Melanie H. Cobb

Mitogen-activated protein kinase pathways.

Nearly all cell surface receptors utilize one or more of the mitogen-activated protein kinase cascades in their repertoire of signal transduction mechanisms. Recent advances in the study of such cascades include the cloning of genes encoding novel members of the cascades, further definition of the roles of the cascades in responses to extracellular signals, and examination of cross-talk between different cascades.

ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF

We recently described the purification and cloning of extracellular signal-regulated kinase 1 (ERK1), which appears to play a pivotal role in converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events. We now describe cloning and characterization of two ERK1-related kinases, ERK2 and ERK3, and provide evidence suggesting that there are additional ERK family members. At least two of the ERKs are activated in response to growth factors; their activations correlate with tyrosine phophorylation, but also depend on additional modifications. Transcripts corresponding to the three cloned ERKs are distinctly regulated both in vivo and in a differentiating cell line. Thus, this family of kinases may serve as intermediates that depend on tyrosine phosphorylation to activate serine/threonine phosphorylation cascades. Individual family members may mediate responses in different developmental stages, in different cell types, or following exposure to different extracellular signals.

MAP kinase pathways.

MAP kinases help to mediate diverse processes ranging from transcription of protooncogenes to programmed cell death. More than a dozen mammalian MAP kinase family members have been discovered and include, among others, the well studied ERKs and several stress-sensitive enzymes. MAP kinases lie within protein kinase cascades. Each cascade consists of no fewer than three enzymes that are activated in series. Cascades convey information to effectors, coordinates incoming information from other signaling pathways, amplify signals, and allow for a variety of response patterns. Subcellular localization of enzymes in the cascades is an important aspect of their mechanisms of action and contributes to cell-type and ligand-specific responses. Recent findings on these properties of MAP kinase cascades are the major focus of this review.

Activation mechanism of the MAP kinase ERK2 by dual phosphorylation.

The structure of the active form of the MAP kinase ERK2 has been solved, phosphorylated on a threonine and a tyrosine residue within the phosphorylation lip. The lip is refolded, bringing the phosphothreonine and phosphotyrosine into alignment with surface arginine-rich binding sites. Conformational changes occur in the lip and neighboring structures, including the P+1 site, the MAP kinase insertion, the C-terminal extension, and helix C. Domain rotation and remodeling of the proline-directed P+1 specificity pocket account for the activation. The conformation of the P+1 pocket is similar to a second proline-directed kinase, CDK2-CyclinA, thus permitting the origin of this specificity to be defined. Conformational changes outside the lip provide loci at which the state of phosphorylation can be felt by other cellular components.

Phosphorylation of the MAP Kinase ERK2 Promotes Its Homodimerization and Nuclear Translocation

The MAP kinase ERK2 is widely involved in eukaryotic signal transduction. Upon activation it translocates to the nucleus of the stimulated cell, where it phosphorylates nuclear targets. We find that nuclear accumulation of microinjected ERK2 depends on its phosphorylation state rather than on its activity or on upstream components of its signaling pathway. Phosphorylated ERK2 forms dimers with phosphorylated and unphosphorylated ERK2 partners. Disruption of dimerization by mutagenesis of ERK2 reduces its ability to accumulate in the nucleus, suggesting that dimerization is essential for its normal ligand-dependent relocalization. The crystal structure of phosphorylated ERK2 reveals the basis for dimerization. Other MAP kinase family members also form dimers. The generality of this behavior suggests that dimerization is part of the mechanism of action of the MAP kinase family.

ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation.

Induction of the human c‐fos proto‐oncogene by mitogens depends on the formation of a ternary complex by p62TCF with the serum response factor (SRF) and the serum response element (SRE). We demonstrate that Elk‐1, a protein closely related to p62TCF in function, is a nuclear target of two members of the MAP kinase family, ERK1 and ERK2. Phosphorylation of Elk‐1 increases the yield of ternary complex in vitro. At least five residues in the C‐terminal domain of Elk‐1 are phosphorylated upon growth factor stimulation of NIH3T3 cells. These residues are also phosphorylated by purified ERK1 in vitro, as determined by a combination of phosphopeptide sequencing and 2‐D peptide mapping. Conversion of two of these phospho‐acceptor sites to alanine impairs the formation of ternary complexes by the resulting Elk‐1 proteins. Removal of these serine residues also drastically diminishes activation of the c‐fos promoter in epidermal growth factor‐treated cells. Analogous mutations at other sites impair activation to a lesser extent without affecting ternary complex formation in vitro. Our results indicate that phosphorylation regulates ternary complex formation by Elk‐1, which is a prerequisite for the manifestation of its transactivation potential at the c‐fos SRE.

