Multiple Gi Proteins Participate in Nerve Growth Factor-Induced Activation of c-Jun N-terminal Kinases in PC12 Cells

Abstract

Nerve growth factor (NGF)-mediated activation of mitogen-activated protein kinases (MAPK) is critical for differentiation and apoptosis of PC12 cells. Since NGF employs stress-activated c-Jun N-terminal kinase (JNK) to regulate both programmed cell death and neurite outgrowth of PC12 cells, we examined NGF-regulated JNK activity and the role of G(i/o) proteins. Induction of JNK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). To discern the participation of various signaling intermediates, PC12 cells were treated with specific inhibitors prior to NGF challenge. NGF-elevated JNK activity was abolished by inhibitors of JNK, p38 MAPK, Src, JAK3 and MEK1/2. NGF-dependent JNK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i1-3) and Galpha(oA). Collectively, these studies indicate that NGF-dependent JNK activity may be mediated via G(i1-3) proteins, JAK3, Src, p38 MAPK and the MEK/ERK cascade.

Quantitative Proteomics Reveals Dynamic Interaction of c-Jun N-terminal Kinase (JNK) with RNA Transport Granule Proteins Splicing Factor Proline- and Glutamine-rich (Sfpq) and Non-POU Domain-containing Octamer-binding Protein (Nono) during Neuronal Differentiation

“…Without the inhibitor, NGF-induced neurite outgrowth was clearly visible at both 6 and 24 h after stimulation. When NGF was not replenished, neurites were largely degenerated after 48 h. Pretreatment of cells with SP600125 decreased NGF-induced neurite outgrowth by ϳ60% at 6 h and 24 h. This result is in line with previous studies showing that JNK is an important mediator for neuronal differentiation in PC12 cells (23,26,39,40). To analyze JNK activity, we quantified phosphorylation levels via Western blotting (Fig.…”

“…These proteins showed increased levels 0.5 h to 2 h after NGF treatment followed by a steady decrease over time below control levels at 24 h. This finding suggests that NGF-dependent JNK activation might be induced via multiple G-proteins. Indeed, it has been shown that several G-proteins are involved in JNK activation during PC12 cell differentiation (25,26) and that Rap1 is involved in NGF-induced PC12 cell differentiation (44,45).…”

“…This leads to a coordinated activation of downstream signaling cascades such as the Ras/Raf/Erk1-2, PI3K, and JNK pathways, resulting in increased expression of genes that are involved in neuronal differentiation (23,24). Furthermore, G-proteins including Ras, Rap, and Cdc42 have been shown to link receptor activity to downstream kinase activation (25)(26)(27).…”

G protein signaling controls the differentiation of multiple cell lineages

“…These include the findings that demonstrate the localization of GPCRs and their signaling components in lipid rafts and caveolae [63], dimerization of GPCRs [64], and trans-activation of receptor tyrosine kinases by GPCRs [65]. In the case of cell differentiation, examples of novel GPCR signaling mechanism include transactivation of TrkA by purinergic P2Y2 receptor [12], and mediation of partial NGF signaling by G i/o proteins [14,17,18]. With new discoveries in the signaling mechanisms of GPCRs and novel ligands for orphan receptors, we will undoubtedly appreciate the significance of G protein-linked signaling in the differentiation of various cell lineages and development of tissues and organs.…”

“…The induction of PI3K/Akt pathway by NGF is in part mediated through the Gβγ subunit released from activated G i/o , which promotes the phosphorylation of translation regulator tuberlin to enhance neuronal survival [14,15]. In a similar manner, G i/o mediates part of the NGF-induced p38 and JNK phosphorylations, which are required for neuronal differentiation [17,18]. These examples suggest bidirectional crosstalk between GPCR and NGF signaling in the regulation of neuronal survival and differentiation.…”