The small GTP-binding protein Ras appears to be required for transformation and differentiation induced by tyrosine kinases. The Ras requirement may be limited to a few tyrosine kinase-regulated signaling pathways or may be universal for all tyrosine kinase actions. Because both Ras and the microtubule-associated protein 2 kinases ERKi and ERK2 have been implicated in events that lead to neurite outgrowth, we explored the possibility that Ras and ERKs may lie on the same signaling pathway. Uzing PC-12 rat adrenal pheochromocytoma cell lines that contain a dominant inhibitory Ras mutant (S17N-RasH), we found that Ras was required for stimulation of the ERK cascade by nerve growth factor but apparently not by the heterotrimeric G protein activator AIF4 . Within this cascade, Ras appears to be upstream of an ERK activator, raising the intrigung psibility that Ras may directly regulate a serine/threonine protein kinase.

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A novel protein kinase activity present in nuclear and cytosolic extracts has been identified and partially purified as a consequence of its tight binding to and phosphorylation of the extracellular signal-regulated protein kinase (ERK) 3. This novel protein kinase is inactivated by treatment with phosphoprotein phosphatase 2A. The ERK3 protein kinase was immunologically distinct from mitogen-activated protein (MAP) kinase/ ERK kinases (MEK) 1 and 2 which phosphorylate the ERK3-related MAP kinases ERK1 and ERK2. This ERK3 kinase phosphorylated a single site on ERK3, Ser 189 , comparable to Thr 183 , one of the two activating phosphorylation sites of ERK2. To test the specificity of the ERK3 kinase, mutants of ERK3 and ERK2 were made in which the phosphorylated residues were exchanged. The double mutant S189T,G191Y ERK3, in which the phosphorylated residues from ERK2 replaced the comparable residues in ERK3, was phosphorylated by the ERK3 kinase but only on threonine. The ERK3 kinase did not phosphorylate ERK2 or ERK2 mutants. These findings indicate that although the ERK3 kinase is highly specific for ERK3, it does not recognize tyrosine, a feature that distinguishes it from MEKs that phosphorylate other ERK/MAP kinase family members.The ERK/MAP 1 kinase pathway is stimulated by numerous hormones and growth factors and its activation is associated with increased proliferative and differentiated functions of cells (1-5). The importance of intracellular processes thought to be regulated by the MAP kinases has focused attention on understanding the control of this pathway. The MAP kinase kinases, also known as MAP/ERK kinases or MEK1 and MEK2, originally discovered by Ahn and Krebs, are dual-specificity protein kinases known to activate the MAP kinases ERK1 and ERK2 in a highly selective manner (6 -8). The MAP kinases, on the other hand, are pleiotropic, phosphorylating many substrates throughout the cell (reviewed in Ref.3). Kinase cascades containing a MEK and an ERK/MAP kinase are present in multiple pathways in yeast and have been reiterated in mammalian cells (1, 9). Although mechanisms regulating the similar, but parallel mammalian pathways are less well characterized, the activation of a multipotential ERK/MAP kinase by a highly specific MEK is the common feature of all the related cascades.ERK1 and ERK2 are phosphorylated on two sites separated by a single residue in the phosphorylation lip at the mouth of their active sites (10, 11). Phosphorylation of both Tyr 185 and Thr 183 on ERK2 and comparable residues on ERK1, catalyzed by the dual specificity protein kinases, MEK1 and MEK2, is required for high activity (10, 12-15). Because of their exquisite specificity, MEK1 and MEK2 are not able to phosphorylate other MAP kinase-related enzymes such as Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) or p38 MAP kinase, even though the phosphorylation sites are in comparable positions in the sequence (1).Much less is known about the protein kinase ERK3. It was cloned in the same cDNA library scree...