A Novel, Nerve Growth Factor-activated Pathway Involving Nitric Oxide, p53, and p21WAF1Regulates Neuronal Differentiation of PC12 Cells

Nitric oxide (NO) modulates nerve-growth-factor- (NGF-) mediated signaling and gene expression. In the present paper, the role of NO in NGF-mediated Akt activation in PC12 and IMR32 cells was investigated. Cells were treated with NGF (50 ng/mL) in the presence or absence of NO synthase (NOS) inhibitors and Akt phosphorylation assessed by western blot analysis. In both cell lines, Akt was phosphorylated within 15 min of NGF treatment. In PC12 cells, this level of phosphorylation was sustained for 60 min, while in IMR32 cells, the activation decreased after 30 min of NGF treatment. The nonselective NOS inhibitor Nω-nitro-L-arginine methylester (L-NAME; 20 mM) had no effect on NGF-mediated Akt phosphorylation in PC12 cells but in combination with NGF, the iNOS selective inhibitor s-methylisothiourea (S-MIU; 2.0 mM) maintained Akt phosphorylation up to 2 h. In IMR32 cells, both L-NAME and S-MIU prolonged the activation of Akt. Pretreatment with 50 μM U0126, a MAP kinase pathway inhibitor, also increased the activation of Akt in both cell lines. These data suggest that NO modulates the duration of phosphorylation of Akt in response to NGF and that this effect may, in part, be mediated by the effects of NO on the Ras-MAP kinase pathway.

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Adapter Protein SH2-Bβ Undergoes Nucleocytoplasmic Shuttling: Implications for Nerve Growth Factor Induction of Neuronal Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.24.9.3633-3647.2004 ◽

2004 ◽

Vol 24 (9) ◽

pp. 3633-3647 ◽

Cited By ~ 24

Author(s):

Linyi Chen ◽

Christin Carter-Su

Keyword(s):

Plasma Membrane ◽

Nerve Growth Factor ◽

Growth Factor ◽

Neurite Outgrowth ◽

Neuronal Differentiation ◽

Pc12 Cells ◽

Nuclear Export ◽

Adapter Protein ◽

Wild Type ◽

Nerve Growth

ABSTRACT The adapter protein SH2-B has been shown to bind to activated nerve growth factor (NGF) receptor TrkA and has been implicated in NGF-induced neuronal differentiation and the survival of sympathetic neurons. However, the mechanism by which SH2-B enhances and maintains neurite outgrowth is unclear. We examined the ability of truncation mutants to regulate neuronal differentiation and observed that certain truncation mutants localized in the nucleus rather than in the cytoplasm or at the plasma membrane as reported for wild-type SH2-Bβ. Addition of the nuclear export inhibitor leptomycin B caused both overexpressed wild-type and endogenous SH2-Bβ to accumulate in the nucleus of both PC12 cells and COS-7 cells as did deletion of a putative nuclear export sequence (amino acids 224 to 233) or mutation of two critical lysines in that sequence. Deleting or mutating the nuclear export signal caused SH2-Bβ to lose its ability to enhance NGF-induced differentiation of PC12 cells. Neither the NGF-induced phosphorylation of ERKs 1 and 2 nor their subcellular distribution was altered in PC12 cells stably expressing the nuclear export-defective SH2-Bβ(L231A, L233A). These data provide strong evidence that SH2-Bβ shuttles constitutively between the nucleus and cytoplasm. However, SH2-Bβ needs continuous access to the cytoplasm and/or plasma membrane to participate in NGF-induced neurite outgrowth. These data also suggest that the stimulatory effect of SH2-Bβ on NGF-induced neurite outgrowth of PC12 cells is either downstream of ERKs or via some other pathway yet to be identified.

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