bFGF Stimulates GAP-43 Phosphorylation at Ser41 and Modifies Its Intracellular Localization in Cultured Hippocampal Neurons Pedro Tejero-Dı´ez, Paloma Rodrı´guez-Sa ´nchez, Noa Beatriz Martı´n-Co ´freces, and F. Javier Dı´ez-Guerra 1 Centro de Biologı ´a Molecular Severo Ochoa, Departamento de Biologı ´a Molecular, Facultad de Ciencias, Universidad Auto ´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Cultured hippocampal neurons have been used to study GAP-43 phosphorylation and subcellular distribution. By immunofluorescence, GAP-43 was found associated with adherent membrane patches that remained attached to the substratum after in situ permeabilization with Non- idet-NP40. This association increases during neuronal de- velopment and is stabilized by the actin cytoskeleton. Basic fibroblast growth factor (bFGF) promotes GAP-43 translocation from the cytosol to adherent membrane patches and, at the same time, stimulates GAP-43 phos- phorylation, mainly at the protein kinase C (PKC) site (Ser41). Inhibition of PKC prevented bFGF-stimulated GAP-43 phosphorylation and translocation, while activa- tion by phorbol esters mimicked bFGF effects, suggesting that phosphorylation at Ser41 regulates GAP-43 subcellu- lar localization. Using biochemical fractionation and phosphorylation analysis, it was found that Ser41 phos- phorylation was highest in cytoskeleton-associated GAP-43 and lowest in membrane-associated GAP-43. It is proposed that GAP-43 is continuously cycling between intracellular compartments depending on its phosphory- lation state and could be taking part in initial adhesive complexes assembled during growth cone advance. INTRODUCTION Growth-associated protein 43 (GAP-43; 2 B50; F1; Neuromodulin) is a nervous tissue-specific protein that is involved in axon elongation and growth cone guid- ance (for reviews, see Coggins and Zwiers, 1991; Be- nowitz and Routtenberg, 1997; Oestreicher et al., 1997). GAP-43 is expressed at high levels in developing neu- rons, transported anterogradely along the axon, and accumulated in growth cones (Meiri et al., 1986; Dani et al., 1991; Yao et al., 1993). Intracellularly, it associates with the plasma membrane through two palmitate res- idues covalently attached to cysteines 3 and 4 (Skene and Vira ´g, 1989; Liu et al., 1994). Thus, most GAP-43 is extracted in membrane-enriched fractions, although smaller amounts can be extracted in soluble form or associated with the actin-rich membrane skeleton (Moss et al., 1990; Meiri and Gordon-Weeks, 1990). GAP-43 is a multiphosphorylated protein (Spencer et al., 1992; Di Luca et al., 1996; Huang et al., 1999). Among its several phosphorylation sites, Ser41 is the best characterized. It is phosphorylated by protein kinase C (PKC), both in vivo and in vitro (Coggins and Zwiers, 1991; Oestreicher et al., 1997), and is located in a region of the molecule 1 To whom correspondence should be addressed. Fax: 34-913978087. E-mail: fjdiez@cbm.uam.es. 2 Abbreviations used: bFGF, basic fibroblast growth factor; FGF-R, basic fibroblast growth factor receptor; BSA, bovine serum albumin; CaM, calmodulin; CKII, casein kinase II; cytoD, cytochalasin D; DIV, days in vitro; DAG lipase, diacylglycerol lipase; DAB, diaminobenzi- dine; DRG, dorsal root ganglia; DMEM, Dulbecco’s modified Eagle’s medium; FITC, fluorescein isothiocyanate; DIGs, glycolipid-enriched membrane domains; GAP-43, growth-associated protein 43; N-CAM, neural cell adhesion molecule; NP-40, Nonidet-P40; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate buffered saline; PLC, phos- pholipase C; PAGE, polyacrylamide gel electrophoresis; PKC, pro- tein kinase C; SDS, sodium dodecyl sulfate. doi:10.1006/mcne.2000.0915, available online at http://www.idealibrary.com on Molecular and Cellular Neuroscience 16, 766–780 (2000) MCN 1044-7431/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 766