Regulation of hypoxic neuronal death signaling by neuroglobin.

The signal transduction pathways involved in neuronal death are not well understood. Neuroglobin (Ngb), a recently discovered vertebrate globin expressed predominantly in the brain, shows increased expression in neurons in response to oxygen deprivation and protects neurons from ischemic and hypoxic death. The mechanism of this neuroprotection is unclear. We examined the surface distribution of raft membrane microdomains in cortical neuron cultures during hypoxia using the raft marker cholera toxin B (CTx-B) subunit. Mechanistically, we demonstrate that hypoxia induces rapid polarization of somal membranes and aggregation of microdomains with the subjacent mitochondrial network. This signaling complex is formed well before neurons commit to die, consistent with an early role in death signal transduction. Neurons from Ngb-overexpressing transgenic (Ngb-Tg) mice do not undergo microdomain polarization or mitochondrial aggregation in response to, and are resistant to death from hypoxia. We link the protective actions of Ngb to inhibition of Pak1 kinase activity and Rac1-GDP-dissociation inhibitor disassociation, and inhibition of actin assembly and death-signaling module polarization.

Phosphorylation of RhoGDI by p21-activated kinase 1.

Rho GTPase activation is partially regulated at the level of guanine nucleotide dissociation inhibitors, or GDIs. The binding of Rho GTPases to GDIs has been shown to dramatically reduce the action of guanine nucleotide exchange factors (GEFs) to initiate Rho GTPase activation. The GDI-GTPase complex thus serves as a major point of regulation of Rho GTPase activity and function. It is likely that specific mechanisms exist to dissociate individual members of the Rho GTPase family from cytosolic Rho GDI complexes to facilitate the activation process. Such dissociation would likely be tightly coupled to GEF-mediated guanine nucleotide exchange and membrane association of the activated GTPase, resulting in effector binding and functional responses. Accumulating evidence suggests that the phosphorylation of either the Rho GTPases themselves and/or phosphorylation of GDIs might serve as a mechanism for regulating the formation and/or dissociation of Rho GTPase-GDI complexes. Indeed, the selective release of Rac1 from RhoGDI complexes induced by the p21-activated kinase-regulated phosphorylation of RhoGDI has been reported. We describe here methods for the analysis of RhoGDI phosphorylation and regulation by p21-activated kinase 1 (Pak1).