| Astrocytes undergo dramatic changes in morphology in response to disease states and brain trauma. Because many of the functions of astrocytes appear to be dependent on their shape and the position of their processes, it is important to determine the mechanisms underlying alterations in cell morphology in order to ultimately understand the functions of these cells. The transformation of flat, polygonal-shaped cells into stellate, process-bearing cells is produced upon treatment of cultured astrocytes with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PK-C), or with cyclic AMP analogs which activate the cyclic AMP-dependent protein kinase (PK-A). The studies of this dissertation were designed to determine if the effects of PMA on astrocyte morphology were mediated by PK-C, and to determine if activators of PK-C and PK-A elicited similar morphological effects through the phosphorylation of common protein substrates. It was demonstrated that the morphological effects of PMA correlated with the activation of PK-C. In addition, longer incubation times with PMA resulted in the loss of PK-C activity and a failure of a second treatment with PMA to elicit changes in astrocyte morphology or protein phosphorylation. These studies demonstrated that the effects of PMA on astrocyte morphology were mediated by PK-C. Phosphoproteins phosphorylated in response to the activation of both PK-C and PK-A included the intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin. Common tryptic fragments of GFAP and vimentin were phosphorylated upon treatment with 8-bromo cyclic AMP and PMA, suggesting that the phosphorylation of these proteins may be a point of convergence between the PK-C and PK-A pathways. In addition, a cytoskeletal-associated kinase was discovered which also phosphorylated GFAP and vimentin. The kinase phosphorylated GFAP and vimentin in the presence of magnesium alone, with an even greater enhancement of phosphorylation in the presence of calcium/calmodulin. The kinase phosphorylated different tryptic fragments of GFAP and vimentin compared to PK-C and PK-A, and also phosphorylated different fragments of GFAP and vimentin depending on the presence or absence of calcium/calmodulin, suggesting that two distinct kinase activities may be present in the preparation. |
