The dephosphorylation of glial fibrillary acidic protein (GFAP) in the immature rat hippocampus is catalyzed mainly by a type 1 protein phosphatase.
We used protein phosphatase inhibitors to study the phosphatase activity involved in the dephosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in a cytoskeletal fraction and in slices prepared from hippocampi of immature rats. Cytoskeletal proteins were labelled with [gamma 32P]ATP in the presence of Ca2+ and calmodulin and then allowed to dephosphorylate through the activity of bound protein phosphatases. Dephosphorylation was inhibited by the protein phosphatase inhibitors okadaic acid and microcystin-LR, but not by EGTA. Maximal inhibition was given by 1 microM okadaic acid and 10 nM microcystin-LR. This difference of two orders of magnitude in the sensitivity of the dephosphorylation to the inhibitors indicates that the bound dephosphorylating activity in the cytoskeletal fraction was due to a type 1 protein phosphatase, rather than protein phosphatase 2A which is equally sensitive to okadaic acid and microcystin-LR. To investigate the dephosphorylation of GFAP in intact tissue we incubated slices with various concentrations of the cell-permeable inhibitor okadaic acid in the presence of [32P] phosphate. Net [32P]-incorporation into GFAP was increased by okadaic acid due to inhibition of dephosphorylation; the minimum effective concentration was 25 nM. Since the IC50 for inhibition of protein phosphatase 2A by okadaic acid is 0.1 nM, this result indicates that in intact tissue GFAP dephosphorylation is primarily due to a type 1 protein phosphatase.
Vinadé
L
L
Departamento de Bioquímica, Instituto de Biociências, UFRGS (Centro), Porto Alegre, RS, Brazil.
Rodnight
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000375:Aging / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001345:Autoradiography; D015415:Biomarkers; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000302:isolation & purification / Q000378:metabolism*; D006624:Hippocampus / Q000201:enzymology* / Q000254:growth & development; D015151:Immunoblotting; D066298:In Vitro Techniques; D008297:Male; D008387:Marine Toxins; D052998:Microcystins; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D054648:Protein Phosphatase 2; D051381:Rats; D017208:Rats, Wistar
D015415:Biomarkers; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D008387:Marine Toxins; D052998:Microcystins; D009419:Nerve Tissue Proteins; D010456:Peptides, Cyclic; D010761:Phosphorus Radioisotopes; D019319:Okadaic Acid; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C057862:cyanoginosin LR