Abstract
The Drosophila mutant turnip was initially isolated based on poor learning performance (Quinn, W.G., Sziber, P.P., and Booker, R. (1979) Nature 277, 212-214). Here we show that turnip is dramatically reduced in protein kinase C (PKC) activity. In addition, turnip flies are deficient in phosphorylation of a 76-kDa head membrane protein (hereafter pp76) which is a major substrate for protein kinase C in homogenates of wild-type flies. Reduced PKC activity, defective pp76 phosphorylation, and most of turnip's learning deficiency co-map genetically to a region on the X-chromosome, 18A5-18D1-2, spanned by the deletion Df(1)JA27. Apparently turnip+ is not a structural gene for PKC because Drosophila PKC genes map elsewhere in the genome. Our results suggest that turnip gene product is required for activation of PKC and that PKC plays a role in associative learning in Drosophila.
Highlights
From the Department of Brain and Cognitive Sciences and the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Biochemical studies on flies with normal, mutant, and deleted turnip genes suggest that the turnip+ gene product is required for activation of Ca’+/phospholipid-stimulatedPKC activity in vitro
Our results suggest that turnip gene product is required for activation of PKC and that PKC plays Materiak-[y-32P]ATP (3,000 Ci/mmol) was from Du Pont-New a role inassociative learning in Drosophila
Summary
Dana-Farber Cancer Institute, 44 Binney St., by Student’s t test (two-tailed). Boston, MA 02115. In Vitro Phosphorylation-Phosphorylation of membrane proteins was carried out in a 50-pl reaction mixture containing 25 mM Tris acetate buffer (pH 7.6), 10 mM magnesium acetate, and 20 p~ ATP (3-5 pCi of [y-”PIATP). Assay for PKC-Membranes were diluted to 1-2 mg of protein/ml in 25 mM Tris acetate buffer (pH 7.6) containing 5 mM EGTA and were incubated for min on ice. The membranes were pelleted by centrifugation in aBeckman Airfuge at p s i . Phosphorylation of endogenous PKC substrates in EGTA membraneextracts was carried out ina 50-pl reaction mixture containing 10 mM magnesium acetate, 20 mM Tris acetate (pH 7.6), and 20 p~ [-y-’2P]ATP (5 pCi) with other additions as indicated. The reaction was started by adding EGTA extracts obtained from membranes containing 80 pg of protein, incubated for 3 min at 26 “C, and stopped with SDS sample buffer. Color was developed in substrate solution (100 mM Tris-HC1 (pH 9.5), 100 mM NaC1, 5 mM MgClz.6H20,50 mg/ml of nitro blue tetrazolium, 3.5 pl/ml 5-bromo-4-chloro-3-indolyl phosphate)
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