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(1/76) Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control.

The activity of the catalytic domain of the orphan MAP kinase ERK5 is increased by Ras but not Raf-1 in cells, which suggests that ERK5 might mediate Raf-independent signaling by Ras. We found that Raf-1 does contribute to Ras activation of ERK5 but in a manner that does not correlate with Raf-1 catalytic activity. A clue to the mechanism of action of Raf-1 on ERK5 comes from the observation that endogenous Raf-1 binds to endogenous ERK5, suggesting the involvement of regulatory protein-protein interactions. This interaction is specific because Raf-1 binds only to ERK5 and not ERK2 or SAPK. Finally, we demonstrate the ERK5/MEK5 pathway is required for Raf-dependent cellular transformation and that a constitutively active form of MEK5, MEK5DD, synergizes with Raf to transform NIH 3T3 cells. These observations suggest that ERK5 plays a large role in Raf-1-mediated signal transduction.  (+info)

(2/76) MEKK3 directly regulates MEK5 activity as part of the big mitogen-activated protein kinase 1 (BMK1) signaling pathway.

Big mitogen-activated protein (MAP) kinase (BMK1), also known as ERK5, is a member of the MAP kinase family whose cellular activity is elevated in response to growth factors, oxidative stress, and hyperosmolar conditions. Previous studies have identified MEK5 as a cellular kinase directly regulating BMK1 activity; however, signaling molecules that directly regulate MEK5 activity have not yet been defined. Through utilization of a yeast two-hybrid screen, we have identified MEKK3 as a molecule that physically interacts with MEK5. This interaction appears to take place in mammalian cells as evidenced by the fact that cellular MEK5 and MEKK3 co-immunoprecipitate. In addition, we show that a dominant active form of MEKK3 stimulates BMK1 activity through MEK5. Moreover, we demonstrate that MEKK3 activity is required for growth factor mediated cellular activation of endogenous BMK1. Taken together, these results identify MEKK3 as a kinase that regulates the activity of MEK5 and BMK1 during growth factor-induced cellular stimulation.  (+info)

(3/76) Involvement of the MKK6-p38gamma cascade in gamma-radiation-induced cell cycle arrest.

The p38 group of kinases belongs to the mitogen-activated protein (MAP) kinase superfamily with structural and functional characteristics distinguishable from those of the ERK, JNK (SAPK), and BMK (ERK5) kinases. Although there is a high degree of similarity among members of the p38 group in terms of structure and activation, each member appears to have a unique function. Here we show that activation of p38gamma (also known as ERK6 or SAPK3), but not the other p38 isoforms, is required for gamma-irradiation-induced G(2) arrest. Activation of the MKK6-p38gamma cascade is sufficient to induce G(2) arrest in cells, and expression of dominant negative alleles of MKK6 or p38gamma allows cells to escape the DNA damage-induce G(2) delay. Activation of p38gamma is dependent on ATM and leads to activation of Cds1 (also known as Chk2). These data suggest a model in which activation of ATM by gamma irradiation leads to the activation of MKK6, p38gamma, and Cds1 and that activation of both MKK6 and p38gamma is essential for the proper regulation of the G(2) checkpoint in mammalian cells.  (+info)

(4/76) MEKK2 associates with the adapter protein Lad/RIBP and regulates the MEK5-BMK1/ERK5 pathway.

MEKK2 and MEKK3 are two closely related mitogen-activated protein kinase (MAPK) kinase kinases. The kinase domains of MEKK2 and MEKK3 are nearly identical, although their N-terminal regulatory domains are significantly divergent. By yeast two-hybrid library screening, we have identified MEK5, the MAPK kinase in the big mitogen-activated protein kinase 1 (BMK1)/ERK5 pathway, as a binding partner for MEKK2. MEKK2 expression stimulates BMK1/ERK5 activity, the downstream substrate for MEK5. Compared with MEKK3, MEKK2 activated BMK1/ERK5 to a greater extent, which might correlate with a higher affinity MEKK2-MEK5 interaction. A dominant negative form of MEK5 blocked the activation of BMK1/ERK5 by MEKK2, whereas activation of c-Jun N-terminal kinase (JNK) was unaffected, showing that MEK5 is a specific downstream effector of MEKK2 in the BMK1/ERK5 pathway. Activation of BMK1/ERK5 by epidermal growth factor and H2O2 in Cos7 and HEK293 cells was completely blocked by a kinase-inactive MEKK3 (MEKK3kin(-)), whereas MEKK2kin(-) had no effect. However, in D10 T cells, expression of MEKK2kin(-) but not MEKK3kin(-) inhibited BMK1/ERK5 activity. Two-hybrid screening also identified Lck-associated adapter/Rlk- and Itk-binding protein (Lad/RIBP), a T cell adapter protein, as a binding partner for MEKK2. MEKK2 and Lad/RIBP colocalize at the T cell contact site with antigen-loaded presenting cells, demonstrating cotranslocation of MEKK2 and Lad/RIBP during T cell activation. MEKK3 neither binds Lad/RIBP nor is recruited to the T cell contact with antigen presenting cell. MEKK2 and MEKK3 are differentially associated with signaling from specific upstream receptor systems, whereas both activate the MEK5-BMK1/ERK5 pathway.  (+info)

(5/76) ERK5 and ERK2 cooperate to regulate NF-kappaB and cell transformation.

