Phorbol myristate acetate and 8-bromo-cyclic AMP-induced phosphorylation of glial fibrillary acidic protein and vimentin in astrocytes: comparison of phosphorylation sites.
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 32P 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 32P 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Harrison
B C
BC
Department of Pharmacology, University of Texas Health Science Center, San Antonio 78284.
Mobley
P L
PL
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002478:Cells, Cultured; D005904:Glial Fibrillary Acidic Protein / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014746:Vimentin / Q000378:metabolism*
D000596:Amino Acids; D005904:Glial Fibrillary Acidic Protein; D014746:Vimentin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D011494:Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate