Inactivation of glycogen synthase kinase-3 (GSK3) by S 9 phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y 216 , on GSK3 is necessary for its activity; however, whether this site can be regulated in cells is unknown. Therefore we examined the regulation of Y 216 phosphorylation on GSK3 in models of neurodegeneration. Nerve growth factor withdrawal from differentiated PC12 cells and staurosporine treatment of SH-SY5Y cells led to increased phosphorylation at Y 216 , GSK3 activity, and cell death. Lithium and insulin, agents that lead to inhibition of GSK3 and adenoviralmediated transduction of dominant negative GSK3 constructs, prevented cell death by the proapoptotic stimuli. Inhibitors induced S 9 phosphorylation and inactivation of GSK3 but did not affect Y 216 phosphorylation, suggesting that S 9 phosphorylation is sufficient to override GSK3 activation by Y 216 phosphorylation. Under the conditions examined, increased Y 216 phosphorylation on GSK3 was not an autophosphorylation response. In resting cells, Y 216 phosphorylation was restricted to GSK3 present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y 216 -phosphorylated GSK3 selectively increased within the nucleus. In rats, Y 216 phosphorylation was increased in degenerating cortical neurons induced by ischemia. Taken together, these results suggest that Y 216 phosphorylation of GSK3 represents an important mechanism by which cellular insults can lead to neuronal death.A berrant cell death within the adult central nervous system is a key mechanism thought to underlie the pathology of several neurodegenerative diseases (1, 2). Survival growth factors protect neurons from a variety of proapoptotic stimuli, and one of the protective mechanisms has been attributed to the activation of the phosphoinositide-3 kinase signal transduction pathway (3). A downstream effector of this signaling pathway is Akt, a kinase that phosphorylates the serine͞threonine kinase GSK3 on S 9 to render it inactive (4, 5), a proposed mechanism by which neurons become resistant to apoptotic stimuli (6-8).A second regulatory site (Y 216 ), which lies within the activation loop between subdomains VII and VIII of the catalytic domain, has been identified on GSK3 and whose phosphorylation is necessary for functional activity (9). Dephosphorylation with a protein tyrosine phosphatase or mutation of Y 216 on GSK3 results in a dramatic decrease in activity (9). It is unclear whether Y 216 is a site for GSK3 autophosphorylation or whether a separate tyrosine kinase phosphorylates this site to activate GSK3 (10).In addition to its role in apoptosis, GSK3 hyperphosphorylates the microtubule-associated protein , a mechanism implicated in paired helical filament formation in Alzheimer's disease (11,12). Despite progress in defining growth factor-dependent pathways that regulate S 9 phosphorylation (13), little is known regarding the regulati...
Basic fibroblast growth factor (bFGF), a potent mitogen for many cell types, is expressed by vascular smooth muscle cells and plays a prominent role in the proliferative response to vascular injury. Basic FGF has also been implicated as a survival factor for a variety of quiescent or terminally differentiated cells. Autocrine mechanisms could potentially mediate both proliferation and cell survival. To probe such autocrine pathways, endogenous bFGF production was inhibited in cultured rat vascular smooth muscle cells by the expression of antisense bFGF RNA. Inhibition of endogenous bFGF production induced apoptosis in these cells independent of proliferation, and apoptosis could be prevented by exogenous bFGF but not serum or epidermal growth factor. The induction of apoptosis was associated with an inappropriate entry into S phase. These data demonstrate that interruption of autocrine bFGF signaling results in apoptosis of vascular smooth muscle cells, and that the mechanism involves disruption of normal cell cycle regulation.
This work investigates the role of cell adhesion molecules in development of synaptic connections and functions through a genetic approach. Fasciclin I (Fas I) is an insect glycoprotein capable of mediating homophilic cell adhesion. It has been shown that Fas I is expressed in motor nerve axons and terminals that innervate larval body-wall muscles in Drosophila. Immunohistochemical analysis of these motor nerve terminals has revealed that nerve terminal arborization, quantified by the numbers of the nerve terminal branches and varicosities, is enhanced in the null mutant fas ITE. In contrast, the number of branches and varicosities are reduced in larvae that overexpress the Fas I molecule resulting from additional copies of the fas I transgene in P(fas I+) or the chromosome duplication in Dp(fas I) mutants. Although arborization is altered, the overall stereotypical pattern of nerve terminal innervation of the body-wall muscle fibers is preserved in all the Fas I mutants examined. The voltage-clamp analysis of excitatory junctional currents (ejcs) at the neuromuscular junction indicates that the amplitude of ejcs is reduced in fas ITE, but increased in P(fas I+) and Dp(fas I) compared to that in wild-type larvae. Further electrophysiological analysis shows that the quantal content and the evoked frequency-dependent response are affected in these mutants, indicating a defective presynaptic function in addition to the anatomic abnormality. Therefore, the cell adhesion molecule Fas I may not be essential for target recognition and synaptogenesis at the larval neuromuscular junction, but may play a role in fine-turning nerve terminal arborization and possibly in modifying, directly or indirectly, development of presynaptic functions.
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