Abstract
Exposure of growing neurons to thrombin or semaphorin 3A stimulates a receptor-mediated signaling cascade that results in collapse of their growth cones. This collapse response necessitates eicosanoid production, as we have shown earlier. The present report investigates whether and which protein kinase C (PKC) isoforms may be activated by such eicosanoids. To examine these questions, we isolated growth cones from fetal rat brain and tested whether thrombin or the eicosanoid, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), could activate endogenous growth cone PKC. We show that both thrombin and 12(S)-HETE stimulate the phosphorylation of the myristoylated alanine-rich protein kinase C substrate, an 87-kDa adhesion site protein. Furthermore, we show both with immunoprecipitated and with recombinant PKC that 12(S)-HETE activation is selective for the epsilon isoform and does not require accessory proteins. Last, we demonstrate that PKC activation is necessary for thrombin-induced growth cone collapse. These data indicate that eicosanoid-mediated repellent effects result from the direct and selective activation of PKCepsilon and suggest the involvement of myristoylated alanine-rich protein kinase C substrate phosphorylation in growth cone collapse.
Highlights
Exposure of growing neurons to thrombin or semaphorin 3A stimulates a receptor-mediated signaling cascade that results in collapse of their growth cones
We isolated growth cones from fetal rat brain and tested whether thrombin or the eicosanoid, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), could activate endogenous growth cone PKC. We show that both thrombin and 12(S)-HETE stimulate the phosphorylation of the myristoylated alanine-rich protein kinase C substrate, an 87-kDa adhesion site protein
Our experiments identify myristoylated alanine-rich protein kinase C substrate (MARCKS) as the primary substrate of thrombin- and 12(S)-HETE-activated phosphorylation in growth cone adhesion sites and show that PKC activation is necessary for growth cone collapse
Summary
Exposure of growing neurons to thrombin or semaphorin 3A stimulates a receptor-mediated signaling cascade that results in collapse of their growth cones. We isolated growth cones from fetal rat brain and tested whether thrombin or the eicosanoid, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), could activate endogenous growth cone PKC We show that both thrombin and 12(S)-HETE stimulate the phosphorylation of the myristoylated alanine-rich protein kinase C substrate, an 87-kDa adhesion site protein. We demonstrate that PKC activation is necessary for thrombin-induced growth cone collapse These data indicate that eicosanoid-mediated repellent effects result from the direct and selective activation of PKC⑀ and suggest the involvement of myristoylated alanine-rich protein kinase C substrate phosphorylation in growth cone collapse. Our experiments identify MARCKS as the primary substrate of thrombin- and 12(S)-HETE-activated phosphorylation in growth cone adhesion sites and show that PKC activation is necessary for growth cone collapse
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