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Evidence for multiple cAMP‐dependent pathways for activation of F98 cells to the reactive state
Author(s) -
Kane Sarah Jo,
García Dana M,
Koke Joseph R
Publication year - 2011
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.25.1_supplement.957.1
Subject(s) - forskolin , nuclear lamina , lamin , glial fibrillary acidic protein , protein kinase a , microbiology and biotechnology , phosphoserine , epitope , biology , cytoplasm , intracellular , monoclonal antibody , chemistry , immunoprecipitation , phosphorylation , biochemistry , nuclear protein , cell culture , in vitro , nucleus , antibody , transcription factor , immunohistochemistry , genetics , immunology , serine , gene
Treating F98 cells in vitro with forskolin induces a reactive response as demonstrated by increased anti‐glial fibrillary acidic protein (GFAP) and monoclonal antibody (mAB) J1‐31 immunostaining. mAB J1‐31 may recognize a phosphoserine‐containing epitope on lamins and GFAP. Intracellular cAMP pathways include cAMP‐dependent protein kinase A (PKA), exchange protein activated by cAMP (Epac), and cyclic nucleotide‐gated channels. Using H89 and PKI to inhibit PKA, we observe decreased labeling of cytoplasmic IFs by mAB J1‐31 in forskolin‐treated F98 cells. Conversely, the same treatment appears to increase both the intensity and number of nuclear epitopes. In F98 cells stimulated with the PKA‐specific analog of cAMP, 6‐Bnz‐cAMP, we find no increase in nuclear labeling by mAB J1‐31, but a striking increase in mAB J1‐31‐labeling of cytoplasmic structures. Using anti‐phosphoserine to probe extracts of forskolin‐stimulated F98 cells separated by SDS‐PAGE, we show PKI was effective in reducing phosphorylation of multiple proteins, including bands at the positions expected for lamins and GFAP which are also labeled by mAB J1‐31. Thus it appears that cAMP acts on nuclear lamins via an Epac (GEF) pathway and on cytoplasmic IFs (GFAP) via a PKA mediated pathway. This work is supported by the Biology Department at Texas State University‐San Marcos and NSF DBI‐0821252 to JRK and DMG.