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J. Biol. Chem., Vol. 279, Issue 38, 40026-40034, September 17, 2004

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Analysis of Drosophila cGMP-dependent Protein Kinases and Assessment of Their in Vivo Roles by Targeted Expression in a Renal Transporting Epithelium^*

Matthew R. MacPherson, Suzanne M. Lohmann, and Shireen-A. Davies¶

From the Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Anderson College, Dumbarton Rd., Glasgow G11 6NU, United Kingdom and the Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg 97080, Germany

cGMP-dependent protein kinase (cGK) forms encoded by the dg2 (for) gene are implicated in behavior and epithelial transport in Drosophila melanogaster. Here, we provide the first biochemical characterization and cellular localization of cGKs encoded by the major transcripts of dg2: dg2P1 and dg2P2. cGMP stimulates kinase activity of DG2P1 (EC₅₀: 0.13 ± 0.039 µM) and DG2P2 (EC₅₀: 0.32 ± 0.14 µM) in Malpighian tubule and S2 cell extracts. DG2P1 and DG2P2 are magnesium-requiring enzymes and were inhibited by 10 and 100 µM of a cGK inhibitor, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp isomer; whereas DG1, the cGK encoded by the D. melanogaster dg1 gene, was unaffected. DG2P1 and DG2P2 were localized in the plasma membrane in S2 cells, whereas DG1 was localized in the cytosol. The D. melanogaster fluid-transporting Malpighian tubule was used as an organotypic model to analyze cGK localization and function in vivo. Targeted expression of DG2P2, DG2P1, and DG1 in tubule cells via the UAS/GAL4 system in transgenic flies revealed differential localization of all three cGKs in vivo: DG2P2 expression at the apical membrane; DG2P1 expression at both the apical and basolateral membranes; and DG1 expression at the basolateral membrane and in the cytosol. Transgenic tubules for all three cGKs displayed enhanced cGK activity compared with wild-type tubules. The physiological impact of targeted expression of individual cGKs in tubule principal cells was assessed by measuring basal and stimulated rates of fluid transport. DG1 expression greatly enhanced fluid transport by the tubule in response to exogenous cGMP, whereas DG2P2 expression significantly increased fluid transport in response to the nitridergic neuropeptide, capa-1. Thus, dg2-encoded proteins are bona fide cGKs, which have differential roles in epithelial fluid transport, as assessed by in vivo studies. Furthermore, a novel epithelial role is suggested for DG1, which is considerably more responsive to cGMP than to capa-1 stimulation.

Received for publication, May 19, 2004 , and in revised form, June 18, 2004.

^* This work was supported by the United Kingdom Biotechnology and Biological Sciences Research Council and the Deutsche Forschungsgemeinschaft (Grant SFB 355). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 44-141-330-2317; Fax: 44-141-330-4878; s.a.davies{at}bio.gla.ac.uk.

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