Protective Role of Phosphorylation in Turnover of Glial Fibrillary Acidic Protein in Mice

Abstract

Glial fibrillary acidic protein (GFAP), the principal intermediate filament (IF) protein of mature astrocytes in the CNS, plays specific roles in astrocyte functions. GFAP has multiple phosphorylation sites at its N-terminal head domain. To examine the role of phosphorylation at these sites, we generated a series of substitution mutant mice in which phosphorylation sites (Ser/Thr) were replaced by Ala, in different combinations.<i>Gfap</i>^{<i>hm3/hm3</i>} mice carrying substitutions at all five phosphorylation sites showed extensive decrease in both filament formation and amounts of GFAP.<i>Gfap^hm1/hm1</i> and<i>Gfap^hm2/hm2</i> mice, which carry substitutions at three of five sites and in different combinations, showed differential phenotypes. Although<i>Gfap^hm3/hm3</i> mice retained GFAP filaments in Bergmann glia in the cerebellum, the (<i>Gfap^hm3/hm3:Vim^−/−</i>) mice lacked GFAP filaments. Pulse-chase experiments of cultured astrocytes indicated that the Hm3-GFAP encoded by<i>Gfap^hm3</i> was unstable particularly in the absence of vimentin, another IF protein. These results revealed the role of phosphorylation in turnover of GFAP and a synergistic role of GFAP and vimentin in the dynamics of glial filaments. The data further suggest that each of the phosphorylated sites has a distinct impact on the dynamics of GFAP.