PROTEIN EXPRESSION AND PURIFICATION 9, 363–371 (1997) ARTICLE NO. PT960710 Purification, Characterization, and in Vitro Phosphorylation of the Neuron-Specific Membrane-Associated Protein SCG10 Bruno Antonsson,* Robert Lu ¨ tjens,* Gilbert Di Paolo,* Daniel Kassel,† ,1 Bernard Allet,* Alain Bernard,* Stefan Catsicas,* and Gabriele Grenningloh* ,2 *Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development S.A., 1228 Plan-les-Ouates, Geneva, Switzerland; and †Glaxo Inc., Research Triangle Park, North Carolina 27709 Received October 24, 1996, and in revised form December 4, 1996 vous system but high levels persist in regions of synap- SCG10 is a neuron-specific, developmentally regu- tic plasticity (4). Up-regulation of SCG10 has also been lated protein which is highly enriched in growth found following lesion experiments (5). cones. Sequence homology indicates that it is related Based on sequence homology SCG10 is related to to the phosphoprotein stathmin or Op18, an in vitro the ubiquitous 19-kDa phosphoprotein stathmin/ and in vivo substrate for several serine/threonine ki- Op18. The two proteins share 74% amino acid identity nases which are involved in a variety of signaling path- in the overlapping region (6,7). A structural feature ways. As a first step to examine the biochemical prop- that distinguishes the two proteins is that SCG10 has erties of SCG10, the protein was expressed in Esche- a N-terminal extension which contains several hy- richia coli and purified to apparent homogeneity. The drophobic residues (1). Whereas stathmin is a soluble purified protein was used in in vitro phosphorylation cytosolic protein (8), SCG10 is membrane-associated, assays. SCG10 was phosphorylated by MAP kinase, localized to the area of the Golgi complex and enriched cAMP-dependent protein kinase, cGMP-dependent in growth cones of developing neurons (1,38). The N- protein kinase, p34 cdc2 kinase, DNA-dependent protein terminal domain is responsible for the membrane as- kinase, Ca 2/ /calmodulin kinase II, and casein kinase sociation of SCG10 (38). Although stathmin is ex- II. The protein was not a substrate for casein kinase I pressed ubiquitously, particularly high levels have and protein kinase C. SCG10 was phosphorylated by been found in brain, mainly in developing neurons src tyrosine kinase, which demonstrates that the pro- where SCG10 expression is also high (1,9). In cultured tein can be phosphorylated in vitro on a tyrosine resi- developing neurons, high levels of both SCG10 and due. Our data suggest that SCG10 is a phosphoprotein stathmin are found, but with distinct subcellular lo- which might be involved in signal transduction in neu- rons.  1997 Academic Press calization (A. Osen-Sand and G. Grenningloh, unpub- lished observations). Given the sequence conservation between SCG10 and stathmin, it is likely that they exert similar specific functions in different subcellular compartments of developing neurons. Recently, stath- SCG10 is a neuron-specific growth-associated pro- min was identified as a regulator of microtubule dy- tein of 21 kDa (1). The levels of SCG10 mRNA and namics (10). Both SCG10 and stathmin are highly con- protein are transiently up-regulated in the developing served between species with only scarce amino acid nervous system (2). In rat, SCG10 expression can be substitutions (7). detected from Embryonic Day 11.5 (3). SCG10 remains Previous studies have identified stathmin as a sub- expressed at low levels in most areas of the adult ner- strate for serine phosphorylation upon activation of several distinct signal transduction pathways (8,11 – 1 Present address: CombiChem, Inc., San Diego, California 92121. 17). Both in vitro and in vivo, stathmin is a substrate 2 To whom correspondence should be addressed at Geneva Biomed- for several protein kinases, including MAP kinase, ical Research Institute, Glaxo Wellcome Research and Development PKA, and p34 cdc2 (18–22). However, it is not known S.A., 14 chemin des Aulx, 1228 Plan-les-Ouates/Geneva, Switzer- land. Fax: 41-22-794 69 65. E-mail: BEA6063@GGR.CO.UK. whether SCG10 is a phosphoprotein. Of the four phos- 363 1046-5928/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.