The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation

Interaction with SV40 small tumor antigen (small t) compromised the ability of multimeric protein phosphatase 2A to inactivate the mitogen-activated protein kinase ERK1 and the mitogen-activated protein kinase kinase MEK1. Transient expression of small t in CV-1 cells activated MEK and ERK but did not affect Raf activity. Small t stimulated the growth of quiescent CV-1 cells almost as effectively as did serum. Coexpression of kinase-deficient ERK2 blocked most, but not all, of the proliferation caused by small t. Activation of the mitogen-activated protein kinase pathway and stimulation of cell growth were dependent on the interaction of small t with protein phosphatase 2A. These findings indicate that SV40 small t is capable of inducing cell growth through blockade of protein phosphatase and deregulation of the mitogen-activated protein kinase cascade.

c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases.

c-Jun transcriptional activity is stimulated by phosphorylation at two N-terminal sites: Ser-63 and -73. Phosphorylation of these sites is enhanced in response to a variety of extracellular stimuli, including growth factors, cytokines, and UV irradiation. New members of the mitogen-activated protein (MAP) kinase group of signal-transducing enzymes, termed JNKs, bind to the activation domain of c-Jun and specifically phosphorylate these sites. However, the N-terminal sites of c-Jun were also suggested to be phosphorylated by two other MAP kinases, ERK1 and ERK2. Despite these reports, we find that unlike the JNKs, ERK1 and ERK2 do not phosphorylate the N-terminal sites of c-Jun in vitro; instead they phosphorylate an inhibitory C-terminal site. Furthermore, the phosphorylation of c-Jun in vivo at the N-terminal sites correlates with activation of the JNKs but not the ERKs. The ERKs are probably involved in the induction of c-fos expression and thereby contribute to the stimulation of AP-1 activity. Our study suggests that two different branches of the MAP kinase group are involved in the stimulation of AP-1 activity through two different mechanisms.

Pharmacological inhibitors of MAPK pathways

Mitogen-activated protein kinases [MAPKs, also called extracellular signal-regulated kinases (ERKs)] are constituents of numerous signal transduction pathways, and are activated by protein kinase cascades. Intense efforts are under way to develop and evaluate compounds that target components of MAPK pathways. In this article, the current status of inhibitors of MAPK pathways will be presented with a focus on the properties of small-molecule inhibitors of p38, MEK1 and MEK2 protein kinases. Several of these inhibitors are effective in animal models of disease and have advanced to clinical trials for the treatment of inflammatory diseases and cancer. The clinical utility of specifically targeting a subset of cellular signaling cascades and signaling cascades that regulate pleiotropic cellular processes are being evaluated. The results of these efforts have broad implications for the treatment of many diseases.

Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins.

Mitogens promote cell growth through integrated signal transduction networks that alter cellular metabolism, gene expression and cytoskeletal organization. Many such signals are propagated through activation of MAP kinase cascades partly regulated by upstream small GTP‐binding proteins. Interactions among cascades are suspected but not defined. Here we show that Rho family small G proteins such as Rac1 and Cdc42hs, which activate the JNK/SAPK pathway, cooperate with Raf‐1 to activate the ERK pathway. This causes activation of ternary complex factors (TCFs), which regulate c‐fos gene expression through the serum response element. Examination of ERK pathway kinases shows that neither MEK1 nor Ras will synergize with Rho‐type proteins, and that only MEK1 is fully activated, indicating that MEKs are a focal point for cross‐cascade regulation. Rho family proteins utilize PAKs for this effect, as expression of an active PAK1 mutant can substitute for Rho family small G proteins, and expression of an interfering PAK1 mutant blocks Rho‐type protein stimulation of ERKs. PAK1 phosphorylates MEK1 on Ser298, a site important for binding of Raf‐1 to MEK1 in vivo. Expression of interfering PAK1 also reduces stimulation of TCF function by serum growth factors, while expression of active PAK1 enhances EGF‐stimulated MEK1 activity. This demonstrates interaction among MAP kinase pathway elements not previously recognized and suggests an explanation for the cooperative effect of Raf‐1 and Rho family proteins on cellular transformation.