Signal transduction is controlled both by regulation of enzyme activation and by organization of enzymatic complexes with nonenzymatic adapters, scaffolds, and anchor proteins. The extracellular signal-regulated kinase (ERK) cascade is one of several evolutionarily conserved mitogen-activated protein (MAP) kinase cascades important in the regulation of growth, apoptosis, and differentiation. A two-hybrid screen was conducted to identify nonenzymatic components of this signaling cascade that might be important in regulating its activity. A protein called MP1 (MEK Partner 1) was identified that bound specifically to MEK1 and ERK1 and facilitated their activation. When overexpressed in cultured cells, MP1 enhanced activation of ERK1 and activation of a reporter driven by the transcription factor Elk-1. Expression of MP1 in cells increased binding of ERK1 to MEK1. MP1 apparently functions as an adapter to enhance the efficiency of the MAP kinase cascade.

Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a β1-integrin (probably αVβ1) and αVβ5. When MCF-7 cells were transfected to express αVβ3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of αVβ3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

Activation of the Ras–MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane–proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf–MEK1–MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogenactivated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-⌬19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-⌬19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serumstarved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.The extracellular signal-regulated protein kinases (ERKs) are ubiquitous protein kinases that function downstream of the ras oncogene and are involved in cell proliferation, movement, and differentiation (35). Ras is activated in response to agonist stimulation and recruits Raf protein kinases to the plasma membrane (28,34,45), where Raf undergoes activation by a mechanism that is incompletely understood. Raf proteins phosphorylate and activate mitogen-activated protein kinase or ERK kinases (MEKs), which phosphorylate the two ERK proteins, ERK1 and ERK2, on a TEY sequence in the catalytic domain, resulting in ERK activation (24, 37). MEKs can serve as cytoplasmic anchors for the ERKs through a direct binding interaction, holding the ERKs in the cytoplasm at times when the signaling pathway is inactive (21). Stimulation of the pathway results in the phosphorylation of the ERKs and their dimerization (26) and translocation to the nucleus and other sites of action (10,23,29), where they phosphorylate the various substrates required for biological responses. Sustained ERK activation and nuclear translocation are required for S-phase entry and proliferation of fibroblasts (6, 36), and both aspects are dependent upon the integration of adhesion and growth factor signals (2, 40, 47). However, the mechanisms by which adhesion and growth factor signals are integrated to modify ERK function are presently unclear.MEK proteins bind directly to the ERKs through a region in the N terminus of MEK (3,4,21). However, other sequ...

To investigate possible involvement of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, we have used indirect immunofluorescence with a highly specific phospho-MAP kinase antibody and found that a portion of the active MAP kinase is localized at kinetochores, asters, and the midbody during mitosis. Although the aster labeling was constant from the time of nuclear envelope breakdown, the kinetochore labeling first appeared at early prometaphase, started to fade during chromosome congression, and then disappeared at midanaphase. At telophase, active MAP kinase localized at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, we identified the kinetochore motor protein CENP-E as a candidate mitotic substrate for MAP kinase. CENP-E was phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules and was found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules.