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Phosphorylation of GFAP and lamin B is an early event in reactive astrocytes and may be stimulated by cAMP
Author(s) -
Patel Mayuri P,
Ramsey Gregory R,
García Dana M,
Koke Joseph R
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.790.1
Subject(s) - forskolin , lamin , intermediate filament , phosphorylation , microbiology and biotechnology , glial fibrillary acidic protein , nuclear lamina , cycloheximide , cytoplasm , cytoskeleton , biology , chemistry , nucleus , in vitro , nuclear protein , cell , biochemistry , protein biosynthesis , transcription factor , immunohistochemistry , gene , immunology
Mammalian astrocytes respond to injury by hypertrophy and cell division. Changes in the intermediate filament (IF) cytoskeleton and the lamin nucleo‐skeleton proceeds by phosphorylation of IF subunits, solubilization and reformation after cell division. Monoclonal AB J1‐31 recognizes a phosphoepitope found on GFAP and lamin B in astrocytes and thus is useful for exploring pathways to the reactive state. F98 (a.k.a. 9L) glioblastoma cells have been used to model gliosis in vitro. To test the hypothesis that a cAMP mediated pathway is involved, we added forskolin to confluent cultures of F98 cells and assayed for phosphorylation of lamin B by measuring binding of mAB J1‐31. Forskolin caused a 2X increase in mAB J1‐31 binding to nuclear structures also labeled by anti‐lamin B, and a lesser increase in cytoplasmic labeling of filaments also labeled by anti‐GFAP. Binding of anti‐lamin B and anti‐GFAP to their respective antigens was unaffected by forskolin. Inhibitors of PKA (H89) were not effective in preventing the forskolin induced phosphorylation of lamin B and GFAP, but inhibitors of Ca +2 influx (verapamil) and protein synthesis (cycloheximide) did significantly reduce the effect of forskolin.