Regulation of protein phosphorylation in astrocyte cultures by external calcium ions: specific effects on the phosphorylation of glial fibrillary acidic protein (GFAP), vimentin and heat shock protein 27 (HSP27).
The effect of external Ca2+ ([Ca2+]e) on the incorporation of [32P] into total protein, cytoskeletal proteins and the heat shock protein HSP27, was studied in primary cultures of astrocytes from the rat hippocampus. Zero [Ca2+]e increased total 32P-incorporation into astrocyte protein and when this was normalized to 100%, incorporation was significantly increased into glial fibrillary acidic protein (GFAP), vimentin (VIM) and HSP27. The difference in total 32P-incorporation between zero [Ca2+]e and 1 mM [Ca2+]e was reversed by incubation of the cells with the protein phosphatase inhibitor okadaic acid in the range 1-10 nM; higher concentrations of okadaic acid (50-100 nM) further increased total 32P-incorporation. In zero [Ca2+]e the non-specific channel blocker Co2+ (1 mM) decreased total 32P-incorporation by approximately 30%. The results were compared with a previous study [S.T. Wofchuk, R. Rodnight, Age-dependent changes in the regulation by external calcium ions of the phosphorylation of glial fibrillary acidic protein in slices of rat hippocampus, Dev. Brain Res. 85 (1995) 181-186] in which it was shown that in immature hippocampal slices zero [Ca2+]e compared with 1 mM [Ca2+]e increased 32P-incorporation into GFAP without changing total incorporation. The difference between the results for total 32P-incorporation obtained in cultured astrocytes and immature brain tissue was found to be related to the concentration of [Ca2+]e in the medium since in slices concentrations of [Ca2+]e higher than 1 mM progressively decreased total incorporation. The difference may reflect a higher Ca2+-permeability of the plasma membrane in cultured astrocytes and/or to the complex structure of the slice tissue. In two-dimensional electrophoresis HSP27, in contrast to GFAP and VIM, was separated into 3 immunodetectable isoforms only two of which were normally phosphorylated. After labelling in the presence of okadaic acid both immunodetectable and phosphorylated HSP27 focussed as a single polypeptide. Phorbol dibutyrate (1 microM) and zero [Ca2+]e stimulated the phosphorylation of both isoforms, but in the case of zero [Ca2+]e the effect on the more acidic isoform was proportionally greater.
Gottfried
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Departamento de Bioquímica, UFRGS, Instituto de Ciências Básicas da Saúde, Rua Ramiro Barcelos 2600-Anexo, 90.035.003, Porto Alegre, RS, Brazil.
Valentim
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Salbego
C
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Karl
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J
Wofchuk
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ST
Rodnight
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R
D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D019319:Okadaic Acid / Q000494:pharmacology; D009924:Organ Culture Techniques; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D014746:Vimentin / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D006360:Heat-Shock Proteins; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D014746:Vimentin; D019319:Okadaic Acid; D003035:Cobalt; D010749:Phosphoprotein Phosphatases; D002118:Calcium