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Phorbol Myristate Acetate and 8‐Bromo‐Cyclic AMP‐Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites
Author(s) -
Harrison Beth C.,
Mobley Philip L.
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb02073.x
Subject(s) - vimentin , glial fibrillary acidic protein , phosphorylation , phosphopeptide , activator (genetics) , gfap stain , microbiology and biotechnology , protein kinase a , protein kinase c , phorbol , chemistry , biochemistry , astrocyte , protein phosphorylation , biology , receptor , endocrinology , immunohistochemistry , immunology , central nervous system
Both the protein kinase C (PK‐C) activator, phorbol 12‐myristate 13‐acetate (PMA), and the cyclic AMP‐dependent protein kinase (PK‐A) activator, 8‐bromo‐cyclic AMP (8‐BR), have been shown to increase 32 P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8‐BR results in the morphological transformation of flat, polygonal‐shaped cells into stellate, process‐bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8‐BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32 P incorporation into all the peptide fragments that were phosphorylated by 8‐BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8‐BR treatment was restricted to serine residues in the N‐terminal domain of these proteins. Studies were also conducted to compare the two‐dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8‐BR with those produced when the proteins were phosphorylated with purified PK‐C or PK‐A. PK‐C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK‐C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells. The increased phosphorylation of vimentin observed in intact cells treated with 8‐BR and with PK‐A using purified vimentin as a substrate resulted in similar phosphopeptide maps. A common major phosphorylated peptide fragment was observed following phosphorylation of GFAP by PK‐A and in intact cells treated with 8‐BR; however, an additional phosphorylated fragment was observed with the purified kinase that was not observed in intact cells. These studies indicate that phosphorylation events mediated by PMA and 8‐BR converge to phosphorylate several of the same regions of GFAP and vimentin, and suggest that these effects are the direct result of the activation of PK‐C and PK‐A.