We have previously demonstrated an involvement of MEK5 and ERK5 in RafBXB-stimulated focus formation in NIH3T3 cells. We find here that MEK5 and ERK5 cooperate with the RafBXB effectors MEK1/2 and ERK1/2 to induce foci. To further understand MEK5-ERK5-dependent signaling, we examined potential MEK5-ERK5 effectors that might influence focus-forming activity. Consistent with results from our focus-formation assays, constitutively active variants of MEK5 and MEK1 synergize to activate NF-kappaB, and MEK5 and ERK5 are required for activation of NF-kappaB by RafBXB. The MEK5-ERK5 pathway is also sufficient to activate both NF-kappaB and p90 ribosomal S6 kinase. Our results support the hypothesis that NF-kappaB and p90 ribosomal S6 kinase are involved in MEK5-ERK5-dependent focus formation and may serve as integration points for ERK5 and ERK1/2 signaling.  (+info)

(6/76) MEK5, a new target of the atypical protein kinase C isoforms in mitogenic signaling.

The MEK5-extracellular signal-regulated kinase (ERK5) tandem is a novel mitogen-activated protein kinase cassette critically involved in mitogenic activation by the epidermal growth factor (EGF). The atypical protein kinase C isoforms (aPKCs) have been shown to be required for cell growth and proliferation and have been reported to interact with the adapter protein p62 through a short stretch of acidic amino acids termed the aPKC interaction domain. This region is also present in MEK5, suggesting that it may be an aPKC-binding partner. Here we demonstrate that the aPKCs interact in an EGF-inducible manner with MEK5 and that this interaction is required and sufficient for the activation of MEK5 in response to EGF. Consistent with the role of the aPKCs in the MEK5-ERK5 pathway, we show that zetaPKC and lambda/iotaPKC activate the Jun promoter through the MEF2C element, a well-established target of ERK5. From all these results, we conclude that MEK5 is a critical target of the aPKCs during mitogenic signaling.  (+info)

(7/76) Differential regulation of mitogen-activated protein kinases ERK1/2 and ERK5 by neurotrophins, neuronal activity, and cAMP in neurons.

Activation of the extracellular signal-regulated kinase 1 (ERK1) and ERK2 by neurotrophins, neuronal activity, or cAMP has been strongly implicated in differentiation, survival, and adaptive responses of neurons during development and in the adult brain. Recently, a new member of the mitogen-activated protein (MAP) kinase family, ERK5, was discovered. Like ERK1 and ERK2, ERK5 is expressed in neurons, and ERK5 stimulation by epidermal growth factor is blocked by the MAP kinase/ERK kinase 1 (MEK1) inhibitors PD98059 and U0126. This suggests the interesting possibility that some of the functions attributed to ERK1/2 may be mediated by ERK5. However, the regulatory properties of ERK5 in primary cultured neurons have not been reported. Here we examined the regulation of ERK5 signaling in primary cultured cortical neurons. Our data demonstrate that, similar to ERK1/2, ERK5 is activated by neurotrophins including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4. BDNF stimulation of ERK5 required the activity of MEK5. Surprisingly, ERK5 was not stimulated by cAMP or neuronal activity induced by glutamate or membrane depolarization. In contrast to ERK1/2, ERK5 strongly activated the transcriptional activity of myocyte enhancer factor 2C (MEF2C) in pheochromocytoma 12 (PC12) cells and was required for neurotrophin stimulation of MEF2C transcription in both PC12 cells and cortical neurons. Furthermore, ERK1/2, but not ERK5, induced transcription from Elk1 and the cAMP/ Ca(2+) response element in PC12 cells. Our data suggest that mechanisms for regulation of ERK5 and downstream transcriptional pathways regulated by ERK5 are distinct from those of ERK1/2 in neurons. Furthermore, ERK5 is the first MAP kinase identified whose activity is stimulated by neurotrophins but not by neuronal activity.  (+info)

(8/76) A novel mitogen-activated protein kinase is responsive to Raf and mediates growth factor specificity.

The proto-oncogene Raf is a major regulator of growth and differentiation. Previous studies from a number of laboratories indicate that Raf activates a signaling pathway that is independent of the classic MEK1,2-ERK1,2 cascade. However, no other signaling cascade downstream of Raf has been identified. We describe a new member of the mitogen-activated protein kinase family, p97, an ERK5-related kinase that is activated and Raf associated when cells are stimulated by Raf. Furthermore, p97 is selectively responsive to different growth factors, providing a mechanism for specificity in cellular signaling. Thus, p97 is activated by the neurogenic factor fibroblast growth factor (FGF) but not the mitogenic factor epidermal growth factor (EGF) in neuronal cells. Conversely, the related kinase ERK5 is activated by EGF but not FGF. p97 phosphorylates transcription factors such as Elk-1 and Ets-2 but not MEF2C at transactivating sites, whereas ERK5 phosphorylates MEF2C but not Elk-1 or Ets-2. Finally, p97 is expressed in a number of cell types including primary neural and NIH 3T3 cells. Taken together, these results identify a new signaling pathway that is distinct from the classic Raf-MEK1,2-ERK1,2 kinase cascade and can be selectively stimulated by growth factors that produce discrete biological outcomes.  (+info)