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 factors^1-4. 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 phenotype^6,7. 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protein S6 kinase-II (refs 9,10), and they phosphorylate identical threonine residues on myelin basic protein (our unpublished results) to those phosphorylated by other MAPKs^11,12. 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.