Nerve Growth Factor Activation of the Extracellular Signal-Regulated Kinase Pathway Is Modulated by Ca²⁺and Calmodulin

Abstract

Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner.

ACKNOWLEDGMENTS

This work was funded by the Comisión Interministerial de Ciencia y Tecnologia through the Plan Nacional de Salud y Farmacia (contract no. 97-0094), Telemarató de TV3 (Edició 1997: Malalties Degeneratives Hereditàries), EU Biotech Program (contract no. BIO4-CT96-0433), and Ajuntament de Lleida. J. Egea is a predoctoral fellow of the Generalitat de Catalunya. S. Peiró is a predoctoral fellow of the Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS).

We thank colleagues in our laboratory for criticism and technical support. The assistance of Dionisio Martin-Zanca, Martí Aldea, and Carme Gallego in many aspects of our work is especially acknowledged. We thank the indicated persons for the generous gifts of the following antibodies: anti-Grb2 (J. Ureña), anti–pan-Ras (O. Bachs and N. Agell), and anti–pan-Trk (203) (D. Martin-Zanca). We thank G. Capellà and C. García for the generous gift of EGF. We also thank F. McKenzie, O. Bachs, and N. Agell for the generous gift of the prokaryotic expression vector containing the GST-RBD construct; G. M. Cooper and A. Aranda for the M-M17-26 cells; and C. E. Marshall and A. López-Rivas for the Glu217-Glu221 MAKK1 mutant construct. We are grateful to J. Fibla for purification of NGF. We thank Isabel Sánchez and Roser Pané for expert technical assistance and A. Porras for helpful technical comments in the PI 3-kinase and Ras GTP loading assays.