GFAP phosphorylation studied in digitonin-permeabilized astrocytes: standardization of conditions.
Cycles of assembly/disassembly of the intermediate filaments of astrocytes are modulated by the phosphorylation of glial fibrillary acidic protein (GFAP). The sites on GFAP are localized at the N-terminal where they are phosphorylated by cAMP-dependent and Ca(2+)-dependent protein kinases. Phosphorylation of GFAP has been investigated in brain slices, astrocyte cultures, cytoskeletal fractions and purified systems. Here we describe a different approach to study GFAP phosphorylation. We show that permeabilization of astrocytes in culture with digitonin allows direct access to the systems phosphorylating GFAP. Conditions for the permeabilization were established with an assay based on the exclusion of Trypan blue. Incubation of permeabilized cells with cAMP and Ca(2+) increased the phosphorylation state of GFAP. Immunocytochemistry with anti-GFAP showed that permeabilized astrocytes retained their typical flat, fibroblast morphology and exhibited well preserved glial filaments. On incubation with cAMP the filaments apparently condensed to form long processes. The results suggest the approach of studying structural changes in glial filaments in parallel to protein phosphorylation, in the presence of specific modulators of protein kinases and phosphatases has considerable potential.
Karl
J
J
Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2600-Anexo, 90035-003, Porto Alegre, Brazil.
Gottfried
C
C
Tramontina
F
F
Dunkley
P
P
Rodnight
R
R
Gonçalves
C A
CA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D007150:Immunohistochemistry; D009414:Nerve Growth Factors / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D012964:Sodium / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D005904:Glial Fibrillary Acidic Protein; D009414:Nerve Growth Factors; D011506:Proteins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D000255:Adenosine Triphosphate; D012964:Sodium; D004072:Digitonin; D011188:Potassium