Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases

MITOGEN activated protein (MAP) kinases (MAPKs) are a family of protein-serine/threonine kinases activated as an early intracellular response to a variety of hormones and growth factors1-4. They are unique in requiring both serine/threonine and tyrosine phosphorylation to become active5 and are the only examples of protein-serine/threonine kinases activated by tyrosine phosphorylation. Nerve growth factor (NGF) promotes differentiation of phaeochromocytoma (PC12) cells, which respond by conversion within hours from a chromaffin-like to a sympathetic neuron-like phenotype6,7. NGF stimulation of PC 12 cells increases the activity of two protein kinases by > 20-fold within minutes8, both strikingly similar to MAPKs. They are inactivated by either protein-tyrosine phosphatases or the protein-serine/threonine phosphatase termed protein phosphatase 2A (ref. 8), they activate protein S6 kinase-II (refs 9,10), and they phosphorylate identical threonine residues on myelin basic protein (our unpublished results) to those phosphorylated by other MAPKs11,12. Immunological data13 indicate that these protein kinases, termed peak-I and peak-II (Fig. la) are probably ERK2 and ERK1, respectively, two widely expressed MAPK isoforms13. Here we identify the 'MAP kinase kinases' (MAPKKs) in PC12 cells which are activated by NGF and report that MAPKKs are dependent on serine/threonine phosphorylation for activity and promote phosphorylation of serine/threonine and tyrosine residues on MAPKs.