Activation of p21ras by nerve growth factor in embryonic sensory neurons and PC12 cells.

N F Ng,E M Shooter

doi:10.1016/s0021-9258(19)74395-6

Abstract

p21ras is believed to be involved in the neuronal differentiation of cells responsive to nerve growth factor (NGF). We show that NGF stimulates the activation of p21ras in embryonic sensory neurons and in PC12 cells. In the initial 5 min of exposure to NGF, the activation is concentration-dependent. In the sensory neurons and PC12 cells, the apparent maximal activation was reached at 50 and 10 ng/ml, respectively, with half-maximal activation at approximately 5 and 2-3 ng/ml, respectively. Kinetic analysis at low concentrations of NGF showed that p21ras activation slowly increases with time in both types of cells, while high concentrations result in rapid activation within 5 min. These results indicate that NGF regulates the activation state of p21ras in these cells and provides evidence suggesting that activation of p21ras is involved in NGF signal transduction. Treatment of PC12 cells with brain-derived neurotrophic factor or neurotrophin-3 (NT-3) failed to activate p21ras, suggesting that binding alone to p75LNGFR is insufficient for ras activation. Treatment with the kinase inhibitor, K252a, which inhibits the NGF tyrosine kinase receptor p140trk, abolished ras activation, suggesting that p140trk is the major mediator of p21ras activation by NGF.

Highlights

(Hempstead et al, 1991) binds NGF with high affinity (Kd = 10-'o-lO-") (Sutter et al, 1979a; Schechterand Bothwell, 1981; Landreth and Shooter,1980; Bernd and Greene, 1984)
We show that the low affinity NGF receptor ~ 7 5 d~oes' n~ot~appear to be involved in thisactivation and suggest that the high affinity NGF receptor involving p140frkis the major mediator of ras activation
The studies presented here show that NGFdependent embryonic sensory neuronsstimulate increased levels of active p21" in response to NGF

Summary

EXPERIMENTAL PROCEDURES

Poly-L-lysine, phosphate-free DME, protease inhibitors, and Protein A-Sepharose were from Sigma. Following digestion with 0.08% trypsin for 15min at 37 "C,the ganglia were diluted 1:3 with 5% heat-inactivated horse serum in DME. The neuron-enriched supernatant was pelleted and resuspended into phosphate-free 5% horse serum/DME, and transferred (0.5 X lo6)to 60-mm plates previously coated sequentially with poly-. In this precoating, plates were treated with poly-L-lysine (1 mg/ml) for at least 1h at 37 "C, washed three times with phosphate-buffered saline, and treated with laminin (10 pg/ml) for 14-16 h followed by three washes with phosphate-free DME. The PC12 cells (1 X IO6) were plated on 100-mm culture dishes 2 days prior to labeling, after which the media was replaced with phosphate-free 5% calf serum, 2.5% horse serum/DME containing the label. The guanine nucleotides GDP and GTPwere quantitated by liquid scintillation countingof the cellulose.The percentage of GTP to the total amount of GTP plus GDP was calculated, and ras activation was represented as percent of control

RESULTS

NGF Concentration

Findings

DISCUSSION