Comp. Biochem. Physiol. Vol. 99B, No. 4, pp. 859-864, 1991 0305-0491/91 $3.00+ 0.00 Printed in Great Britain © 1991 PergamonPresspie EFFECT OF PHORBOL ESTER ON PROTEIN PHOSPHORYLATION IN THE CENTRAL NERVOUS SYSTEM OF THE LEECH HIRUDO MEDICINALIS: A TWO-DIMENSIONAL ELECTROPHORETICAL ANALYSIS M. GARCIA-GIL,* D. BOTTAI, M. CANNIZZARO and M. BRUNELLI Department of Physiology and Biochemistry, University of Pisa, Via S. Zeno 31, 1-56100, Pisa, Italy (Tel: 50 553517) (Received 11 February 1991) Abstract--1. Proteins of different regions of the Hirudo medicinalis central nervous system have been analyzed by means of two-dimensional electrophoresis. 2. SubceUular distribution of phosphoproteins has been studied in leech segmental ganglia. 3. Phorbol 12,13-dibutyrate, a protein kinase C activator, stimulates the phosphorylation of a number of proteins whose isoelectric points and mol. wts are presented. 4. Putative roles for these phosphoproteins are discussed. INTRODUCTION The leech central nervous system consists of two major ganglia (one cephalic and one caudal) and a chain of 21 nearly identical segmental ganglia. All these ganglia are interconnected by bundles of fibers (connectives). Each segmental ganglion contains about 400 neurons, many of which are identified cells (Muller et al., 1981). The caudal ganglion is origi- nated by the fusion of seven segmental ganglia. The cephalic ganglion is composed by two different por- tions: a subesophageal one, equivalent to the fusion of four segmental ganglia, and a supraesophageal one, formed by the fusion of two ganglia. The cells of the supraesophageal portion are primarily neuro- secretory (Sawyer, 1986). The leech has proved to be a useful model for the study of synaptogenesis (Nicholls and Garcia-Hernandez, 1989), neuro- development (Weisblast, 1985; Stewart et al., 1986), axotomy and regeneration (Macagno et al., 1985; Pellegrino et al., 1984), and the cellular mechanisms underlying swimming (Kristan, 1983), shortening (Belardetti et al., 1982), feeding (Lent, 1985) and learning (Brunelli et al., 1985; Debski and Friesen, 1985; Sahley and Ready, 1988; Catarsi et al., 1990). Many of the mentioned neural functions are regu- lated in different systems by phosphorylation of proteins (Nairn et al., 1985; Nishizuka, 1984, 1986; Kaczmarek, 1987). Therefore, it is important to identify and characterize the phosphoproteins of the leech central nervous system. Recently, protein kinase C (PKC) has been proposed to play a crucial role in neuronal plasticity. It has been shown that GAP-43, a PKC substrate, is particularly abundant in growth cones (De Graan et al., 1985), and that it is correlated *Author to whom correspondence should be addressed. Abbreviations used: IEF, isoelectrofocusing; PKC, protein kinase C; pI, isoelectric point; SDS--PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCA, trichloroacetic acid. with neurotransmitter release (Dekker et al., 1989) and with long term potentiation in the hippocampus (Akers and Routtenberg, 1985). Moreover, PKC is involved in learning processes in invertebrates (Alkon and Nelson, 1990; Sacktor and Schwartz, 1990) and in vertebrates (Bank et al., 1988; Olds et al., 1989). We have previously studied different kinase activities in homogenates of leech central nervous system (Garcia Gil et al., 1989, 1991). In this paper we have analyzed, in intact segmental ganglia, the effect of an activator of PKC, using a higher resolution technique for the study of proteins. MATERIALSAND METHODS Specimens of adult H. medicinalis were purchased from Ricarimpex (Audenge, France). Phorbol 12,13-dibutyrate and tool. wt standards were from Sigma. Electrophoresis reagents were from LKB or Biorad. [32P]ortophosphoric acid (10mCi/ml) was from Amersham. Autoradiography films were from Kodak. Leeches were anesthetized in 10% ethanol and ganglia isolated as previously described (Belardetti et al., 1982). Labeling of ganglia Ganglia were labeled in leech physiological solution con- taining 0.2 mCi/ml [32P]ortophosphoric acid, 115 mM NaCl, 4mM KC1, 1.8mM CaCl2, 10raM glucose in 10mM Tris-maleate pH 7.4, for 2 hr, at room temperature, and treated with 100 nM phorbol 12,13-dibutyrate for 5 min. Control samples contained the same amount of dimethyl- sulfoxide as phorbol-treated samples 0%0). Homogenization Ganglia were washed three times with ice cold 50 mM Tris 7.4, I mM EGTA, 1 mM EDTA, and then homogenized at 4°C as previously described (Garcia Gil et al., 1991) in 50 mM Tris pH 7.4 containing I mM EDTA, i mM EGTA, 5 mM 2-mercaptoethanol, 5 #g/ml leupeptin, 1/~g/ml pep- statin A and 0.I mM phenylmethylsulfonylfluoride. Subcellular localization After removal of poorly homogenized material by centrifigation (1000g x 1 min), cytosolic and membrane ¢sPs ~/4-J 859