During CNS development, neuroblasts proliferate within germinal zones of the neuroepithelium, and then migrate to their final positions. Although many neurons are thought to migrate along processes of radial glial fibers, increasing evidence suggests environmental factors also influence nerve cell movement. Extracellular matrix molecules are thought to be involved in guiding neuronal migration, and molecules such as NGF and GABA exert trophic effects on immature neurons. The nature of the signals that initiate and direct neuroblast migration, however, is unknown. In vitro, NGF and GABA promote neurite outgrowth from cultured cells, and NGF induces axonal chemotaxis (directed migration along a chemical gradient). At earlier developmental stages, these molecules could influence neuroblast movement. Therefore, we investigated whether these molecules induce embryonic neuronal migration. Using an in vitro microchemotaxis assay, we show that rat embryonic spinal cord neurons migrate toward picomolar NGF and femtomolar GABA beginning at embryonic day 13 (E13). Cells exhibit chemotactic responses to NGF while GABA stimulates chemokinesis (increased random movement). GABA effects are mimicked by muscimol and inhibited by bicuculline and picrotoxin, suggesting GABA motility signals are mediated by GABA receptor proteins. Expression of GABA receptors by embryonic cord cells has been previously reported (Mandler et al., 1990; Walton et al., 1993). We used polymerase chain reaction analysis to demonstrate the presence of NGF and trk mRNA in E13 and E14 cord cells, indicating the cells express message for both NGF and high-affinity NGF receptors. Immunohistochemistry of E13 spinal cord sections indicates that NGF and GABA colocalize in fibers close to the target destinations of migrating neurons, suggesting diffusible gradients of these molecules provide chemoattractant signals to migratory cells. Thus, in vitro, neuroblast migration is induced by specific signaling molecules that are present in the developing spinal cord, and may stimulate migration of embryonic neurons prior to synaptogenesis.

Protein kinase C (PKC) is a multigene family of serine/threonine kinases that are central to many signal transduction pathways. Among the PKC isozymes, only PKCe has been reported to exhibit full oncogenic potential. PKCe also displays unique substrate specificity and intracellular localization. To examine the interrelationship between the biological effects and domain structure of PKCe, NIH 3T3 cells were stably transfected to overexpress different epitopetagged fragments of PKCe. The overexpressed proteins each contain the E-tag peptide at the C terminus to allow ready detection with an antibody specific for the tag. The holo-PKCe was found to localize with the Golgi network and other compartments, whereas the zinc-finger domain localized exclusively at the Golgi. Golgi-specific glycosaminoglycan sulfation was strongly inhibited in cells overexpressing either holo-PKCE or its zinc-finger domain, while the secretion of sulfated glycosaminoglycans into the medium was impaired in cells expressing the PKCE zinc-finger domain. Thus, these results suggest that PKCE may be involved in specifically regulating Golgi-related processes. Further, the results indicate that PKCE domains other than the kinase domain may also have biological activity and that the zinc-finger domain may function as a subcellular localization signal.Protein kinase C (PKC) consists of a family of more than 10 closely related phospholipid-dependent protein phosphotransferase isozymes (1, 2). The various PKC isozymes show considerable diversity in their domain structure, regulatory properties, and biological effects (1, 2). Although overexpression of most of the PKC isozymes has some effect on the morphology and growth characteristics of cells, only PKCs has been reported to exhibit full oncogenic potential (3, 4). PKCs also has been implicated in regulating other biological processes, such as antiviral resistance (5), neuropeptide signal transduction (6), and transporter regulation (7). PKCS has unique substrate specificity (8), and a portion of PKCs can always be detected in a membrane-associated state (3, 4, 7). NIH 3T3 cells have been reported to contain PKCs (2, 9). To study the interrelationship of the function, subcellular localization, and domain organization of PKCs, we used NIH 3T3 cell lines overexpressing holo-PKCs and various truncated derivatives of PKCs. For purposes of uniform detection, the C-terminal 12 amino acids of PKCS were added to the C termini of all constructs as an antibody epitope tag. The zinc-finger domain of PKCs was found to contain all the information necessary for exclusive localization to the Golgi. Further, sulfate uptake and Golgi-specific sulfation of glycosaminoglycan (GAG) chains were inhibited in cell lines overexpressing either PKCs or its zinc-finger domain, indicating that PKCs is involved in modulating Golgi function.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 s...

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