PHORBOL ESTERS AND PKC SIGNALING REGULATE PROLIFERATION, VIMENTIN CYTOSKELETON ASSEMBLY AND GLUTAMINE-SYNTHETASE ACTIVITY OF CHICK-EMBRYO CEREBRUM ASTROCYTES IN CULTURE

We have recently shown that expression of specific protein kinase C (P KC) isoforms correlates with cell fate in neural chicken embryo cells. Therefore we investigated the effects of PKC activation by phorbol es ters on acquisition of the astrocytic phenotype, using cultured embryo nic cortical astrocytes, derived from 15-day-old chick embryos (E15CH) , as a model. Short term treatment with the phorbol ester 12-tetradeca noylphorbol-13-acetate (TPA), which activates PKC-alpha/beta in E15CH, caused association of PKC with the cytoskeleton. In vitro kinase assa ys of cytoskeleton-associated PKC demonstrated phosphorylation of many cytoskeletal proteins. Phosphorylation was blocked by protein kinase inhibitors (H8), and enhanced by phosphatase inhibitors (calyculin A). Among these PKC substrates, a most prominent 60-kDa protein was ident ified as vimentin. Assembly of vimentin into the cytoskeleton depends on cell type and state of differentiation. To establish that TPA (PKC) regulates assembly of vimentin into the cytoskeleton of astrocytes, w e used pulse-chase (20/5 min) labeling with [S-35]methionine, and immu noprecipitations with an anti-vimentin mAb from extractable and cytosk eletal fractions. These studies revealed that 20 min treatment with TP A leads to a 3-fold increase in the rate of newly synthesized full-len gth vimentin assembly (posttranslational assembly). Furthermore, TPA i ncreased cotranslational assembly of vimentin. The protein kinase A ac tivator forskolin, did not have such effects on vimentin assembly. Lon g-term TPA treatment, which correlates with a prolonged phospholipase D (PLD) activation, was mitogenic and caused dramatic changes in the m orphology of astrocytes. In addition these fibrous, polarized astrocyt es had decreased activity of the astrocyte specific enzyme, glutamine synthetase, but had increased abundance of vimentin protein. These stu dies provide biochemical evidence on acquisition of a different astroc ytic phenotype after activation of the PKC/PLD pathway, in the chick e mbryo. Therefore PKC and PLD activation is pivotal for the acquisition and maintenance of phenotypes in chick embryonic astrocytes.