Ž . Developmental Brain Research 104 1997 11–17 Research report Evidence for a role for calcium ions in the dephosphorylation of glial ž / fibrillary acidic protein GFAP in immature hippocampal slices and in astrocyte cultures from the rat Lucia Vinade, Carlos A. Gonc ¸alves, Susana Wofchuk, Carmem Gottfried, Richard Rodnight ) ´ ( ) Departamento de Bioquımica, Instituto de Ciencias Basicas da Saude, UFRGS Centro , 90.046.900, Porto Alegre RS, Brazil ´ ˆ ´ ´ Accepted 17 June 1997 Abstract 2q Ž . Evidence was sought for a role for Ca in the dephosphorylation of the astrocyte marker glial fibrillary acidic protein GFAP in immature hippocampal slices. Although previous work showed that the main phosphatase dephosphorylating GFAP in this preparation is 2q 2q w 32 x a Ca -independent type 1 enzyme, a role for Ca was suggested by the observation that the incorporation of P phosphate into GFAP in immature slices is inhibited by external Ca 2q . This inhibition is strikingly different to the situation in mature slices where GFAP phosphorylation is completely dependent on Ca 2q . Pure astrocyte cultures were probed by immunoblotting for the presence of the Ca 2q -dependent phosphatase calcineurin. An enzyme content, amounting to about 2% of that found in fresh hippocampal tissue, was Ž . Ž . detected for both the catalytic a and regulatory b subunits. The direct or indirect association of calcineurin with GFAP was suggested by observations showing that FK506, a specific inhibitor of calcineurin, increased the phosphorylation state of GFAP in immature slices and of GFAP and vimentin in astrocyte cultures. q 1997 Elsevier Science B.V. Keywords: Glial fibrillary acidic protein; Protein phosphorylation; Calcineurin; FK506; Astrocyte development; Calcium ion 1. Introduction Ž . Proteins of the intermediate filaments IF are major components of the cytoskeleton in most mammalian cells w x 19,35 . Recent work has shown that intact IFs exist in dynamic equilibrium with a small soluble pool of continu- w x ously exchanging disassembled subunits 9,27,34,38 . This equilibrium is influenced by a number of factors and especially by the phosphorylation state of the filament protein. In vitro phosphorylation of polymerized IF pro- ww x teins results in their depolymerization 1,9,14 , reviewed w xx in 15,16 and phosphorylation of disassembled subunits inhibits their assembly into filaments, with subunit ex- change being suppressed in proportion to the extent of w x phosphorylation 28 . In intact cells, increasing the phos- phorylation state of IF proteins results in filament reorgani- zation. For example the micro-injection of cyclic AMP Ž . Ž . cAMP protein kinase PKA into fibroblasts increased the phosphorylation of the IF protein vimentin and modi- ) Corresponding author. Fax: q55-51-316-5535 w x fied the organization of the filaments 18 ; similar changes in filament organization were obtained by increasing the phosphorylation state of vimentin by exposure to protein w x phosphatase inhibitors 10,20 . Moreover, the regulation of IF dynamics by phosphorylation assumes special impor- tance during the reorganization of the cytoskeleton that occurs in development and in differentiating and dividing cells. In particular, the role of multisite protein phosphory- lation of IF proteins has been intensively studied during the cell cycle where, in many cells, the reorganization of the cytoskeleton that precedes and continues during mito- sis is accompanied by the temporally regulated site-specific phosphorylation and dephosphorylation of IF proteins w x 5,6,12,16,36 . In the case of the astrocyte marker glial Ž . wx fibrillary acidic protein GFAP 8 elegant studies using site and phosphorylation-specific antibodies have shown that, as cells enter the cycle, phosphorylation of each site occurs at a different time point and with a spatially differ- ent distribution pattern and is followed by dephosphoryla- w x tion during cytokinesis 15,23,29,32 . While considerable progress has been made in identify- ing the protein kinases that phosphorylate IFs in vivo 0165-3806r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.