Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal‐Associated Intermediate Filament Protein Kinase Activity in Astrocytes

Abstract

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping phosphoproteins in Mycoplasma genitalium and Mycoplasma pneumoniae

“…This may be analogous to eukaryotic cells where phosphorylation and dephosphorylation of cytoskeletal elements has been associated with changes in cell morphology [49]. …”

In vitro differentiation of cultured human CD34+ cells into astrocytes

“…[11] All Type III intermediate filament proteins like GFAP are involved in the cytoskeleton structure and provide mechanical support to astrocytes [12] and GFAP has been used as a marker in determining the stellate morphology of astrocytes, which is maintained by the phosphorylation carried out by specific serine/ threonine kinases. [13][14][15] Thus, differentiated astrocytes generated from autologous stem cells probably may be used in the treatment of some of the neurodegenerative diseases and spinal cord injuries. The present study is aimed in the development of astrocytes from human CD34+ stem cells.…”

“…The kinetics of serine/threonine kinase was determined [13] by using ATP 2 mM-14 mM and 250 μg/mL lysozyme, 0.1 M Tris HCl pH 7.5 and cytosolic fraction as an enzyme source and incubated at 37°C for 10 min. Then, the reaction was stopped by the addition of 0.5 mL of ferric chloride reagent; further 1 mL of acidified molybdate color reagent was added and reduced phospho-molybdenum complex was measured at 820 nm in cyber lab spectrophotometer, USA.…”

Heterologous expression of acetylcholinesterase affects proliferation and glial cytoskeleton of adherent chicken retinal cells

“…9), possibly representing a 60-kDa form of vimentin, as well as an N-terminal fragment. Intermediate filament kinase(s) can phosphorylate intact vimentin (60 kDa), as well as its N-terminal (41-kDa) and C-terminal (18-kDa) fragments (Harrison and Mobley 1992). This phosphorylation -mediated by cdc2-kinase during mitosis, or other kinases such as PKC -can reorganize cytoplasmic intermediary filaments (Takai et al 1996).…”