiative: Interplay between Molecular Conformations and Biological Function. doi:10.1016/j.cbpa.2008.04.335

vating a transepithelial chloride conductance. The MT can also synthesize TA when its precursor, L-tyrosine, is present in the peritubular fluid. The goal of this study was to identify two of the gene products necessary for TA signaling: tyrosine decarboxylase (tdc), the enzyme that converts tyrosine to TA, and the TA receptor (TAR). The Drosophila genome contains two tdc genes: tdc1 and tdc2 (Cole et al., 2005). The tdc1 gene is highly expressed in the MT while tdc2 is not. MTs from tdc1 mutant flies show no electrophysiological or diuretic responses to tyrosine but normal responses to TA; in contrast, mutation of tdc2 does not alter tyrosine sensitivity. Overexpressing tdc1 in the principal cells, but not the stellate cells, of tdc1 mutants, rescues tyrosine sensitivity, suggesting that TA synthesis occurs in the principal cells. The TAR in the MT is unlikely to be encoded by the canonical TAR gene tyrR, which is expressed in the MT at very low levels. In contrast, the recently identified TAR gene CG7431 (Cazzamali et al., 2005) is abundantly expressed in the MT. Mutation of CG7431 results incomplete insensitivity to TA, suggesting that CG7431 encodes the TAR in the MT. Rescue experiments are underway to determine the cell type in which this receptor functions. Supported by Marquette University and NSF0744619. doi:10.1016/j.cbpa.2008.04.337

A11.29 Segmental bidirectional transport of H+ in the adult Drosophila midgut S. Shanbhag, S. Tripathi (Tata Institute of Fundamental Research, Colaba, Mumbai) The perfused midgut of adult Drosophila is amenable to stereological analysis of its epithelial ultrastructure and electrophysiological measurement of ion transport, as was done earlier for the larva (S. Shanbhag, S. Tripathi J. Membrane Biol. 206, 61–72, 2005). The anterior (≈2 mm), middle (≈1 mm) and posterior (≈2 mm) segments are distinctive. The anterior segment is absorptive with a real amplication of the basal membrane of 43.4 ± 2.7, n = 11. The middle segment has alternating absorptive (apically amplified 8.9 ± 0.8 times, n = 9 and basally by 94.3 ± 7.2 times, n = 26) and secretory (apically amplified by 93.7 ±2.8 times, n = 9, and basally by 10.0 ± 1.2 times, n = 20) cells. Posterior segment cells have an extensively dilated basal extracellular labyrinth, twice those of anterior segment cells, indicating more fluid re absorption in the posterior segment. Intraluminal pH was determined using pH-indicator dyes. Acidification was detected in the middle midgut (pH b 4.0). The luminal pH of anterior and posterior adult midgut is 8.0 to 10.0. Inclusion of Acetazolamide (104 M) in the food dissipated these gradients. The transepithelial (23 ± 1.4 to 14 ± 1.4 mV, n = 94), apical (40.9 ± 2.7 to 68 ± 2.2 mV, n = 111), and basal (54 ± 1.8 to 63 ± 3.2 mV, n = 111) potentials, and transepithelial resistance (384 ± 23 to 526 ± 31 Ω∙cm2, n = 88), gives short circuit currents of 53 ± 5 to 21 ±2 µA∙cm2 (n = 88). The plasma membrane transporters driving these fluxes are yet to be identified. doi:10.1016/j.cbpa.2008.04.336

A11.30 Molecular dissection of tyraminergic communication in the Drosophila Malpighian tubule E. Blumenthal (Marquette University) The biogenic amine tyramine (TA) acts as a diuretic agent in the Drosophila Malpighian tubule (MT), increasing urine secretion by acti-

A11.31 Phosphorylation of the V-ATPase subunit C by protein kinase A O. Vitavska (University of Osnabrueck); M. Voss (University of Potsdam); B. Walz (University of Potsdam); O. Baumann (University of Potsdam); H. Wieczorek (University of Osnabrueck) V-ATPases are regulated by the reversible disassembly V1VO holoenzyme into a cytosolic V1 complex and a membrane bound VO complex. The signalling cascades triggering these events are largely unknown. We report that the V1 subunit C of the tobacco hornworm Manduca sexta interacts with protein kinase A (PKA) and is the only V-ATPase subunit that is phosphorylated by PKA. Subunit C can be phosphorylated as single polypeptide as well as a part of the V1 complex, but not as a part of the V1VO holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V1 complex from which subunit Chad been removed before. Using salivary gland extracts of the blowfly Calliphora vicina in which V-ATPase reassembly and activity is regulated by the neurohormone serotonin via PKA, we show that 8-CPT-cAMP causes phosphorylation of subunit C. Similarly, incubation of intact salivary glands with 8-CPT-cAMP or serotonin leads to the phosphorylation of subunit C but this is abolished by the PKA inhibitor H89. These data suggest that subunit C binds to and serves as a substrate for PKA and that this phosphorylation may be a regulatory switch for the formation of the active V1VO holoenzyme. doi:10.1016/j.cbpa.2008.04.338